Diet formulations

Abstract

Provided herein are poultry diets comprising a phytase polypeptide or fragment thereof, wherein the diet is free or substantially free of inorganic phosphate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 950,747 filed on December 19, 2019 and U.S. Provisional Patent Application No. 63 / 075,972 filed on September 9, 2020, the disclosures of each of which are incorporated by reference in their entirety.

[0003] Incorporation by Reference

[0004] The sequence listing provided in the form of a file of 130KB in size and named "20191217_NB41781-US-PSP_Sequence Listing_ST25", created and concurrently filed on December 17, 2019, is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to poultry diets containing no or substantially no or reduced inorganic phosphates and / or no meat and bone meal and containing engineered phytase polypeptides and their use for enhancing animal performance. Background Art

[0006] Phytase is the most commonly used exogenous enzyme in monogastric animal feeds. Phytase can reduce the anti-nutritional effects of phytate and improve the digestibility of phosphorus, calcium, amino acids and energy, as well as reduce the negative impact of inorganic phosphorus excretion on the environment.

[0007] Phytate is the major storage form of phosphorus in cereals and legumes. However, monogastric animals such as pigs, poultry and fish cannot effectively metabolize or absorb phytate (or phytic acid) in their diets and therefore excrete it, leading to phosphorus pollution in areas of intensive livestock production. In addition, phytic acid also acts as an anti-nutrient in monogastric animals by chelating metal agents such as calcium, copper and zinc, and forming insoluble complexes with proteins and amino acids in various sections of the digestive tract.

[0008] Phytate is usually hydrolyzed to produce lower inositol phosphates and inorganic phosphates by the action of exogenous phytase. Phytase is used as an additive for animal feed, wherein they improve the availability of organic phosphorus for animals and reduce the phosphate pollution of the environment (Wodzinski RJ, Ullah A H. Adv Appl Microbiol. [Progress in applied microbiology] 42, 263-302 (1996)). However, despite the effect of the phytase provided in animal feed, in order to provide enough phosphates for the growth and health of these animals, inorganic phosphorus must still be added to their diets, particularly in young broilers of 0-10 days old. Such interpolation may be expensive, and further increases the pollution problem.

[0009] Therefore, there is a need to develop animal diets, particularly for poultry, which are substantially or completely free of inorganic phosphates while still ensuring normal growth characteristics at all growth stages compared to phytase-containing diets supplemented with inorganic phosphate sources (such as monocalcium phosphate and / or meat and bone meal). Summary of the invention

[0010] In particular, animal diets containing phytase are provided herein, which contain no or substantially no exogenously added inorganic phosphate or contain significantly reduced exogenously added inorganic phosphate. When fed to poultry, these diets ensure normal growth at all developmental stages compared to diets supplemented with inorganic phosphate.

[0011] Therefore, in one aspect, the present invention provides a poultry diet comprising (i) an engineered phytase polypeptide or a fragment thereof containing phytase activity; and (ii) no or substantially no inorganic phosphorus. In some embodiments, the phytase polypeptide or a fragment thereof containing phytase activity has at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the phytase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, NO:36 and SEQ ID NO:37. In some embodiments of any of the embodiments disclosed herein, the diet further comprises one or more of: (a) a direct-fed microbial comprising at least one bacterial strain, (b) at least one other enzyme, and / or (c) an essential oil. In some embodiments of any of the embodiments disclosed herein, the engineered phytase polypeptide or fragment thereof is present in an amount of at least about 0.1 g / ton of feed. In some embodiments of any of the embodiments disclosed herein, the diet further comprises about 0.62% to 0.72% in the starter diet, about 0.54% to 0.64% in the growing diet, and / or about 0.42% to 0.55% calcium in the finishing diet. In some embodiments, the finishing diet comprises about 0.46% to 0.55% calcium. In other embodiments, the finishing diet comprises about 0.42% to 0.50% calcium. In some embodiments of any of the embodiments disclosed herein, the diet comprises about 1.18% to 1.22% amino acids (e.g., digestible lysine) in a starter diet, about 1.06% to 1.10% in a grower diet, and / or about 0.88% to 1.0% in a finisher diet.In some embodiments, the fattening diet comprises about 0.96% to 1.0% amino acids (e.g., digestible lysine). In other embodiments, the fattening diet comprises about 0.88% to 0.92% amino acids (e.g., digestible lysine). In some embodiments of any of the embodiments disclosed herein, the diet comprises about 2824 to 2950 kcal / kg in a starter diet, about 2924 to 3050 kcal / kg in a growth diet, and / or about 2970 to 3120 kcal / kg metabolizable energy in a fattening diet. In some embodiments, the fattening diet comprises about 2970 to 3100 kcal / kg metabolizable energy. In other embodiments, the fattening diet comprises about 2994 to 3120 kcal / kg metabolizable energy. In some embodiments of any of the embodiments disclosed herein, for all stages (starter, growth and fattening diets), the diet comprises about 0.13% to 0.17% sodium. In some embodiments of any of the embodiments disclosed herein, the diet contains a phytate source comprising one or more of corn, wheat, soybean meal, rapeseed, rice and / or wheat bran. In some embodiments of any of the embodiments disclosed herein, the diet further comprises oat husks. In some embodiments of any of the embodiments disclosed herein, the diet lacks meat and / or bone meal. In some embodiments of any of the embodiments disclosed herein, the diet further comprises one or more additional feed enzymes selected from the group consisting of xylanases, proteases, amylases and glucoamylases. In some embodiments of any of the embodiments disclosed herein, the diet is a starter diet. In some embodiments of any of the embodiments disclosed herein, the diet is a growing diet. In some embodiments of any of the embodiments disclosed herein, the diet is a fattening diet. In some embodiments of any of the embodiments disclosed herein, the poultry is a broiler or laying hen.

[0012] In other aspects, there is provided herein a method for improving animal performance in terms of one or more indicators, the method comprising administering to the animal an effective amount of any diet disclosed herein. In some embodiments, the one or more indicators are selected from the group consisting of: increased feed efficiency, increased weight gain, reduced feed conversion rate, improved digestibility of nutrients or energy in feed, improved nitrogen retention, improved ability to avoid the negative effects of necrotic enteritis, and improved immune response. In some embodiments of any embodiments disclosed herein, the animal is poultry. In some embodiments of any embodiments disclosed herein, the poultry is selected from the group consisting of turkeys, ducks, chickens, geese, pheasants, quails, and emus. In some embodiments, the chicken is selected from the group consisting of laying hens and broilers. In some embodiments, the egg production of laying hens is increased by up to 12.5% ​​compared to the egg production of laying hens fed with a diet containing no inorganic phosphate that is not supplemented with any phytase disclosed herein. In other embodiments, the egg quality is increased by up to 15.6%, compared to the egg quality of laying hens fed with a diet containing no inorganic phosphate that is not supplemented with any phytase disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A and Figure 1B Bar graphs depicting the effect of the interaction between diet (PC / IPF) and sex (F / M) on FCR (g / g) during (a) days 0 to 22 and (b) days 0 to 42. Means with different superscript letters are significantly different at P < 0.05; comparisons of treatment means were performed by Tukey's test. IPF = inorganic phosphate free; F = female; M = male.

[0014] Sequence Description

[0015] The following sequence complies with 37 CFR §§1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures—the Sequence Rules”) and conforms to World Intellectual Property Organization (WIPO) Standard ST.25 (2009), and Rules 5.2 and 49.5(a-bis) of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT), and Section 208 and Annex C of the Administrative Regulations regarding sequence listings. The symbols and formats used for nucleotide and amino acid sequence data follow the rules set forth in 37 CFR §1.822.

[0016] SEQ ID NO: 1 corresponds to the predicted mature sequence of the engineered phytase PHY-13594.

[0017] SEQ ID NO: 2 corresponds to the predicted mature sequence of the engineered phytase PHY-10931.

[0018] SEQ ID NO: 3 corresponds to the predicted mature sequence of the engineered phytase PHY-10957.

[0019] SEQ ID NO: 4 corresponds to the predicted mature sequence of the engineered phytase PHY-11569.

[0020] SEQ ID NO: 5 corresponds to the predicted mature sequence of the engineered phytase PHY-11658.

[0021] SEQ ID NO: 6 corresponds to the predicted mature sequence of the engineered phytase PHY-11673.

[0022] SEQ ID NO: 7 corresponds to the predicted mature sequence of the engineered phytase PHY-11680.

[0023] SEQ ID NO: 8 corresponds to the predicted mature sequence of the engineered phytase PHY-11895.

[0024] SEQ ID NO: 9 corresponds to the predicted mature sequence of the engineered phytase PHY-11932.

[0025] SEQ ID NO: 10 corresponds to the predicted mature sequence of the engineered phytase PHY-12058.

[0026] SEQ ID NO: 11 corresponds to the predicted mature sequence of the engineered phytase PHY-12663.

[0027] SEQ ID NO: 12 corresponds to the predicted mature sequence of the engineered phytase PHY-12784.

[0028] SEQ ID NO: 13 corresponds to the predicted mature sequence of the engineered phytase PHY-13177.

[0029] SEQ ID NO: 14 corresponds to the predicted mature sequence of engineered phytase PHY-13371

[0030] SEQ ID NO: 15 corresponds to the predicted mature sequence of the engineered phytase PHY-13460.

[0031] SEQ ID NO: 16 corresponds to the predicted mature sequence of the engineered phytase PHY-13513.

[0032] SEQ ID NO: 17 corresponds to the predicted mature sequence of the engineered phytase PHY-13637.

[0033] SEQ ID NO: 18 corresponds to the predicted mature sequence of the engineered phytase PHY-13705.

[0034] SEQ ID NO: 19 corresponds to the predicted mature sequence of the engineered phytase PHY-13713.

[0035] SEQ ID NO: 20 corresponds to the predicted mature sequence of the engineered phytase PHY-13747.

[0036] SEQ ID NO: 21 corresponds to the predicted mature sequence of the engineered phytase PHY-13779.

[0037] SEQ ID NO: 22 corresponds to the predicted mature sequence of the engineered phytase PHY-13789.

[0038] SEQ ID NO: 23 corresponds to the predicted mature sequence of the engineered phytase PHY-13798.

[0039] SEQ ID NO: 24 corresponds to the predicted mature sequence of the engineered phytase PHY-13868.

[0040] SEQ ID NO: 25 corresponds to the predicted mature sequence of the engineered phytase PHY-13883.

[0041] SEQ ID NO: 26 corresponds to the predicted mature sequence of the engineered phytase PHY-13885.

[0042] SEQ ID NO: 27 corresponds to the predicted mature sequence of the engineered phytase PHY-13936.

[0043] SEQ ID NO: 28 corresponds to the predicted mature sequence of the engineered phytase PHY-14004.

[0044] SEQ ID NO: 29 corresponds to the predicted mature sequence of the engineered phytase PHY-14215.

[0045] SEQ ID NO: 30 corresponds to the predicted mature sequence of the engineered phytase PHY-14256.

[0046] SEQ ID NO: 31 corresponds to the predicted mature sequence of the engineered phytase PHY-14277.

[0047] SEQ ID NO: 32 corresponds to the predicted mature sequence of the engineered phytase PHY-14473.

[0048] SEQ ID NO: 33 corresponds to the predicted mature sequence of the engineered phytase PHY-14614.

[0049] SEQ ID NO: 34 corresponds to the predicted mature sequence of the engineered phytase PHY-14804.

[0050] SEQ ID NO: 35 corresponds to the predicted mature sequence of the engineered phytase PHY-14945.

[0051] SEQ ID NO: 36 corresponds to the predicted mature sequence of the engineered phytase PHY-15459.

[0052] SEQ ID NO: 37 corresponds to the predicted mature sequence of the engineered phytase PHY-16513. DETAILED DESCRIPTION

[0053] There is a growing global awareness of sustainability regarding the proper use of limited resources such as inorganic phosphorus (P) and the reduction of P pollution. Although poultry has the lowest environmental impact of all animal proteins, in commercial practice, it still uses significant amounts of inorganic phosphates. The inventors of the present application surprisingly discovered that the use of the next generation biosynthetic bacterial 6-phytase in broiler diets can completely eliminate the need to supplement the diet with one or more inorganic phosphate sources and / or can significantly reduce the need to supplement the diet with one or more inorganic phosphate sources. All animals fed a diet without inorganic phosphate supplemented with phytase had comparable or better growth and feed conversion than animals fed a diet containing exogenously added inorganic phosphate. In addition, tests showed that these animals did not show symptoms of bone loss or demineralization characteristic of animals experiencing phosphate deficiency. In contrast, the bone breaking strength of animals fed a diet without inorganic phosphate supplemented with phytase did not deteriorate compared to the control. This represents the first report of a 100% plant, completely inorganic-free, commercially relevant broiler diet showing normal growth characteristics at all growth stages. Thus, the use of the inorganic phosphate-free diets supplemented with phytase disclosed herein provides both economic advantages in the form of reduced feed costs, particularly for large, industrial-scale flocks, and significant environmental benefits due to reduced phosphate pollution as a byproduct of large-scale poultry production.

[0054] All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.

[0055] In this disclosure, a number of terms and abbreviations are used. Unless otherwise specifically stated, the following definitions apply.

[0056] As used herein, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. For example, the terms "a / an", "the", "one or more", and "at least one" may be used interchangeably herein.

[0057] The terms "and / or" and "or" are used interchangeably herein and refer to a specific disclosure that each of two specified features or components has or does not have the other. Thus, the term "and / or" as used in the phrase "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in the phrase "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] Words using the singular include the plural and vice versa.

[0059] The terms "comprises / comprising", "includes / including", "having" and variations thereof are used interchangeably and mean "including but not limited to". It should be understood that wherever an aspect is described with the language "comprising", similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0060] The term "consisting of" means "including and limited to."

[0061] The term "consisting essentially of" means the specified materials of a composition or the specified steps of a process, as well as those additional materials or steps that do not materially affect the basic characteristics of the materials or process.

[0062] Throughout the application, various embodiments can be presented in the form of range.It should be understood that the description of range form is only for convenience and simplicity, and should not be construed as an inflexible limitation of the scope of embodiments described herein.Therefore, the description of scope should be considered to have all possible sub-ranges disclosed and the individual numerical values ​​in the scope.For example, the description of scope such as 1 to 6 should be considered to have the individual numerical values ​​disclosed in the scope, such as 1 to 2, 1 to 3, 1 to 4 and 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6 etc., and the individual numerical values ​​in the scope, for example 1, 2, 3, 4, 5 and 6.No matter how the width of the scope, this is applicable.

[0063] As used herein, the term "about" can allow for a certain degree of variability in values ​​or ranges, for example, within 10%, within 5%, or within 1% of a stated value or stated limit of a range.

[0064] The term "phytase" (inositol hexaphosphate phosphohydrolase) refers to a class of phosphatases that catalyze the hydrolysis of phytic acid (inositol hexaphosphate or IP6), an indigestible organic form of phosphorus found in cereals and oil seeds, and release inorganic phosphorus in a usable form.

[0065] As used herein, "poultry" refers to birds such as turkeys, ducks, chickens, broilers, and laying hens.

[0066] The reference book "Commercial Poultry Nutrition" (3rd edition, 2005, ISBN 0-9695600-5-2; incorporated herein by reference) is a standard textbook in the field of nutritional aspects of chicken production. The following is a summary of the background believed to be relevant to the present invention.

[0067] The main ingredients for delivering energy in poultry diets are corn, soybeans, soybean oil and amino acids. Corn is the main contributor to metabolizable energy. Endosperm starch, mainly composed of amylopectin, and germ, which is mainly oil, constitute the energy value of corn. The typical energy value of individual corn expressed in kcal / kg at 85% dry matter is in the range of 3014 to 3313. The energy level of starter and growth diets is usually in the range of 3000 to 3100 Kcal / kg. In many countries, wheat is also commonly used in poultry diets. Such diets have energy levels similar to those described above. Soybeans as a protein source have become the world standard for other protein sources to compare with. The amino acid profile of soybeans is excellent for most types of poultry, and when combined with corn or sorghum, methionine is usually the only factor of insufficient amount. In addition, fats and oils provide concentrated energy sources in the diet, and even relatively small changes in levels can have a significant effect on dietary ME. Finally, the diet is supplemented with synthetic amino acids such as methionine and lysine. Other important sources used are barley, sorghum and other cereals, as well as their by-products that contribute to energy.

[0068] Chickens for optimized commercial broiler production are usually fed different diets depending on their age. For example, chickens for broiler production can be fed with three or four diets. These diets are usually referred to as "starter," "growth," and "finishing." A "pre-starter" diet is also possible. Therefore, the engineered phytase disclosed herein can be included in a starter diet only, a grower diet only, a fattening diet only, a combination of any two stages, or a combination of all stages at different dosage levels, as long as they do not contain or are substantially free of inorganic phosphate.

[0069] "Inorganic phosphorus" and "inorganic phosphate" are used interchangeably herein to refer to dietary supplements that are typically added to poultry feed to ensure that the animal receives sufficient phosphate to meet the animal's nutritional requirements. However, the poultry diets disclosed herein are free or substantially free of inorganic phosphate when the diet also comprises an appropriate dose of an engineered phytase polypeptide disclosed herein and the diet contains sufficient phytate as a substrate. The expressions "substantially none or substantially no" used herein to describe the amount of inorganic phosphate in the dietary formulations disclosed herein mean that any amount present is a trace amount, an amount that is inadvertently included, and / or an amount that is less than about 0.1% in the diet.

[0070] "Starter," "growing" and "finishing" diets are often distinguished by crude protein content, which is often provided by ingredients such as soybean meal (SBM). For example, a starter diet for broilers may optionally contain a crude protein content of about 22-25% by weight, such as 22%, 23%, 24% or 25%, with 23% or 25% being preferred. In another example, a grower diet for broilers may optionally contain a crude protein content of about 21-23% by weight, such as 21%, 22% or 23%, with 22% being preferred. In another example, a finishing diet for broilers may optionally contain a crude protein content of about 19-23% by weight, such as 19%, 20%, 21%, 22% or 23%, with 19%, 20% or 21% being preferred.

[0071] Additionally or alternatively, "starter," "growing," and "finishing" can be distinguished by metabolizable energy (ME) content, which is generally lowest for starter diets and highest for finishing diets, with growing diets having levels in between. For example, a starter diet for broilers may have an ME of about 3000 or 3025 kcal / kg (±50, 40, 30, 20, 10, 5 or less kcal / kg). In another example, a growing diet for broilers may have an ME of about 3100 kcal / kg or 3150 kcal / kg (±50, 40, 30, 20, 10, 5 or less kcal / kg). In another example, a growing diet for broilers may have an ME of about 3200 kcal / kg (±50, 40, 30, 20, 10, 5 or less kcal / kg).

[0072] The poultry diets described herein can be vegetarian or non-vegetarian products. Vegetarian products do not contain meat or fish products. Non-vegetarian diets can contain one or both of fish products (such as fish meal) or meat products (such as meat derivatives or other meat products that do not contain inorganic phosphates).

[0073] The terms "mixer liquid application" and "MLA" are used interchangeably herein and refer to animal feed production in which heat-sensitive compounds, particularly enzymes, can be applied to animal feed in liquid form prior to conditioning and pelleting and remain functional in the feed after conditioning and pelleting.

[0074] The terms "feed", "animal feed" or "diet" are used interchangeably herein to refer to any natural or artificial diet, meal or the like, or a component of such a diet, intended or suitable for consumption, ingestion, digestion by non-human animals, respectively. Preferably, the term "feed" is used to refer to products fed to animals when raising livestock.

[0075] As used herein, "feed additive" refers to one or more ingredients, substances (e.g., cells) used alone or together in nutrition, for example to improve the quality of food (e.g., animal feed), improve the performance and / or health of animals and / or enhance the digestibility of food or substances in food.

[0076] As used herein, the term "food" is used in a broad sense - and encompasses any form of food and food products for humans as well as food (ie, feed) for animals.

[0077] The food or feed may be in solution form or in solid form, depending on the use and / or mode of application and / or mode of administration. In some embodiments, the enzymes mentioned herein may be used as food or feed material or in the preparation or production of food or feed material.

[0078] As used herein, the term "food or feed ingredient" includes preparations that are added or can be added to food or foods, and includes preparations that can be used at low levels in various products. The food ingredient can be in solution form or solid form, depending on the use and / or application mode and / or administration mode. The enzymes described herein can be used as food or feed ingredients or for preparation or production. These enzymes may or may not be added to food supplements. Feed compositions for monogastric animals generally include compositions comprising plant products that contain phytates. Such compositions include, but are not limited to, corn meal, soybean meal, rapeseed meal, cottonseed meal, corn, wheat, barley, and sorghum-based feeds.

[0079] As used herein, the term "granulation" refers to pellets, which may be solid, round, spherical and cylindrical tablets, particularly feed pellets and the production of solid extruded animal feeds. An example of a known feed pelleting manufacturing process generally includes mixing the food or feed ingredients together at room temperature for at least 1 minute, transferring the mixture to a surge bin, conveying the mixture to a steam conditioner (i.e., conditioning), optionally transferring the steam-conditioned mixture to an expander, transferring the mixture to a pelletizer or extruder, and finally transferring the pellets to a pellet cooler. (Fairfield, D. 1994. Chapter 10, Pelleting Cost Center. In Feed Manufacturing Technology IV. (McEllhiney, ed.), American Feed Industry Association, Arlington, Va., pp. 110-139).

[0080] The term "pellet" refers to a composition of animal feed (usually derived from cereals) that has been subjected to a heat treatment, such as steam treatment (i.e., conditioning), and pressed or extruded by a machine. The pellets may be incorporated with enzymes in the form of liquid formulations or dry formulations. Dry formulations may be coated or uncoated and may be in granular form. The term "granule" is used for particles composed of enzymes (such as phytases, e.g., any of the engineered phytases disclosed herein) and other chemicals (such as salts and sugars), and may be formed using any of a variety of techniques (including fluidized bed granulation methods to form layered granules).

[0081] The term phytase activity in relation to determination in solid or liquid formulations means 1 FTU (phytase unit) which is defined as the amount of enzyme required to liberate 1 micromole of inorganic orthophosphate from 5.0 mM sodium phytate substrate (from rice) within one minute under reaction conditions of 37°C, pH 5.5, as also defined in the ISO 2009 phytase assay (a standard assay for determining phytase activity, see International Standard ISO / DIS 30024:1-17, 2009).

[0082] Alternatively, as used herein, one unit of phytase (U) may be defined as the amount of enzyme that releases 1 micromole of inorganic orthophosphate from 0.2 mM sodium phytate substrate (from rice) in one minute in a malachite green assay at 25°C and pH 5.5 or 3.5, respectively.

[0083] As used herein, the term "specific activity" is the number of enzyme units per milliliter divided by the (total) protein concentration in mg / ml (mg / ml). Therefore, specific activity values ​​are usually expressed in units / mg. Alternatively, the term "specific activity" is the number of enzyme units per milliliter divided by the phytase concentration in mg / ml.

[0084] As used herein, the term "differential scanning calorimetry" or "DSC" is a thermal analysis technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured as a function of temperature. Both the sample and the reference are maintained at nearly the same temperature throughout the experiment. Typically, the temperature program for a DSC analysis is designed so that the sample holder temperature increases linearly as a function of time. The reference sample should have a well-defined heat capacity over the temperature range to be scanned.

[0085] The term "probiotic" means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and / or activity of one or a limited number of beneficial bacteria.

[0086] As used herein, the term "direct-fed microorganism" ("DFM") is a source of live (viable) microorganisms that, when applied in sufficient quantities, can confer a benefit, i.e., a probiotic, on its recipient. A DFM may contain one or more such microorganisms, such as bacterial strains. Classes of DFM include bacillus, lactic acid bacteria, and yeast. Thus, the term DFM encompasses one or more of: direct-fed bacteria, direct-fed yeast, direct-fed yeast, and combinations thereof.

[0087] Bacilli are unique, spore-forming, Gram-positive rods. These spores are very stable and can withstand environmental conditions such as heat, moisture, and a range of pH. These spores germinate into active, vegetative cells when ingested by the animal and can be used in meal and pellet diets. Lactic acid bacteria are Gram-positive cocci that produce lactic acid, which has an antagonistic effect on pathogens. Since lactic acid bacteria appear to be somewhat heat-sensitive, they are not used in pellet diets. Types of lactic acid bacteria include Bifidobacterium, Lactobacillus, and Streptococcus.

[0088] The terms "probiotics", "probiotic cultures" and "DFM" are used interchangeably herein and define live microorganisms (including, for example, bacteria or yeast) that beneficially affect a host organism (i.e., by conferring one or more demonstrable health benefits such as health, digestion, and / or performance benefits) when, for example, ingested in sufficient quantities or applied topically. Probiotics can improve the microbial balance of one or more mucosal surfaces. For example, a mucosal surface can be the intestine, urinary tract, respiratory tract, or skin. As used herein, the term "probiotic" also encompasses live microorganisms that can stimulate beneficial arms of the immune system and simultaneously reduce inflammatory responses in mucosal surfaces (e.g., the intestine). Although there is no lower or upper limit for probiotic intake, it has been shown that at least 10 6 -10 12 , preferably at least 10 6 -10 10 , preferably 10 8 -10 9 cfu as a daily dose will be effective to achieve beneficial health effects in the subjects.

[0089] As used herein, the term "CFU" means "colony forming unit" and is a measure of viable cells, wherein a colony represents a collection of cells derived from a single progenitor cell.

[0090] The term "isolated" means a substance in a form or environment that does not exist in nature, and does not reflect the degree to which an isolate has been purified, but rather indicates separation or separation from a native form or natural environment. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance that is at least partially removed from one or more or all of the naturally occurring components with which it is naturally associated, including but not limited to any host cell, enzyme, engineered enzyme, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to that found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated. The terms "isolated nucleic acid molecule," "isolated polynucleotide," and "isolated nucleic acid fragment" will be used interchangeably and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or altered nucleotide bases. An isolated nucleic acid molecule in the form of a DNA polymer may be composed of one or more fragments of cDNA, genomic DNA, or synthetic DNA.

[0091] The terms "purify," "purified," and purification mean to remove unwanted components, material contaminants, admixtures, or imperfections to render substantially pure or clean. For example, purification, as applied to nucleic acids or polypeptides, generally refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces substantially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., weight percentage on a molar basis). In a related sense, a molecule in a composition is enriched when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other specific substance or component in a composition at a relative or absolute concentration greater than that of the starting composition.

[0092] The terms "peptide", "protein" and "polypeptide" are used interchangeably herein and refer to polymers of amino acids linked together by peptide bonds. "Protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. Single-letter and 3-letter codes of amino acids defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. Single letter X refers to any of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. Mutations can be named by the single letter code of the parent amino acid, followed by the position number, and then the single letter code of the variant amino acid. For example, mutation of glycine (G) at position 87 to serine (S) is represented as "G087S" or "G87S". When describing a modification, the position following the amino acid listed in brackets indicates a list of amino acid substitutions at that position that are listed by any of the amino acids. For example, 6 (L, I) means that position 6 can be substituted by leucine or isoleucine. Sometimes in a sequence, a slash ( / ) is used to define a substitution, for example, F / V indicates that either phenylalanine or valine may be present at that position.

[0093] As used herein, with respect to amino acid residue positions, "corresponding to" or "corresponds to" or "corresponds to" refers to the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or identical to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related protein or reference protein.

[0094] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by a strain of the organism in question, but also to proteins encoded by a DNA sequence isolated from such a strain and produced in a host organism containing such a DNA sequence. In addition, the term refers to proteins encoded by a DNA sequence of synthetic and / or cDNA origin and having the identifying characteristics of the protein in question.

[0095] The term "amino acid" refers to the basic chemical building block of a protein or polypeptide. The following abbreviations used herein to identify specific amino acids can be found in Table A.

[0096] Table A. Single-letter and three-letter amino acid abbreviations

[0097]

[0098]

[0099] Those of ordinary skill in the art will recognize that the amino acid sequences disclosed herein may be modified while retaining the functions associated with the disclosed amino acid sequences. For example, it is well known in the art that genetic alterations that result in the production of chemically equivalent amino acids at a given site but do not affect the functional properties of the encoded protein are common.

[0100] The term "codon optimized," when it refers to the coding region of a gene or nucleic acid molecule for transformation of a variety of hosts, refers to altering the codons in the coding region of a gene or nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

[0101] As used herein, the term "transformation" refers to the transfer or introduction of a nucleic acid molecule into a host organism. The nucleic acid molecule can be introduced as a linear or circular form of DNA. The nucleic acid molecule can be an autonomously replicating plasmid, or it can be integrated into the genome of a production host. Production hosts containing transforming nucleic acids are referred to as "transformed" or "recombinant" or "transgenic" organisms or "transformants."

[0102] The terms "recombinant" and "engineered" refer to the artificial combination of two originally separated nucleic acid sequence fragments, for example, by chemical synthesis or by manipulating separated nucleic acid fragments through genetic engineering techniques. For example, DNA in which one or more fragments or genes from different molecules, from another part of the same molecule or an artificial sequence have been inserted naturally or through laboratory manipulation, resulting in the introduction of new sequences in the gene and subsequently in the organism. The terms "recombinant", "transgenic", "transformed", "engineered", "genetically engineered" and "modified for exogenous gene expression" are used interchangeably herein.

[0103] The terms "recombinant construct," "expression construct," "recombinant expression construct," and "expression cassette" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that do not exist together in nature. For example, a construct may comprise regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from that which occurs in nature. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector depends on the method that will be used to transform the host cell, as is well known to those skilled in the art. For example, a plasmid vector may be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells. The skilled artisan will also recognize that different independent transformation events may result in different expression levels and patterns (Jones et al., (1985) EMBO J [Journal of the European Molecular Biology Association] 4:2411-2418; De Almeida et al., (1989) Mol Gen Genetics [Molecular General Genetics] 218:78-86), and therefore typically screen multiple events to obtain strains that display the desired expression levels and patterns. Such screening can be accomplished using standard molecular biology, biochemistry, and other assays, including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzyme or activity assays, and / or phenotypic analysis.

[0104] The terms "production host," "host," and "host cell" are used interchangeably herein and refer to any plant, organism, or cell of any plant or organism, whether human or non-human, into which a recombinant construct can be introduced stably or transiently to express a gene. The term encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during reproduction.

[0105] The term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of nucleotides or amino acids that match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Primer [Sequence Analysis Primers] (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Methods for determining identity and similarity are codified in publicly available computer programs.

[0106] As used herein, "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. Useful algorithms include the BLAST algorithm (see, Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787, 1993). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res [Nucleic Acids Research], 25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res [Nucleic Acids Research], 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: neighborhood word threshold = 11; E-value cutoff = 10; scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap opening = 5; and gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap opening = 11; and gap extension = 1. Amino acid sequence identity percentage (%) values ​​are determined by dividing the number of identical residues matched by the total number of residues in the "reference" sequence. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0107] As used herein, "homologous proteins" or "homologous phytases" refer to proteins that have significant similarities in primary, secondary and / or tertiary structure. When aligning proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold (E-value cutoff) of 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein database search programs. Nucleic Acids Res 1997 Group 1; 25(17): 3389-402). Using this information, protein sequences can be grouped.

[0108] Sequence alignments and calculations of percent identity can be performed using the Megalign program of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, WI), the AlignX program of Vector NTI v.7.0 (Informax, Inc., Bethesda, MD), or the EMBOSS open source software package (EMBL-EBI; Rice et al., Trends in Genetics 16, (6): 276-277 (2000)). Multiple alignments of sequences can be performed using the CLUSTAL method of alignment (such as CLUSTALW; e.g., version 1.83) (Higgins and Sharp, CABIOS [Bioinformatics], 5:151-153 (1989); Higgins et al., Nucleic Acids Res. [Nucleic Acids Research] 22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res [Nucleic Acids Research] 31(13):3497-500 (2003), obtained from the European Molecular Biology Laboratory via the European Bioinformatics Institute) with default parameters. Suitable parameters for CLUSTALW protein alignments include gap existence penalty=15, gap extension=0.2, matrix=Gonnet (e.g., Gonnet250), protein ENDGAP=-1, protein GAPDIST=4, and KTUPLE=1. In one embodiment, fast or slow alignments use the default settings in the case of slow alignments. Alternatively, the parameters for using the CLUSTALW method (e.g., version 1.83) can be modified to also use KTUPLE=1, GAP PENALTY=10, GAP EXTENSION=1, MATRIX=BLOSUM (e.g., BLOSUM64), WINDOW=5, and SAVED TOP DIAGONAL=5. Alternatively, the method from Multiple sequence alignments were derived using MAFFT version 10.2.4 with default settings, scoring matrix BLOSUM62, gap open penalty 1.53 and offset value 0.123).

[0109] The MUSCLE program (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput [MUSCLE: multiple sequence alignment with high accuracy and high throughput] Nucl. Acids Res. [Nucleic Acids Research] (2004) 32(5): 1792-1797) is another example of a multiple sequence alignment algorithm.

[0110] Figure 2 depicts a phylogenetic or evolutionary tree showing the relatedness among various phytases, including engineered phytase polypeptides and fragments thereof, based on amino acid sequence similarities and differences.

[0111] The term "engineered phytase polypeptide" means a polypeptide that does not occur in nature and that has phytase activity.

[0112] Note that a fragment of an engineered phytase polypeptide is a portion or subsequence of an engineered phytase polypeptide that is capable of functioning like the engineered phytase polypeptide, ie, it retains phytase activity.

[0113] The term "vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, expression cassettes, and the like.

[0114] As used herein, "expression vector" means a DNA construct comprising a DNA sequence that is operably linked to a suitable control sequence capable of achieving DNA expression in a suitable host. Such control sequences may include a promoter that achieves transcription, an optional operator sequence that controls transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and sequences that control transcription and translation termination.

[0115] As used herein, the term "expression" refers to the production of a functional end product (eg, mRNA or protein) in either precursor or mature form. Expression may also refer to the translation of mRNA into a polypeptide.

[0116] The expression of a gene involves the transcription of the gene and the translation of mRNA into a precursor or mature protein. A "mature" protein refers to a polypeptide that has been processed post-translationally; that is, a polypeptide from which any signal sequence, prepeptide, or propeptide present in the primary translation product has been removed. A "precursor" protein refers to the primary product of mRNA translation; that is, the propeptide and propeptide are still present. The propeptide and propeptide can be, but are not limited to, intracellular localization signals. "Stable transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, including both the nuclear and organelle genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus of a host organism or into an organelle containing DNA, resulting in gene expression without integration or stable inheritance.

[0117] Thus, in one embodiment, a recombinant construct is described that comprises a regulatory sequence functional in a production host operably linked to a nucleotide sequence encoding an engineered phytase polypeptide and fragments thereof as described herein.

[0118] The recombinant construct may include a regulatory sequence that functions in a production host, which is operably connected to a nucleotide sequence encoding any engineered phytase polypeptide described herein and fragments thereof. In addition, the production host is selected from the group consisting of bacteria, fungi, yeast, plants or algae. A preferred production host is the filamentous fungus Trichoderma reesei.

[0119] Alternatively, it is possible to use cell-free protein synthesis as described in Chong, Curr Protoc Mol Biol. 2014; 108: 16.30.1-16.30.11.

[0120] Also described herein is a method for producing an engineered phytase polypeptide or fragment thereof, the method comprising:

[0121] (a) transforming a production host with a recombinant construct described herein; and

[0122] (b) culturing the production host of step (a) under conditions such that the engineered phytase polypeptide or fragment thereof is produced.

[0123] Optionally, the engineered phytase polypeptide or fragment thereof may be recovered from the production host.

[0124] In another aspect, a culture supernatant containing phytase may be obtained by any of the methods disclosed herein.

[0125] In another embodiment, a polynucleotide sequence encoding any of the engineered phytase polypeptides or fragments thereof as described herein is described.

[0126] The possible initiation control region or the promoter that can be included in the expression vector are numerous and are familiar to those skilled in the art." constitutive promoter " is a kind of promoter that is active under most environments and development conditions. " inducible " or " repressible " promoter refers to promoter that is active under environment or development regulation. In certain embodiments, due to the variation of environmental factors, promoter is inducible or repressible, and these environmental factors include but are not limited to the presence of carbon, nitrogen or other nutrients availability, temperature, pH, osmotic pressure, one or more heavy metals, the concentration of one or more inhibitors, pressure or the combination of aforementioned factors as known in the art. In certain embodiments, inducible or repressible promoter can be induced or repressed by metabolic factors, such as the level of some carbon sources as known in the art, the level of some energy sources, the level of some catabolites or the combination of aforementioned factors.

[0127] In one embodiment, the promoter is a native promoter of the host cell. For example, in some cases, when Trichoderma reesei is the host, the promoter can be a native Trichoderma reesei promoter, such as the cbhl promoter deposited in GenBank under Accession No. D86235. Other suitable non-limiting examples of promoters that can be used for fungal expression include cbh2, egl1, egl2, egl3, egl4, egl5, xyn1 and xyn2, the repressible acid phosphatase gene (phoA) promoter of Penicillium chrysogenus (see, e.g., Graessle et al., (1997) Appl. Environ. Microbiol., 63:753-756), the glucose-repressible PCK1 promoter (see, e.g., Leuker et al., (1997), Gene, 192:235-240), the maltose-inducible, glucose-repressible MET3 promoter (see Liu et al., (2006), Eukary. Cell, 5:638-649), the pKi promoter and the cpc1 promoter. Other examples of useful promoters include promoters from A. awamori and A. niger glucoamylase genes (see, e.g., Nunberg et al., (1984) Mol. Cell Biol. 15 4: 2306-2315 and Boel et al., (1984) EMBO J. 3: 1581-1585). In addition, the promoter of the Trichoderma reesei xln1 gene may be useful (see, e.g., EPA 137280A1).

[0128] The DNA fragment controlling transcription termination can also be derived from various natural genes of preferred production host cells. In certain embodiments, including a termination control region is optional. In certain embodiments, the expression vector includes a termination control region derived from a preferred host cell.

[0129] The terms "production host", "production host cell", "host cell" and "host strain" are used interchangeably herein and refer to a suitable host of an expression vector or DNA construct comprising a polynucleotide encoding a phytase polypeptide or fragment thereof. The choice of production host may be selected from the group consisting of bacteria, fungi, yeast, plants and algae. Typically, the choice will depend on the gene encoding the engineered phytase polypeptide or fragment thereof and its source.

[0130] In particular, the host strain is preferably a filamentous fungal cell.In a preferred embodiment of the present invention, "host cell" means cells and protoplasts produced by filamentous fungal strains, and in particular cells of Trichoderma species or Aspergillus species.

[0131] The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina (see Alexopoulos, CJ (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungi of the present invention differ from yeasts morphologically, physiologically and genetically. Vegetative growth of filamentous fungi is by hyphal elongation, and carbon catabolism is obligately aerobic. In the present invention, the filamentous fungal parent cell can be, but is not limited to, a cell of the following species: Trichoderma (e.g., Trichoderma reesei (formerly classified as T. longibrachiatum and currently also known as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, Trichoderma harzianum); Penicillium species, Humicola species (e.g., Humicola insolens and Humicola grisea) grisea), Chrysosporium species (e.g., C. lucknowense), Gliocladium species, Aspergillus species (e.g., A. oryzae, A. niger, and A. awamori), Fusarium species, Neurospora species, Hypocrea species, and Emericella species (see also Innis et al., (1985) Sci. [Science Citation Index] 228:21-26).

[0132] As used herein, the term "Trichoderma" or "Trichoderma species" refers to any genus of fungi previously or currently classified as Trichoderma.

[0133] The expression cassette may be included in a production host, particularly a cell of a microbial production host. The production host cell may be a microbial host found in the fungus family, and grows under a wide range of temperature, pH value, and solvent tolerance. For example, any one of an expected bacterium, yeast, plant, algae, or fungus (such as a filamentous fungus) may be suitably used as a host for an expression vector.

[0134] Including an expression cassette in a production host cell can be used to express the protein of interest so that it can be located intracellularly, extracellularly, or a combination of both. Extracellular expression makes it easier to recover the desired protein from the fermentation product compared to methods for recovering proteins produced by intracellular expression.

[0135] Methods for converting nucleic acids into filamentous fungi (such as Aspergillus spp., e.g. A. oryzae or A. niger, H. grisea, H. insolens and Trichoderma reesei) are well known in the art. Suitable procedures for transforming Aspergillus host cells are described in, e.g., EP 238023.

[0136] Suitable procedures for transforming Trichoderma host cells are described in, for example, Steiger et al. 2011, Appl. Environ. Microbiol. [Application and Environmental Microbiology] 77: 114-121. The uptake of DNA into the host Trichoderma species strain depends on the calcium ion concentration. Typically, about 10mM CaCl2 to 50mM CaCl2 is used in the uptake solution. In addition to the need for calcium ions in the uptake solution, other compounds that are typically included are buffer systems such as TE buffer (10MmTris, pH 7.4; 1mM EDTA) or 10mM MOPS (pH 6.0) buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that polyethylene glycol plays a role in fusing cell membranes, thereby allowing the contents of the culture medium to be delivered to the cytoplasm of the Trichoderma species strain and transferring the plasmid DNA to the nucleus. This fusion often leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0137] Usually, the transformation uses 5 Up to 10 7 / mL, preferably 2×10 6 / mL density of Trichoderma species protoplasts or cells subjected to osmotic treatment. These protoplasts or cells of 100 μL volume are mixed with the desired DNA in an appropriate solution (e.g., 1.2M sorbitol; 50mM CaCl2). Typically, a high concentration of PEG is added to the uptake solution. 0.1 to 1 times the volume of 25% PEG 4000 can be added to the protoplast suspension. However, adding about 0.25 times the volume to the protoplast suspension is preferred. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. can also be added to the uptake solution and help transformation. Similar procedures can be used for other fungal host cells. (See, for example, U.S. Patent Nos. 6,022,725 and 6,268,328, both of which are incorporated herein by reference).

[0138] Preferably, a genetically stable transformant is constructed using a vector system whereby the nucleic acid encoding the phytase polypeptide or fragment thereof is stably integrated into the host strain chromosome. The transformant is then purified by known techniques.

[0139] After the expression vector has been introduced into the cells, the transfected or transformed cells are cultured under conditions that favor expression of the gene under the control of the promoter sequence.

[0140] Typically, cells are cultured in standard culture media containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION, Aubert, JP et al., eds., Academic Press, pp. 71-86, 1988 and IImen, M. et al., (1997) Appl. Environ. Microbiol. 63: 1298-1306). Common commercially prepared culture media (e.g., yeast malt extract (YM) broth, Luria-Bertani (LB) broth, and Sabouraud Dextrose (SD) broth can also be used in the present invention.

[0141] The culture conditions are also standard (e.g., the culture is incubated at about 28° C. in a suitable medium in a shaking culture or fermenter until the desired phytase expression level is reached). Preferred culture conditions for a given filamentous fungus are known in the art and can be found in the scientific literature and / or from sources of fungi such as the American Type Culture Collection and the Fungal Genetics Stock Center.

[0142] After fungal growth has been established, the cells are exposed to conditions effective to cause or allow expression of phytase, and in particular phytase as defined herein. In the case where the phytase coding sequence is under the control of an inducible promoter, an inducer (e.g., a sugar, a metal salt or an antimicrobial agent) is added to the culture medium at a concentration effective to induce expression of phytase. Engineered phytase polypeptides or fragments thereof secreted from the host cells can be used as a whole broth formulation with minimal post-production processing.

[0143] Preparation of spent whole fermentation broth of a recombinant microorganism resulting in expression of an engineered phytase polypeptide or fragment thereof can be accomplished using any culture method known in the art.

[0144] The term "spent whole fermentation broth" is defined herein as the unfractionated contents of the fermentation material including culture medium, extracellular proteins (e.g., enzymes), and cell biomass. It should be understood that the term "spent whole fermentation broth" also encompasses cell biomass that has been lysed or permeabilized using methods well known in the art.

[0145] After fermentation, a fermentation broth is obtained, and the microbial cells and various suspended solids (including residual crude fermentation material) are removed by conventional separation techniques to obtain a phytase solution. Typically, filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, ultrafiltration after centrifugation, extraction or chromatography are used.

[0146] It is possible to optionally recover the desired protein from the production host.In another aspect, the engineered phytase polypeptide or fragment thereof is obtained by using any method known to those skilled in the art containing culture supernatant.

[0147] Examples of such techniques include, but are not limited to, affinity chromatography (Tilbeurgh et al., (1984) FEBS Lett. 16:215), ion exchange chromatography (Goyal et al., (1991) Biores. Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol. 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336; and Ellouz et al., (1987) Chromatography 396:307), including ion exchange using materials having high resolution capabilities (Medve et al., (1998) J. Chromatography 396:307). A [Journal of Chromatography A] 808: 153), hydrophobic interaction chromatography (see, Tomaz and Queiroz, (1999) J. Chromatography A [Journal of Chromatography A] 865: 123); two-phase partitioning (see, Brumbauer et al., (1999) Bioseparation [Biological Separation] 7: 287); ethanol precipitation; reversed phase HPLC, silica gel or cation exchange resin chromatography such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration (e.g., Sephadex G-75). The desired degree of purification will vary depending on the use of the engineered phytase polypeptide or fragment thereof. In some embodiments, purification will not be necessary.

[0148] On the other hand, it may be necessary to concentrate the solution containing the engineered phytase polypeptide or its fragment in order to optimize the recovery. Using unconcentrated solution requires increasing the incubation time in order to collect the enzyme precipitate through enrichment or purification. Use conventional concentration techniques to concentrate the enzyme-containing solution until the desired enzyme level is obtained. The concentration of the enzyme-containing solution can be achieved by any of the techniques discussed in this article. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and / or ultrafiltration.

[0149] In addition, the concentration of the desired protein product can be carried out using, for example, a precipitant such as a metal halide precipitant. The metal halide precipitant sodium chloride can also be used as a preservative. The metal halide precipitant is used in an amount that effectively precipitates the engineered phytase polypeptide or its fragment. After conventional testing, it will be apparent to those of ordinary skill in the art to select at least an effective amount and an optimal amount of metal halide to effectively cause precipitation of the enzyme, as well as precipitation conditions for maximizing recovery, including incubation time, pH, temperature and enzyme concentration. Typically, at least about 5% w / v (weight / volume) to about 25% w / v of metal halide is added to the concentrated enzyme solution, and typically at least 8% w / v.

[0150] Another alternative to precipitating enzymes is to use organic compounds. Exemplary organic compound precipitation agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of organic compound precipitation agents can be carried out before, simultaneously with, or after the addition of metal halide precipitation agents, and the addition of two precipitation agents, organic compounds, and metal halides can be carried out sequentially or simultaneously. Typically, organic precipitation agents are selected from the group consisting of: alkali metal salts of 4-hydroxybenzoic acid (such as sodium salts or potassium salts), and straight or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 12 carbon atoms, and blends of two or more of these organic compounds. Other organic compounds also include, but are not limited to, methyl 4-hydroxybenzoate (called methyl p-hydroxybenzoate), propyl 4-hydroxybenzoate (called propyl p-hydroxybenzoate). For further description, see, for example, U.S. Patent No. 5,281,526. The addition of an organic compound precipitation agent provides the advantage of high flexibility in precipitation conditions in terms of pH, temperature, concentration, precipitation agent, protein concentration and incubation time. Typically, at least about 0.01% w / v and no more than about 0.3% w / v of an organic compound precipitation agent is added to the concentrated enzyme solution.

[0151] After the incubation period, the enzyme through enrichment or purification is then separated from the pigment and other impurities of dissociation, and collected by conventional separation techniques such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, filter press, cross-membrane microfiltration, cross-flow membrane microfiltration, etc. The further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enzyme precipitate through enrichment or purification can be obtained by washing with water containing a metal halide precipitation agent or with a water washing containing a metal halide and an organic compound precipitation agent.

[0152] Sometimes it is advantageous to delete genes from an expression host, where the gene defect can be cured by the expression vector. Where it is desired to obtain a fungal host cell with one or more inactivated genes, known methods (e.g., methods disclosed in U.S. Pat. No. 5,246,853, U.S. Pat. No. 5,475,101, and WO 92 / 06209) can be used. Gene inactivation can be accomplished by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene inoperative for its intended purpose (such that the gene is prevented from expressing a functional protein).

[0153] Any cloned gene from Trichoderma species or other filamentous fungal hosts can be deleted, such as cbh1, cbh2, egl1 and egl2 genes. In some embodiments, gene deletion can be completed by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art. Then, within the desired gene coding region, the deletion plasmid is cut at one or more appropriate restriction enzyme sites, and the gene coding sequence or part thereof is replaced with a selective marker. Flanking DNA sequences (preferably between about 0.5 and 2.0 kb) from the locus of the gene to be deleted are retained on either side of the marker gene. Appropriate deletion plasmids will generally have unique restriction enzyme sites present therein so that the fragment containing the deleted gene (including the flanking DNA sequences) and the selective marker gene can be removed as a single linear piece.

[0154] Depending on the host cell used, post-transcriptional and / or post-translational modifications may be performed. A non-limiting example of post-transcriptional and / or post-translational modifications is "chopping" or "truncating" of the polypeptide. In another case, such chopping may result in obtaining a mature phytase polypeptide and further removing the N or C-terminal amino acid to produce a truncated form of the phytase that retains enzymatic activity.

[0155] Other examples of post-transcriptional or post-translational modifications include, but are not limited to, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine or threonine phosphorylation. Those skilled in the art will recognize that the type of post-transcriptional or post-translational modification that a protein may undergo may depend on the host organism in which the protein is expressed.

[0156] Further sequence modifications may occur to the polypeptide after expression. This includes, but is not limited to, oxidation, deglycosylation, saccharification, etc. It is known that saccharification can affect the activity of phytase when incubated with glucose or other reducing sugars, particularly at temperatures above 30°C and neutral or alkaline pH. Protein engineering to eliminate lysine residues can be used to prevent such modifications. Such examples can be found in US 8,507,240. For example, yeast expression can produce highly glycosylated polypeptides, resulting in a significant increase in molecular weight. In addition, WO 2013 / 119470, with an international publication date of August 15, 2013 (incorporated herein by reference), relates to phytases with increased stability (which is believed to be due to increased glycosylation).

[0157] As used herein, the term "glycosylation" refers to the attachment of a polysaccharide to a molecule, such as a protein. Glycosylation can be an enzymatic reaction. Attachment can be formed by a covalent bond. The phrase "highly glycosylated" refers to a molecule (such as an enzyme) being glycosylated at many sites and all or nearly all available glycosylation sites (e.g., N-linked glycosylation sites). Alternatively or in addition thereto, the phrase "highly glycosylated" can refer to a wide range of glycolytic branches (such as, the size and number of glycolytic moieties associated with a specific N-linked glycosylation site) at all or substantially all N-linked glycosylation sites. In some embodiments, the engineered phytase polypeptide is glycosylated at all or substantially all consensus sequence N-linked glycosylation sites (i.e., NXS / T consensus sequence N-linked glycosylation sites).

[0158] As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide, or the carbohydrate portion of a glycoconjugate (such as a glycoprotein). Glycans can be homopolymers or heteropolymers of monosaccharide residues. They can be straight-chain or branched molecules.

[0159] Phytases may have varying degrees of glycosylation. Such glycosylation is known to increase stability during storage and application.

[0160] The activity of any of the engineered phytase polypeptides or fragments thereof disclosed herein can be determined as discussed above.

[0161] It is believed that applying a robust engineered phytase polypeptide or fragment thereof to the feed in liquid form is beneficial compared to applying such phytase in the form of coated granules. Such coated granules are the current commercial method for making phytase products suitable for high temperature conditioning and pelleting. The benefits of liquid application of the robust enzyme include: 1) the enzyme will begin to work immediately after ingestion by the animal because the enzyme does not have to be released from the coated granules before interacting with the feed, 2) the distribution of the enzyme throughout the feed is improved, thus ensuring a more consistent delivery of the enzyme to the animal, which is especially important for young animals that eat small amounts of feed, 3) uniform distribution in the feed makes it easier to measure the enzyme in the feed, and 4) in the case of a robust phytase (such as the engineered phytase polypeptides and fragments disclosed herein), it can begin to degrade phytate already present in the feed.

[0162] In other words, the novel engineered phytase polypeptides and fragments thereof are so robust that no special coating or formulation is required prior to conditioning and pelleting to apply them to feed, because they have been engineered to withstand the stresses of conditioning and pelleting used in industrial feed production. Thus, the robustness of the novel engineered phytase polypeptides and fragments thereof described herein allows them to be applied in the form of uncoated granules or particles or in an uncoated and unprotected form when placed in a liquid.

[0163] It should be noted that the engineered phytase polypeptides and fragments thereof can be formulated inexpensively on solid carriers without particular need for protective coatings and remain active throughout the conditioning and pelleting process. A protective coating providing additional thermal stability when applied in solid form may be beneficial for obtaining pelleting stability when required in certain areas where harsh conditions are used or if conditions permit (e.g. in the case of super-conditioned feeds above 90°C).

[0164] The disclosed engineered phytase polypeptides or fragments thereof are derived using a combination of methods and techniques known in the art of protein engineering, including phylogenetic analysis, site-evaluated libraries, combinatorial libraries, high throughput screening, and statistical analysis.

[0165] In one aspect, the present disclosure relates to an engineered phytase polypeptide or fragment thereof that also has at least 82% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0166] One skilled in the art will understand that such at least 82% sequence identity also includes 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0167] One skilled in the art will understand that such at least 79% sequence identity also includes 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0168] The following may also be mentioned, as in some embodiments, it is provided:

[0169] a) an engineered phytase polypeptide or fragment thereof that further has at least 81% (such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 2, 3, 8, 10, 12, 18, 19, 24, 26, 27, 28, 30, 31, 32, 33, and / or 36.

[0170] b) an engineered phytase polypeptide or fragment thereof that further has at least 82% (such as 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 1, 4, 5, 7, 9, 11, 14, 15, 17, 21, 25, 34, and / or 35;

[0171] c) an engineered phytase polypeptide or fragment thereof that also has at least 83% (such as 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 13;

[0172] d) an engineered phytase polypeptide or fragment thereof that further has at least 79% (such as 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 6 and / or 22; and / or

[0173] e) an engineered phytase polypeptide or fragment thereof that further has at least 80% (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16, 20, 23, 29, and / or 37.

[0174] In a further aspect, the polypeptide comprises the core domain of the engineered phytase polypeptide or is a core domain fragment of the engineered phytase polypeptide. "Core domain fragment" is defined herein as a polypeptide that lacks one or more amino acids from the amino and / or carboxyl termini of the polypeptide. As used herein, the phrase "core domain" refers to a polypeptide region that encompasses the amino acids necessary for maintaining the structure and function of the polypeptide (such as phytic acid hydrolysis). The amino acids in the core domain may be further modified to improve thermal stability or catalytic activity under various conditions (such as, but not limited to, pH). In some non-limiting embodiments, the core domain of the engineered phytase polypeptide disclosed herein or its fragment corresponds to amino acid positions 14-325 of SEQ ID NO:1. In other non-limiting embodiments, the core domain corresponds to amino acid positions 13-326, 12-327, 11-328, 10-329, 9-330, 8-331, 7-332, 6-333, 5-334, 4-335, 3-336, 2-337, or 1-338 of SEQ ID NO: 1. In other embodiments, the N-terminus of the core domain corresponds to amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO: 1, and the C-terminus of the core domain corresponds to SEQ ID NO: Amino acid position 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363 404, 405, 406, 407, 408, 409, 410, 411, 412, or 413.

[0175] Therefore, this article also provides:

[0176] f) an engineered phytase polypeptide or a core domain fragment thereof having at least 78% (such as 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:6, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1;

[0177] g) an engineered phytase polypeptide or a core domain fragment thereof having at least 79% (such as 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 2, 8, 27 and / or 37, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1;

[0178] h) an engineered phytase polypeptide or a core domain fragment thereof having at least 81% (such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:3, 10, 12, 18, 25, 26, 28, 30, 32 and / or 35, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1;

[0179] i) an engineered phytase polypeptide or a core domain fragment thereof having at least 82% (such as 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 1, 4, 5, 7, 9, 11, 13-17, 21, 22, 31, 33, 34 and / or 36, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1;

[0180] j) an engineered phytase polypeptide or a core domain fragment thereof having at least 83% (such as 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 19, 20, 23 and / or 24, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1; and / or

[0181] k) an engineered phytase polypeptide or a core domain fragment thereof having at least 84% (such as 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:29, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1.

[0182] On the other hand, any of the engineered polypeptides disclosed herein or fragments thereof comprises a specific activity of at least about 100 U / mg at pH 3.5. Specific activity ranges (U / mg at pH 3.5) include, but are not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.

[0183] On the other hand, some of the engineered polypeptides or fragments thereof disclosed herein comprise a specific activity of at least about 100 U / mg at pH 5.5. Specific activity ranges (U / mg at pH 5.5) include, but are not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.

[0184] In still another aspect, any of the engineered phytase polypeptides disclosed herein or fragments thereof may be stable in liquid form at a pH of about 3.0 or lower. This is particularly relevant when the engineered phytase polypeptides described herein or fragments thereof pass through the digestive tract of an animal, as discussed below.

[0185] In another embodiment, an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides or fragments thereof described herein is described.

[0186] Importantly, feed additive enzymes (e.g., phytase) are subject to very harsh conditions when passing through the animal digestive tract, i.e., the presence of low pH and digestive enzymes. Pepsin is one of the most important proteolytic digestive enzymes present in the gastrointestinal tract of monogastric animals. Pepsin has low specificity and high pH tolerance in acidic regions (pH 1.5-6.0 is stable until pH 8.0). The engineered phytase polypeptides described herein or fragments thereof tolerate pepsin to a great extent, and pepsin is essential for good in vivo performance.

[0187] The inorganic phosphate-free diet comprising any of the engineered phytase polypeptides or fragments thereof described herein may be used (i) alone or (ii) in combination with a direct-fed microbial comprising at least one bacterial strain or (iii) in combination with at least one other enzyme or (iv) in combination with a direct-fed microbial comprising at least one bacterial strain and at least one other enzyme, or (v) any of (i), (ii), (iii) or (iv) further comprises at least one other feed additive component, and optionally, the engineered phytase polypeptide or fragment thereof is present in an amount of at least 0.1 g / ton of feed (such as at least about 0.1 g / ton of feed). .1 g / ton, 0.2 g / ton, 0.3 g / ton, 0.4 g / ton, 0.5 g / ton, 0.6 g / ton, 0.7 g / ton, 0.8 g / ton, 0.9 g / ton, 1 g / ton, 1.1 g / ton, 1.2 g / ton, 1.3 g / ton, 1.4 g / ton, 1.5 g / ton, 1.6 g / ton, 1.7 g / ton, 1.8 g / ton, 1.9 g / ton, 2 g / ton, 2.1 g / ton, 2.2 g / ton, 2.3 g / ton, 2.4 g / ton, 2.5 g / ton, 2.6 g / ton, 2.7 g / ton, 2.8 g / ton, 2.9 g / ton, 3 g / ton or more).

[0188] In some non-limiting embodiments, the phytase is present in the diet in a range of about 200 FTU / kg to about 1000 FTU / kg feed, more preferably about 300 FTU / kg feed to about 750 FTU / kg feed, more preferably about 400 FTU / kg feed to about 500 FTU / kg feed. In one embodiment, the phytase is present in the feed in an amount of more than about 200 FTU / kg feed, suitably more than about 300 FTU / kg feed, suitably more than about 400 FTU / kg feed. In one embodiment, the phytase is present in the feed in an amount of less than about 1000 FTU / kg feed, suitably less than about 750 FTU / kg feed. In some embodiments, the phytase is present in the feed additive composition in an amount ranging from about 40 FTU / g to about 40,000 FTU / g composition, more preferably from about 80 FTU / g composition to about 20,000 FTU / g composition, and even more preferably from about 100 FTU / g composition to about 10,000 FTU / g composition, and even more preferably from about 200 FTU / g composition to about 10,000 FTU / g composition. In one embodiment, the phytase is present in the feed additive composition in an amount exceeding about 40 FTU / g composition, suitably exceeding about 60 FTU / g composition, suitably exceeding about 100 FTU / g composition, suitably exceeding about 150 FTU / g composition, suitably exceeding about 200 FTU / g composition. In one embodiment the phytase is present in the feed additive composition in an amount of less than about 40,000 FTU / g of the composition, suitably less than about 20,000 FTU / g of the composition, suitably less than about 15,000 FTU / g of the composition, suitably less than about 10,000 FTU / g of the composition.

[0189] In some non-limiting embodiments, "1 FTU" (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from a substrate in one minute under the reaction conditions defined in the ISO 2009 phytase assay (a standard assay for determining phytase activity), and 1 FTU can be found in the International Standard ISO / DIS 30024:1-17, 2009. In one embodiment, the enzyme is classified using the EC classification described above, and the EC classification specifies an enzyme having that activity when tested in the assay for determining 1 FTU taught herein.

[0190] The terms "feed additive", "feed additive component" and / or "feed additive ingredient" are used interchangeably herein.

[0191] Feed additives can be described as products used in animal nutrition with the aim of improving feed quality and the quality of animal-derived food, or improving animal performance and health, for example by providing enhanced digestibility of feed materials.

[0192] Feed additives fall into a variety of categories, such as sensory additives, which stimulate the animal's appetite, making them naturally want to eat more. Nutritional additives provide specific nutrients that may be lacking in the animal's diet. Animal husbandry additives improve the overall nutritional value of the animal's diet through additives in the feed.

[0193] As used herein, "a diet free of inorganic phosphates" refers to a diet that contains no or substantially no inorganic phosphates added, for example, as a feed additive.

[0194] Examples of such feed additives include, but are not limited to, probiotics, essential oils (such as, but not limited to, thymol and / or cinnamaldehyde), fatty acids, short-chain fatty acids (such as propionic acid and butyric acid, etc.), vitamins, minerals, amino acids, etc.

[0195] The feed additive composition or formulation may further comprise at least one component selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben and propylparaben.

[0196] At least one other enzyme (i.e., in addition to any of the engineered phytase polypeptides or fragments thereof disclosed herein) may be included in the feed additive composition or formulation disclosed herein, which may include, but is not limited to, a xylanase, an amylase, another phytase, a β-glucanase, and / or a protease.

[0197] Xylanase is the name of a class of enzymes that degrade the linear polysaccharide β-1,4-xylan to xylose, thereby breaking down hemicellulose, one of the main components of plant cell walls. Xylanases, such as endo-β-xylanase (EC 3.2.1.8), hydrolyze the xylan backbone.

[0198] In one embodiment, the xylanase can be any commercially available xylanase. Suitably, the xylanase can be an endo-1,4-Pd-xylanase (classified as EG3.2.1.8) or a 1,4β-xylosidase (EG3.2.1.37). In one embodiment, the present disclosure relates to a composition comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and an endo-xylanase (e.g., endo-1,4-Pd-xylanase) and another enzyme combination. All EC enzyme classifications mentioned herein relate to the classification provided in the Enzyme Nomenclature—Recommendations (1992)—ISBN 0-12-226164-3 of the International Union of Biochemistry and Molecular Biology Nomenclature Committee, which is incorporated herein by reference.

[0199] In another embodiment, the xylanase may be a xylanase from Bacillus, Trichodermna, Therinomyces, Aspergillus, Humicola, and Penicillium. In another embodiment, the xylanase may be a xylanase from Axtra or Avizyme In one embodiment, the xylanase may be a mixture of two or more xylanases. In another embodiment, the xylanase is an endo-1,4-β-xylanase or a 1,4-β-xylosidase.

[0200] In one embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and a xylanase. In one embodiment, the inorganic phosphate-free diet comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and greater than 750 xylanase units / g composition.

[0201] In one embodiment, the inorganic phosphate-free diet comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000 and greater than 8000 xylanase units / g of composition.

[0202] It is understood that one xylanase unit (XU) is the amount of enzyme which releases 0.5 μmol reducing sugar equivalents (calculated as xylose by the dinitrosalicylic acid (DNS) assay - reducing sugar method) per minute from oat-spelt-xylan substrate at pH 5.3 and 50° C. (Bailey et al., Journal of Biotechnology, Vol. 23, (3), May 1992, 257-270).

[0203] Amylases are enzymes that hydrolyze starch into shorter chain oligosaccharides such as maltose. The glucose moiety can then be transferred from maltose to monoglycerides or glycosyl monoglycerides more easily than from the original starch molecule. The term amylase includes alpha-amylases (EG 3.2.1.1), G4-forming amylases (EG 3.2.1.60), beta-amylases (EG 3.2.1.2), and gamma-amylases (EC 3.2.1.3). Amylases can be of bacterial or fungal origin, or chemically modified or protein engineered mutants.

[0204] In one embodiment, the amylase can be a mixture of two or more amylases. In another embodiment, the amylase can be an amylase (e.g., α-amylase) from Bacillus licheniformis and an amylase (e.g., α-amylase) from Bacillus amyloliquefaciens. In one embodiment, the α-amylase can be an amylase (e.g., α-amylase) from Axtra or Avizyme In another embodiment, the amylase may be a pepsin-resistant alpha-amylase, such as a pepsin-resistant Trichoderma (such as Trichoderma reesei) alpha-amylase. A suitable pepsin-resistant alpha-amylase is taught in UK Application No. 101 1513.7 (incorporated herein by reference) and PCT / IB2011 / 053018 (incorporated herein by reference).

[0205] It will be appreciated that one amylase unit (AU) is the amount of enzyme that releases 1 mmol of glycosidic bonds per minute from a water-insoluble cross-linked starch polymer substrate at pH 6.5 and 37° C. (This may be referred to herein as the assay for determining 1 AU).

[0206] In one embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and an amylase. In one embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof, a xylanase and an amylase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and greater than 750 amylase units / g composition.

[0207] In one embodiment, the inorganic phosphate-free diet comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500- and more than 15,000 amylase units / g of composition.

[0208] As used herein, the term protease is synonymous with peptidase or proteinase. Protease can be subtilisin (EG3.4.21.62) or bacillus protease (EG3.4.24.28) or alkaline serine protease (EG3.4.21.x) or keratinase (EG3.4.XX). In one embodiment, protease is subtilisin. Suitable proteases include those of animal, plant or microbial origin.

[0209] Chemically modified or protein engineered mutants are also suitable. The protease may be a serine protease or a metalloprotease. For example, an alkaline microbial protease or a trypsin-like protease. In one embodiment, the present invention provides a composition comprising any one of the engineered phytase polypeptides disclosed herein or its fragment and one or more proteases.

[0210] Examples of alkaline proteases are subtilisins, especially those derived from Bacillus species, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see, e.g., WO 89 / 06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and Fusarium proteases (see, e.g., WO 89 / 06270 and WO 94 / 25583). Examples of useful proteases also include, but are not limited to, variants described in WO 92 / 19729 and WO 98 / 20115.

[0211] In one embodiment, the protease is selected from the group consisting of subtilisin, bacillus protease, alkaline serine protease, keratinase and Nocardiopsis protease.

[0212] It is understood that one protease unit (PU) is the amount of enzyme that releases one microgram of phenolic compounds (expressed as tyrosine equivalents) from a substrate (0.6% casein solution) in one minute at pH 7.5 (40 mM Na2PO4 / lactate buffer) and 40° C. This may be referred to as an assay for determining 1 PU.

[0213] In one embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and a protease. In another embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and a xylanase and a protease. In another embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and an amylase and a protease. In another embodiment, the present disclosure relates to an inorganic phosphate-free diet comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and a xylanase, an amylase and a protease.

[0214] In one embodiment, the inorganic phosphate-free diet comprises about 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 protease units / g of composition.

[0215] In one embodiment, the inorganic phosphate-free diet comprises about 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500- 7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and more than 15000 protease units / g composition.

[0216] In other embodiments, the diet may have reduced (such as significantly reduced) inorganic phosphate levels relative to those recommended by the National Research Council (NRC) or broiler breeders. In some embodiments, the diet contains an inorganic phosphate level of from 0.2% to about 75% relative to the level recommended by the National Research Council (NRC) or broiler breeders, such as about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% inorganic phosphate levels.

[0217] The at least one direct fed microbial (DFM) may comprise at least one viable microorganism, such as a viable bacterial strain or a viable yeast or a viable fungus. Preferably, the DFM comprises at least one viable bacterium.

[0218] It is possible that the DFM may be a spore-forming bacterial strain, and thus the term DFM may consist of or contain spores, such as bacterial spores. Thus, as used herein, the term "viable microorganism" may include microbial spores, such as endospores or conidia. Alternatively, the DFM in the feed additive composition described herein may not consist of or contain microbial spores, such as endospores or conidia.

[0219] The microorganism may be a naturally occurring microorganism, or it may also be a transformed microorganism.

[0220] The DFM as described herein may comprise a microorganism from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera, and combinations thereof.

[0221] Preferably, the DFM comprises one or more bacterial strains selected from the following Bacillus species: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilis and Bacillus amyloliquefaciens.

[0222] As used herein, "Bacillus" includes all species within the "Bacillus" genus as known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. brevis, and B. thuringiensis. It will be recognized that the genus Bacillus continues to undergo taxonomic recombination. Thus, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus (now named "Geobacillus stearothermophilus") or Bacillus polymyxa (now named "Paenibacillus polymyxa"). The production of resistant endospores under stressful environmental conditions is considered to be a defining characteristic of the genus Bacillus, although this property also applies to the more recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0223] In another aspect, the DFM may be further combined with the following Lactococcus species: Lactococcus cremoris and Lactococcus lactis and combinations thereof.

[0224] The DFM may further be combined with the following Lactobacillus species: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum ... reuteri, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactobacillus jensenii, and any combination thereof.

[0225] In another aspect, the DFM may be further combined with the following Bifidobacterium species: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis and Bifidobacterium angulatum, and any combination thereof.

[0226] Bacteria of the following species may be mentioned: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, Enterococci, Enterococcus species and Pediococcus species, Lactobacillus species, Bifidobacterium species, Lactobacillus acidophilus, Pediococcus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii, Lactobacillus farinaceus, Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius subsp. salivarius. salivarius ssp. Salivarius), Propionibacterium species, and combinations thereof.

[0227] The direct fed microbial comprising one or more bacterial strains described herein may be of the same type (genus, species and strain), or may comprise a mixture of genera, species and / or strains.

[0228] Alternatively, the DFM may be combined with one or more of the products disclosed in WO 2012110778 or the microorganisms contained in these products and summarized as follows: Bacillus subtilis strain 2084 accession number NRRL B-50013, Bacillus subtilis strain LSSAO1 accession number NRRL B-50104 and Bacillus subtilis strain 15A-P4 ATCC accession number PTA-6507 (from Enviva Formerly known as ); Bacillus subtilis strain C3102 (from ); Bacillus subtilis strain PB6 (from ); Bacillus pumilus (8G-134); Enterococcus NCIMB 10415 (SF68) (from ); Bacillus subtilis strain C3102 (from and ); Bacillus licheniformis (from ); Enterococci and Pediococci (from Poultry ); Lactobacillus, Bifidobacterium and / or Enterococcus (from ); Bacillus subtilis strain QST 713 (from ); Bacillus amyloliquefaciens CECT-5940 (from and Plus); Enterococcus faecium SF68 (from ); Bacillus subtilis and Bacillus licheniformis (from ); Lactobacillus 7 Enterococcus faecium (from ); Bacillus strains (from ); Saccharomyces cerevisiae (from ); Enterococci (from Biomin ); Pediococcus acidilactici, Enterococcus, Bifidobacterium animalis subsp. animalis, Lactobacillus reuteri, Lactobacillus salivarius subsp. salivarius (from Biomin ); Lactobacillus fumigans (from ); Enterococci (from Oralin ); Enterococci (2 strains), Lactococcus lactis DSM 1103 (from Probios-pioneer ); Lactobacillus rhamnosus and Lactobacillus farnesii (from ); Bacillus subtilis (from ); Enterococci (from ); Saccharomyces cerevisiae (from Levucell SB ); Saccharomyces cerevisiae (from Levucell SC 0& ME); Pediococcus acidilactici (from Bactocell); Saccharomyces cerevisiae (from (Previously )); Saccharomyces cerevisiae NCYCSc47 (from SC47); Clostridium butyricum (from ); Enterococci (from Fecinor and Fecinor ); Saccharomyces cerevisiae NCYC R-625 (from ); Saccharomyces cerevisiae (from ); Enterococci and Lactobacillus rhamnosus (from ); Bacillus subtilis and Aspergillus oryzae (from ); Bacillus cereus (from ); Bacillus cereus var. toyoi NCIMB40112 / CNCM I-1012 (from ), or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from YC) and Bacillus subtilis (from ).

[0229] DFM can be PRO combination, the latter of which is commercially available from Danisco Corporation. Enviva It is a combination of Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAO1 Accession No. NRRL B-50104, and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507 (as taught in US 7,754,469 B - incorporated herein by reference).

[0230] The DFMs described herein may also be combined with yeast from the genus: Saccharomyces species.

[0231] Preferably, the DFM described herein comprises a microorganism that is generally recognized as safe (GRAS) and preferably GRAS approved.

[0232] One of ordinary skill in the art will readily recognize specific microbial species and / or strains from within the genera described herein that are used in the food and / or agricultural industries and that are generally considered suitable for animal consumption.

[0233] In some embodiments, it is important that the DFM is thermotolerant, i.e. heat resistant. This is particularly true when the feed is pelleted. Thus, in another embodiment, the DFM may be a thermotolerant microorganism, such as a thermotolerant bacterium, including, for example, Bacillus species.

[0234] In other aspects, it may be desirable for the DFM to comprise spore-forming bacteria, such as Bacillus, e.g., Bacillus species. Bacillus are able to form stable endospores when growth conditions are unfavorable and are highly resistant to heat, pH, moisture and disinfectants.

[0235] The DFMs described herein can reduce or prevent the intestinal establishment of pathogenic microorganisms such as Clostridium perfringens and / or Escherichia coli and / or Salmonella species and / or Campylobacter species. In other words, the DFM can be anti-pathogenic. As used herein, the term "anti-pathogenic" means that the DFM resists the effect (negative effect) of pathogens.

[0236] As described above, the DFM may be any suitable DFM. For example, the following assay "DFM Assay" may be used to determine the suitability of a microorganism as a DFM. The DFM Assay as used herein is explained in more detail in US 2009 / 0280090. For the avoidance of doubt, a DFM selected as an inhibitory strain (or anti-pathogenic DFM) according to the "DFM Assay" taught herein is a suitable DFM for use according to the present disclosure, i.e. a DFM for use in a feed additive composition according to the present disclosure.

[0237] Each tube is inoculated with a representative pathogen (eg, bacteria) from a representative cluster.

[0238] Supernatants from potential DFMs grown aerobically or anaerobically were added to inoculated tubes (except for controls where no supernatant was added) and incubated. After incubation, the optical density (OD) of each pathogen in the control tubes and supernatant-treated tubes was measured.

[0239] Colonies of the (potential DFM) strain producing a lower OD compared to the control (not containing any supernatant) can then be classified as suppressive strains (or anti-pathogenic DFM). Thus, the DFM assay as used herein is explained in more detail in US 2009 / 0280090.

[0240] Preferably, the representative pathogen used in this DFM assay may be one (or more) of the following: Clostridium, such as Clostridium perfringens and / or Clostridium difficile, and / or Escherichia coli and / or Salmonella species and / or Campylobacter species. In a preferred embodiment, the assay is performed with one or more of the following: Clostridium perfringens and / or Clostridium difficile and / or Escherichia coli, preferably Clostridium perfringens and / or Clostridium difficile. More preferably Clostridium perfringens.

[0241] Anti-pathogenic DFMs include one or more of the following bacteria and are described in WO 2013029013:

[0242] Bacillus subtilis strain 3BP5 accession number NRRL B-50510,

[0243] Bacillus amyloliquefaciens strain 918 ATCC accession number NRRL B-50508 and

[0244] Bacillus amyloliquefaciens strain 1013 ATCC accession number NRRL B-50509.

[0245] The DFM may be prepared as a culture and carrier (if used) and may be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the time may be increased or decreased. The components are blended so that a uniform mixture of the culture and carrier is obtained. The final product is preferably a dry, flowable powder. The DFM comprising one or more bacterial strains may then be added to the animal feed or feed premix, added to the animal's water, or administered in other ways known in the art (preferably simultaneously with the enzymes described herein).

[0246] The proportion of individual strains included in the DFM mixture may vary from 1% to 99% and preferably from 25% to 75%.

[0247] A suitable dose of DFM in animal feed may be about 1 × 10 3 CFU / g feed is about 1×10 10 CFU / g feed, preferably about 1×10 4 CFU / g feed is about 1×10 8 CFU / g feed, preferably about 7.5×10 4 CFU / g feed is about 1×10 7 CFU / g feed range.

[0248] In another aspect, the dose of DFM in the feed exceeds about 1×10 3 CFU / g feed, suitably more than about 1×10 4CFU / g feed, suitably more than about 5×10 4 CFU / g feed, or suitably more than about 1×10 5 CFU / g feed.

[0249] The DFM dosage in the diet without inorganic phosphate was about 1 × 10 3 CFU / g composition is about 1×10 13 CFU / g composition, preferably 1×10 5 CFU / g composition is about 1×10 13 CFU / g composition, more preferably about 1×10 6 CFU / g composition is about 1×10 12 CFU / g composition and most preferably about 3.75×10 7 CFU / g composition is about 1×10 11 CFU / g composition. In another aspect, the dose of DFM in the feed additive composition is greater than about 1×10 5 CFU / g composition, preferably greater than about 1×10 6 CFU / g composition and most preferably greater than about 3.75×10 7 CFU / g composition. In one embodiment, the dose of DFM in the feed additive composition is greater than about 2×10 5 CFU / g composition, suitably greater than about 2×10 6 CFU / g composition, suitably greater than about 3.75×10 7 CFU / g composition.

[0250] In another aspect, an inorganic phosphate-free diet for use in animal feed is disclosed, the inorganic phosphate-free diet comprising at least one polypeptide having phytase activity as described herein, alone or in combination with at least one direct-fed microorganism, or in combination with at least one other enzyme, or in combination with at least one direct-fed microorganism and at least one other enzyme, wherein the feed additive composition comprises particles in any form such as granular particles. Such granular particles can be produced by a method selected from the group consisting of high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spray coating, spray drying, freeze drying, granulation, spray cooling, vortex disk atomization, agglomeration, tableting, or any combination of the above methods.

[0251] Furthermore, the particles of the granular feed additive composition may have an average diameter greater than 50 microns and less than 2000 microns.

[0252] Those skilled in the art will appreciate that animal feeds may include plant materials for poultry, swine, ruminants, aquaculture and pets, such as corn, wheat, sorghum, soybeans, canola, sunflower, or a mixture of any of these plant materials or plant protein sources. It is expected that animal performance parameters, such as growth, feed intake and feed efficiency, as well as improved uniformity, reduced ammonia concentrations in animal sheds and thus improved animal welfare and health will all be improved.

[0253] Thus, a method for improving the nutritional value of animal feed is disclosed, wherein any of the engineered phytases or fragments thereof as described herein may be added to the animal feed.

[0254] The phrase "effective amount" as used herein refers to the amount of an active agent (such as a phytase, e.g. any of the engineered phytase polypeptides disclosed herein) required to confer improved performance on one or more parameters to an animal, either alone or in combination with one or more other active agents (such as but not limited to one or more additional enzymes, one or more DFMs, one or more essential oils, etc.).

[0255] As used herein, the term "animal performance" can be determined by any indicator such as, but not limited to, feed efficiency and / or weight gain of the animal, and / or by feed conversion ratio and / or by digestibility of nutrients in the feed (e.g., amino acid digestibility or phosphorus digestibility) and / or digestible or metabolizable energy in the feed, and / or by nitrogen retention and / or by the ability of the animal to avoid the negative effects of disease or by the immune response of the subject.

[0256] Animal performance characteristics may include, but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; egg size; egg weight; egg quality; egg production rate; mineral absorption; mineral excretion, mineral retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake (ADFI); average daily gain (ADG); retention and / or secretion of any one or more of copper, sodium, phosphorus, nitrogen and calcium; amino acid retention or absorption; mineralization, bone mineralization carcass yield and carcass quality.

[0257] "Improved animal performance in one or more parameters" means increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio and / or improved digestibility of nutrients or energy in the feed and / or improved nitrogen retention and / or improved ability to avoid the negative effects of necrotic enteritis and / or improved immune response in the subject due to the use of a feed comprising the feed additive composition as described herein, compared to a feed not comprising the feed additive composition described herein.

[0258] Preferably, by "improved animal performance" is meant an increase in feed efficiency and / or an increase in weight gain and / or a decrease in feed conversion ratio. As used herein, the term "feed efficiency" refers to the amount of weight gain an animal experiences when the animal is fed ad libitum or a specified amount of food over a period of time. As used herein, "improvement in an indicator" or "improved indicator" refers to an improvement in at least one parameter listed under an indicator defined for an animal.

[0259] By "increased feed efficiency" is meant that the use of the feed additive composition according to the invention in feed results in increased weight gain per unit of feed intake compared to animals fed in the absence of the feed additive composition.

[0260] As used herein, the term "feed conversion ratio" refers to the amount of feed fed to an animal to increase the animal's body weight by a specified amount.

[0261] Improved feed conversion means lower feed conversion ratio.

[0262] "Lower feed conversion ratio" or "improved feed conversion ratio" means that the use of the feed additive composition in the feed results in a lower amount of feed required to be fed to the animal to increase the animal's weight by a specified amount compared to the amount of feed required to increase the animal's weight by the same amount when the feed did not contain the feed additive composition.

[0263] The improvement in performance parameters may be relative to a control wherein the feed used does not contain phytase.

[0264] The term tibia ash refers to a quantitative method for bone mineralization. This parameter gives an indication whether phosphorus is deficient (e.g., levels should be low in a phosphorus-deficient negative control diet) or sufficient (e.g., levels in a phytase treatment comparable to a positive control diet that meets the phosphorus requirements of broilers).

[0265] The term "phosphorus-deficient diet" refers to a diet in which the phosphorus level is insufficient to meet the nutritional requirements of the animal, for example, a diet formulated with phosphorus levels far below those recommended by the National Research Council (NRC) or broiler producers. The mineral levels in the animal's feed are below those required for optimal growth. If the diet lacks phosphorus, calcium is also not absorbed by the animal. Excessive Ca can lead to poor phosphorus (P) digestibility and the formation of insoluble mineral-phytate complexes. Deficiencies in both P and Ca can lead to reduced bone integrity, subnormal growth, and ultimately weight loss.

[0266] The term "mineralization" or "mineralization" encompasses the deposition of minerals or the release of minerals. Minerals may be deposited or released from the animal. Minerals may be released from feed. Minerals may include any mineral essential to the animal's diet, and may include calcium, copper, sodium, phosphorus, iron, and nitrogen.

[0267] As used herein, nutrient digestibility means the fraction of nutrients that disappear from the gastrointestinal tract or a specific segment of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility can be measured as the difference between the nutrients administered to a subject and the nutrients discharged in the feces of the subject, or the difference between the nutrients administered to a subject and the nutrients retained in the digesta in a specified segment of the gastrointestinal tract (e.g., the ileum).

[0268] As used herein, nutrient digestibility can be measured as follows: by the difference between the amount of nutrient intake over a period of time and the amount of nutrient excreted as completely collected by excreta; or using an inert marker that is not absorbed by the animal and allows researchers to calculate the amount of nutrients that disappear in the entire gastrointestinal tract or a segment of the gastrointestinal tract. Such inert markers can be titanium dioxide, chromium oxide, or acid-insoluble ash. Digestibility can be expressed as a percentage of the nutrient in the feed, or as a mass unit of digestible nutrient / mass unit of nutrient in the feed.

[0269] As used herein, nutrient digestibility encompasses phosphorus digestibility, starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility. Digestible phosphorus (P) can be defined as ileal digestible P, which is the proportion of total P intake that is absorbed by the animal in the terminal ileum, or fecal digestible P, which is the proportion of total P intake that is not excreted in the feces.

[0270] As used herein, the term "survival rate" means the number of subjects that survive. The term "improved survival rate" is another way of saying "reduced mortality rate."

[0271] As used herein, the term "carcass yield" means the amount of carcass measured as a proportion of live weight after a commercial or experimental slaughter process. The term carcass means the body of an animal that has been slaughtered for consumption and from which the head, viscera, limb parts, and feathers or skin have been removed. As used herein, the term meat yield means the amount of edible meat measured as a proportion of live weight, or the amount of a specified cut of meat measured as a proportion of live weight.

[0272] The terms "carcass quality" and "meat quality" are used interchangeably and refer to compositional quality (fat to lean ratio) as well as palatability factors such as visual appearance, smell, firmness, juiciness, tenderness and flavor. For example, producing poultry without "woody breast". In the United States, muscle abnormalities in a small number of chickens lead to the quality problem of woody breast, a condition that causes the chicken breast to be difficult to touch and often pale in color and of poor texture. Woody breast does not pose any health or food safety risks to people, and the health of the chicken itself is not negatively affected.

[0273] "Increased weight gain" refers to an increase in body weight of an animal fed a feed comprising the feed additive composition compared to an animal fed a feed not containing the feed additive composition.

[0274] The terms "animal feed composition", "feed", "feedstock" and "fodder" are used interchangeably and may comprise one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and / or large grains, such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, distillers dried grains with solubles (DDGS), particularly corn-based distillers dried grains with solubles (cDDGS), wheat bran, semolina, wheat short meal, rice bran, rice hulls, oat hulls, palm kernels and citrus pulp; c) proteins obtained from sources such as soy, sunflower, peanut, lupin, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; and / or e) minerals and vitamins.

[0275] Suitably, a premix as referred to herein may be a composition consisting of minor ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. A premix is ​​typically a composition suitable for blending into a commercial ration.

[0276] As used herein, the term "contacting" refers to applying any of the engineered phytase polypeptides or fragments thereof (or a composition comprising any of the engineered phytase polypeptides or fragments thereof) indirectly or directly to a product (e.g., feed). Examples of usable application methods include, but are not limited to, treating the product in a material comprising a feed additive composition, applying directly by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or immersing the product in a formulation of the feed additive composition. In one embodiment, the feed additive composition of the present invention is preferably mixed with a product (e.g., a feed). Alternatively, the feed additive composition may be included in an emulsion or original ingredient of the feed. For some applications, it is important that the composition is available on or available for the surface of the product to be affected / treated. This allows the composition to impart performance benefits.

[0277] Any of the engineered phytase polypeptides or fragments thereof described herein (or compositions comprising such engineered phytase polypeptides or fragments thereof) can be used with a controlled amount of the enzyme to spread, coat and / or impregnate a product (e.g., a diet or feed or an original ingredient of a feed that is free or substantially free of inorganic phosphorus).

[0278] On the other hand, the feed additive composition can be homogenized to produce a powder. The powder can be mixed with other components known in the art. The powder or a mixture comprising the powder can be forced through a die, and the resulting strands are cut into suitable pellets of varying lengths.

[0279] Optionally, the granulation step may include a steam treatment or conditioning stage before forming the pellets. The mixture comprising the powder may be placed in a conditioner, for example, a mixer with steam injection. The mixture is heated in the conditioner to a specified temperature, such as 60°C-100°C, a typical temperature would be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time may vary from a few seconds to a few minutes. It should be understood that any of the engineered phytase polypeptides or fragments thereof described herein (or a composition comprising any of the engineered phytase polypeptides or fragments thereof) is suitable for addition to any appropriate feed material.

[0280] In other embodiments, the particles may be introduced into a feed pelleting process, wherein the feed pre-treatment process may be carried out at between 70°C and 95°C, such as between 85°C and 95°C for up to several minutes.

[0281] In some embodiments, any of the engineered phytase polypeptides or fragments thereof may be present in the feed in the range of 1 ppb (parts per billion) to 10% (w / w) based on pure enzyme protein. In some embodiments, the engineered phytase polypeptide or fragment thereof is present in the feed in the range of 1-100 ppm (parts per million). A preferred dosage may be 1-20 g of the engineered phytase polypeptide or fragment thereof per ton of feed product or feed composition, or a final dosage of 1-20 ppm of the engineered phytase polypeptide or fragment thereof in the final feed product.

[0282] Preferably, the engineered phytase polypeptide or fragment thereof should be present in the feed at least about 50-10,000 FTU / kg, equivalent to about 0.1 to 20 mg of engineered phytase polypeptide or fragment thereof protein / kg.

[0283] Ranges can include, but are not limited to, any combination of the lower and upper limits discussed above.

[0284] The formulation and / or preparation comprising any one of the engineered phytase polypeptides or fragments thereof and compositions described herein can be prepared in any suitable manner to ensure that the formulation comprises active phytase. Such formulations can be liquids, dry powders or granules, which can be uncoated and unprotected, or can involve the use of heat-protective coatings, depending on processing conditions. As described above, engineered phytase polypeptides and fragments thereof can be formulated inexpensively on solid carriers without the need for protective coatings in particular, and remain active throughout the conditioning and granulation process. When certain areas where harsh conditions are used require or if conditions permit (e.g., in the case of ultra-conditioned feeds above 90°C), a protective coating providing additional heat stability when applied in solid form may be useful for obtaining granulation stability.

[0285] The feed additive composition described herein may be formulated as a dry powder or granules as described in WO 2007 / 044968 (referred to as TPT granules) or in WO 1997 / 016076 or WO 1992 / 012645 (each of which is incorporated herein by reference).

[0286] In one embodiment, the feed additive composition can be formulated as a granule for a feed composition, the granule comprising: a core; an active agent (e.g., a phytase, such as any of the engineered phytase polypeptides disclosed herein); and at least one coating, wherein the active agent of the granule remains at least 50% active, at least 60% active, at least 70% active, at least 80% active after being subjected to one or more conditions selected from the following: a) a feed pelleting process, b) a steam heating feed pretreatment process, c) storage, d) storage as an ingredient in an unpelleted mixture, and e) storage as an ingredient in a feed base mixture or feed premix comprising at least one compound selected from the following: trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that produce an acidic or alkaline feed base mixture or feed premix.

[0287] With respect to the granules, at least one coating may comprise a moisture hydrating material that accounts for at least 55% w / w of the granules; and / or at least one coating may comprise two coatings. The two coatings may be a moisture hydrating coating and a moisture barrier coating. In some embodiments, the moisture hydrating coating may account for 25% w / w to 60% w / w of the granules and the moisture barrier coating may account for 2% w / w to 15% w / w of the granules. The moisture hydrating coating may be selected from inorganic salts, sucrose, starch and maltodextrin, and the moisture barrier coating may be selected from polymers, gums, whey and starch.

[0288] In other embodiments, the particles may be introduced into a feed pelleting process, wherein the feed pre-treatment process may be carried out at between 70°C and 95°C, such as between 85°C and 95°C for up to several minutes.

[0289] The feed additive composition can be formulated as a granule for animal feed, the granule comprising: a core; an active agent, the active agent of the granule remaining at least 80% active after storage and after a steam-heated pelleting process of which the granule is an ingredient; a moisture barrier coating; and a moisture hydration coating comprising at least 25% w / w of the granule, the granule having a water activity of less than 0.5 prior to the steam-heated pelleting process.

[0290] The granules may have a moisture barrier coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may comprise 25% to 45% w / w of the granules and the moisture barrier coating may comprise 2% to 10% w / w of the granules.

[0291] Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumable contains one or more of: a buffer, salts, sorbitol and / or glycerol.

[0292] Furthermore, the feed additive composition may be formulated by applying (eg, spraying) the enzyme onto a carrier substrate such as ground wheat.

[0293] In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, a premix may include one or more feed components, such as one or more minerals and / or one or more vitamins.

[0294] In one embodiment, the direct fed microbial ("DFM") and / or engineered phytase polypeptide or fragment thereof is formulated with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate, and mixtures thereof.

[0295] It should be noted that any of the engineered phytase polypeptides and fragments thereof may be used in grain applications, such as in grain processing for non-food / feed applications such as ethanol production.

[0296] Examples

[0297] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D Edition, John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a general dictionary of many of the terms used in the present disclosure to those of skill.

[0298] The present disclosure is further defined in the following examples. It should be understood that these examples, although indicating certain embodiments, are given by way of illustration only. From the above discussion and examples, those skilled in the art will be able to ascertain the essential characteristics of the present disclosure and that various changes and modifications may be made to adapt to various uses and conditions without departing from the spirit and scope of the present disclosure.

[0299] Example 1: Next generation biosynthetic bacterial 6-phytase for complete replacement of inorganic phosphate in commercial diets Benefits

[0300] The efficacy of the phytase variant PHY-13885 used in commercial diets to completely replace inorganic P was evaluated in a broiler chicken study at Texas A&M University (USA).

[0301] A total of 10 treatments were tested, and these were divided into two parts of the study. In the first part of the study, 4 test groups were fed a diet with reduced calcium (Ca) and complete replacement of inorganic P. In the second part of the study, 4 test groups were fed a diet with further reduced digestible amino acids (AA), metabolizable energy (ME) and sodium (Na). Two positive controls were used in the data analysis of the two parts of the study.

[0302] Part 1: Comparison of four test diets with reduced Ca and complete replacement of inorganic P with two positive controls:

[0303] Six treatments were used. A positive control diet (PC1, treatment 1) was formulated to meet the nutritional requirements of broilers, nutritionally adequate but not over-specified. The diet was based on corn, wheat, soybean meal, rapeseed, rice and wheat bran to provide sufficient substrate (phytate) in 4 stages: starter 0-10 days of age, grower 11-21 days of age, finisher 1, 21-35 days of age, and finisher 2, 35-42 days of age. A negative control basal diet was formulated that did not contain any inorganic phosphorus (P) and in which Ca was reduced (about 0.2%-0.3% units per stage relative to PC1, Table 1a, b). The basal diet was supplemented with the next generation biosynthetic bacterial 6-phytase: 1) 1000 FTU / kg in all 4 phases (treatment 2, NC1a); 2) 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2 (NC1b, treatment 3); 3) 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2 (NC1c, treatment 4); 4) the same as NC1b, but with 71 kcal / kg less ME and supplemented with 2000 XU / kg Danisco xylanase (NC1d, treatment 5). A second positive control diet was prepared in which ME was reduced by 71 kcal / kg relative to PC1 and 2000 XU / kg Danisco xylanase was added (treatment 6, PC2).

[0304] For each of the 6 treatments, 10 replicates of 24 birds / pen (Ross 308; 50% males and 50% females) were used. Birds were fed a crumb (starter phase) or pelleted diet (growth phase and finisher phase) ad libitum at all stages. On day 21 and at the end of the trial, 4 birds (2 males and 2 females) were randomly selected to determine the defatted shank ash content. At the end of the trial, carcass quality was measured from randomly selected birds (6 birds / pen).

[0305] Table 1a: Composition and calculated nutrients for the starter and grower phases of Experiment 1, Part 1

[0306]

[0307]

[0308]

[0309] Table 1b: Composition and calculated nutrient composition of the finishing 1 and finishing 2 phases of Experiment 1, Part 1

[0310]

[0311]

[0312]

[0313]

[0314] *Phytase produced by fermentation of a fungal (Trichoderma reesei) production strain (DuPont Nutrition and Biosciences) expressing a biosynthetic variant of a consensus sequence bacterial phytase gene.

[0315] Data were analyzed using one-way ANOVA, and treatment means were separated using the Tukey HSD test. P < 0.05 was considered a statistically significant difference.

[0316] The performance results are summarized in Table 2.

[0317] Table 2. Effects of complete replacement of inorganic P with phytase in a reduced Ca diet on broiler performance, trial 1, part 1

[0318]

[0319]

[0320]

[0321] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0322] *PC1: positive control diet that is nutritionally adequate but not over-specified; NC1a: supplemented with phytase at 1000 FTU / kg in all 4 phases. NC1b: supplemented with phytase at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1c: supplemented with phytase at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1d: NC1b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0323] *Tukey test shows identical superscripts

[0324] **FCRc: FCR corrected for body weight, e.g. BW difference is 2.85kg: FCRc = FCR (0-42d)+(((2.85-BW d42，kg )*1000) / 32) / 100, 2.85kg BW for Ross 308 mixed gender.

[0325] Results showed that phytase or in combination with xylanase at different dosing strategies in diets formulated with low Ca, adequate phytate substrate and oat hulls to support gizzard development could completely replace all inorganic P and maintain broiler performance compared to PC and Ross 308 broiler grower targets (Tables 2 and 14).

[0326] There was no deterioration in bone breaking strength of the treatments compared to the control, instead at 42 days, bone strength was significantly higher (P<0.05) for the highest starting phytase dose (3000 FTU) and the 2000 FTU phytase + xylanase combination compared to PC without any enzyme (Table 4).

[0327] Table 4. Effects of complete replacement of inorganic P with phytase in a reduced Ca diet on bone ash and bone breaking strength in broilers, Experiment 1, Part 1

[0328]

[0329]

[0330] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0331] *PC1: positive control diet that is nutritionally adequate but not overspecified; NC1a: supplemented with phytase at 1000 FTU / kg in all 4 phases. NC1b: supplemented with phytase at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1c: supplemented with phytase at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1d: NC1b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0332] All test groups maintained carcass characteristics. However, treatments supplemented with phytase at 3000 FTU / kg (starter), 2000 FTU / kg (growing), 1000 FTU / kg (finishing 1 and 2) showed greater (P<0.05) breast meat yield compared to both controls (Table 5).

[0333] Table 5. Effects of complete replacement of inorganic P with phytase in a reduced Ca diet on broiler carcass characteristics, trial 1, part 1

[0334]

[0335]

[0336] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0337] *PC1: positive control diet that is nutritionally adequate but not overspecified; NC1a: supplemented with phytase at 1000 FTU / kg in all 4 phases. NC1b: supplemented with phytase at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1c: supplemented with phytase at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1d: NC1b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0338] Part 2: Comparison of four test diets with further reductions in digestible AA, ME and Na in addition to Ca reduction, complete replacement of inorganic P, and two positive controls.

[0339] In the four test groups (NC2a, NC2b, NC2c, NC2d), the same enzyme content as NC1a, NC1b, NC1c and NC1d was used, however, the substrate specific substrate (for These diets were formulated with further reductions in digestible AA, ME and Na using phytase (developed with phytase from PHY and produced by DuPont Nutrition & Biosciences) as shown in Tables 6a, b.

[0340] Table 6a: Composition and calculated nutrients for the starter and grower phases of Experiment 1, Part 2

[0341]

[0342]

[0343]

[0344]

[0345] Table 6b: Ingredients and calculated nutrient composition of the Finishing 1 and Finishing 2 phases of Experiment 1, Part 2

[0346]

[0347]

[0348]

[0349] *Phytase produced by fermentation of a fungal (Trichoderma reesei) production strain (DuPont Nutrition Biosciences) expressing a biosynthetic variant of a consensus sequence bacterial phytase.

[0350] When phytase and phytase + xylanase were added to diets with reduced digestible AA, ME and Na, all test groups maintained performance parameters during each phase and 0-42 d when compared to the control without enzyme (PC1) (Table 7).

[0351] Table 7. Effects of supplementation of diets reduced in Ca, digestible AA, ME and Na with phytase to completely replace inorganic P on broiler performance, trial 1, part 2

[0352]

[0353]

[0354]

[0355] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0356] *PC1: nutritionally adequate but not overspecified positive control diet; all NC2 diets were reduced in Ca, digestible animal acid, ME, and Na compared to PC1. NC2a: phytase supplemented at 1000 FTU / kg in all 4 phases. NC2b: phytase supplemented at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2c: phytase supplemented at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2d: NC2b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0357] **FCRc: body weight corrected FCR, e.g. FCRc=FCR(0-42d)+(((2.85-BWd42, kg)*1000) / 32) / 100. A BW of 2.85 kg is used for Ross 308 mixed sex.

[0358] When phytase and phytase + xylanase were added to diets with reduced digestible AA, ME and Na in addition to Ca reduction and complete replacement of inorganic P, all test groups maintained tibia ash and tibia breaking strength at d 21 and 42 when compared to the control without enzyme (PC1) (Table 8).

[0359] Table 8. Effects of supplementation of diets reduced in Ca, digestible AA, ME and Na with complete replacement of inorganic P with phytase on tibia ash and tibia breaking strength in broiler chickens, Experiment 1, Part 2

[0360]

[0361]

[0362] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0363] *PC1: nutritionally adequate but not overspecified positive control diet; all NC2 diets were reduced in Ca, digestible animal acid, ME, and Na compared to PC1. NC2a: phytase supplemented at 1000 FTU / kg in all 4 phases. NC2b: phytase supplemented at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2c: phytase supplemented at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2d: NC2b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0364] When phytase and phytase + xylanase were added to diets with reduced digestible AA, ME and Na in addition to Ca reduction and complete replacement of inorganic P, all test groups maintained carcass characteristics when compared to the enzyme-free control (PC1), however, the combined treatment with phytase (2000, 1500 and 1000 FTU in starter, grower and finisher) + xylanase (2000 U / kg) reduced fat pad production (Table 9).

[0365] Table 9. Effect of complete replacement of inorganic P with phytase supplemented into diets reduced in digestible Ca, AA, ME and Na on carcass yield (%) in broilers, Experiment 1, Part 2

[0366]

[0367]

[0368] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0369] *PC1: nutritionally adequate but not overspecified positive control diet; all NC2 diets were reduced in Ca, digestible animal acid, ME and Na compared to PC1. NC2a: phytase supplemented at 1000 FTU / kg in all 4 phases. NC2b: phytase supplemented at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2c: phytase supplemented at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2d: NC2b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0370] Example 2: Next generation biosynthetic bacterial 6-phytase for complete replacement of inorganic phosphate in commercial diets Further evaluation of the efficacy of

[0371] The experiments described in Example 1 were repeated in a second study to increase the reliability of the findings. The second study was also conducted at Texas A&M university (USA).

[0372] Ten treatments were tested and the design was identical to Experiment 1. The only modifications were: 1) only male Ross 308 broilers were used, 26 birds per pen; 2) oat hulls were maintained at 1% and soybean hulls were used (Table 10a, b).

[0373] The data were analyzed in the same manner as in Experiment 1, with the first part where the experimental group was formulated with only reduced minerals (e.g., total replacement of Ca and inorganic P) compared to the control. The second part where the experimental group was further reduced in digestible AA, ME, and Na compared to the control group.

[0374] Part 1: Comparison of four test diets with reduced Ca, complete replacement of inorganic P and two positive controls.

[0375]

[0376] Table 10b. Ingredients and calculated nutrient composition of the finishing 1 and finishing 2 phases of Experiment 2

[0377]

[0378] The phytase was produced by fermentation of a fungal (Trichoderma reesei) production strain (DuPont Nutrition Biosciences) expressing a biosynthetic variant of a consensus sequence bacterial phytase.

[0379] No significant differences were found in performance between the positive control and test groups during the starter phase. During 0-21 days of age, NC1a, NC1c, and NC1d, PC2 improved (P<0.05) BW and decreased FCR compared to PC1. During the overall 0-42 days of age, a similar response was observed for BW, with decreased FCR in NC1a, b, c compared to PC1 (P<0.05) (Table 11). The body weight corrected FCRc was lower than PC1 in the test diet (P<0.05).

[0380] These results confirm the findings of the first trial: phytase or in combination with xylanase in diets with different dosing strategies, formulated with low Ca, adequate phytate substrate and oat hulls to support gizzard development, can completely replace all inorganic P and maintain broiler performance compared to PC and Ross 308 broiler breeder recommendations.

[0381] Table 11. Effects of complete replacement of inorganic P with phytase in a reduced Ca diet on broiler performance, trial 2, part 1

[0382]

[0383]

[0384]

[0385] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0386] *PC1: positive control diet that is nutritionally adequate but not overspecified; NC1a: supplemented with phytase at 1000 FTU / kg in all 4 phases. NC1b: supplemented with phytase at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1c: supplemented with phytase at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC1d: NC1b with 71 kcal / kg ME reduced and 2000 XU / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 XU / kg Danisco xylanase added.

[0387] **FCRc: FCR corrected for body weight, corrected to a BW of 3.11 kg (e.g. FCRc = FCR(0-42d) + (((3.11 - BWd42, kg) * 1000) / 32) / 100). 3.11 kg is for Ross 308 male broilers.

[0388] Part 2: Comparison of four test diets with further reductions in digestible AA, ME and Na in addition to Ca reduction, complete replacement of inorganic P, and two positive controls.

[0389] In four test groups (NC2a, NC2b, NC2c, NC2d), the same enzyme inclusions as NCla, NC1b, NC1c and NC1d were used, however, these diets were formulated with further reductions in digestible AA, ME and Na based on substrate-specific matrices for a given dosage level as shown in Table 12a,b.

[0390] Table 12a: Ingredients and calculated nutrient composition of the starter and grower phases of Experiment 2, Part 1

[0391]

[0392]

[0393]

[0394]

[0395] Table 12b: Ingredients and calculated nutrient composition of the finishing 1 and finishing 2 phases of Experiment 2

[0396]

[0397]

[0398]

[0399]

[0400] *Phytase produced by fermentation of a fungal (Trichoderma reesei) production strain (DuPont Nutrition Biosciences) expressing a biosynthetic variant of a consensus sequence bacterial phytase gene.

[0401] When phytase and phytase + xylanase were added to diets with reduced digestible AA, ME and Na, all test groups maintained performance parameters during each phase and 0-42 d when compared to the control without enzyme (PC1) (Table 13).

[0402] Table 13. Effects of complete replacement of inorganic P with phytase and reduction of digestible AA, ME and Na on broiler performance, trial 2, part 2

[0403]

[0404]

[0405] a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

[0406] *PC1: nutritionally adequate but not over-specified positive control diet; all NCs further reduce digestible animal acid, ME and Na. NC2a: phytase supplemented at 1000 FTU / kg in all 4 phases. NC2b: phytase supplemented at 2000 FTU / kg in the starter phase, 1500 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2c: phytase supplemented at 3000 FTU / kg in the starter phase, 2000 FTU / kg in the grower phase, and 1000 FTU / kg in the finishing phases 1 and 2; NC2d: NC2b with 71 kcal / kg ME reduced and 2000 U / kg Danisco xylanase supplemented; PC2: 71 kcal / kg ME reduced relative to PC1 with 2000 U / kg Danisco xylanase added.

[0407] **FCRc: FCR corrected for body weight, corrected to a BW of 3.11 kg (e.g. FCRc = FCR(0-42d) + (((3.11 - BWd42, kg) * 1000) / 32) / 100). 3.11 kg is for Ross 308 male broilers.

[0408] Comparison with Ross 308 feeder target:

[0409] The final BW of broilers fed the PC1 diet was very close to the Ross 308 performance target (Table 14). Starting from day 21, the phytase treatment outperformed the Ross 308 performance target, with an average increase of 3.4% and 3.6% in final BW at day 42 for the NC1 diet in trials 1 and 2, respectively.

[0410] Table 14. Comparison of Ross 308 performance targets (BW, kg) with the average BW of Tests 1 and 2

[0411]

[0412] In conclusion, the results from both studies demonstrate that with proper diet formulation and the use of an effective phytase, all inorganic P can be replaced by phytase without MBM and broiler performance is maintained. This is the first report of a commercially relevant broiler diet that is 100% plant-based and completely free of inorganic P with normal growth characteristics at all growth stages.

[0413] Economic benefit calculation

[0414] The data from this study showed that complete replacement of inorganic P resulted in significant feed cost savings. With complete replacement of inorganic P and reduction of Ca, feed cost savings were up to $7.7 / MT. With further reductions in digestible AA, ME and Na, feed cost savings were up to $19.8 / MT. When a combination of xylanase and phytase was used, feed costs could be reduced by $23.5 / MT using the ME matrix values ​​for xylanase (Table 15).

[0415] Table 15, Estimated cost savings

[0416]

[0417]

[0418] *Feed costs are based on current US prices and include enzyme costs

[0419] Feed costs / kg body weight gain can be reduced by up to 0.046 USD, saving integrators more than one million USD at an annual production of 500,000 MT of broiler live weight.

[0420] Example 3: Novel conjugate as a complete replacement for inorganic phosphate in broiler diets containing xylanase Bacterial 6-phytase variants

[0421] This study evaluated the performance of a novel consensus 6-phytase variant (PHY-13885, “PhyG”) in broiler diets by applying it to diets containing xylanase and a gradient of decreasing phytate-P levels from starter to finisher in the absence of supplemental inorganic phosphate (Pi).

[0422] Materials and methods

[0423] Birds, housing, and experimental design: The study was conducted at the Mas Bové research institute of the Agrifood Research and Technology (IRTA) in Catalonia, Spain. All experimental procedures were performed in accordance with European Directive 2010 / 63 / EU (2010) and the Spanish guidelines for the care and use of animals in research (BOE No. 252, Royal Decree (Real Decreto) 2010 / 2005). A 2 × 2 factorial arrangement was used, including two diets and two sexes. A total of 1,248 mixed-sex Ross 308 broiler chicks were obtained from a commercial hatchery and randomly allocated to 24 pens (52 birds per pen, stocking density ≤16 kg / m at 42 days of age). 2 ), each dietary treatment had 12 pens, including six male pens and 6 female pens. Birds were assigned to each treatment in a randomized complete block design, which included 6 blocks corresponding to the locations in the animal shed. The pens were lined with fresh wood shavings disinfected with glutaraldehyde. The treatment diets were provided as stage diets in the form of crumbs during the starter phase (days 0 to 11) and in the form of pellets during the grower phase (days 11 to 22) and the fattening phase (days 22 to 35 and 35 to 42). Diet and water were provided ad libitum during the study. The pens were placed in an environmentally controlled broiler shed with a lighting regimen of LD24:0h during days 1 to 2, LD18:6h during days 3 to 7, and LD14:10h thereafter. The temperature was initially maintained at 32°C to 34°C, then gradually decreased to 19°C to 21°C during days 3 to 28, and then maintained at this temperature, consistent with breeder recommendations.

[0424] Treatment diets: The diets were composite diets based on corn, soybean meal, wheat, rapeseed meal, rice bran, soybean oil and wheat bran and were formulated according to the phases (Table 16). Oat hulls were included to stimulate gizzard development, and soybean hulls were used as filler material. The positive control diet (PC) was formulated according to breeder recommendations (Aviagen NL, 2014) to provide adequate levels of all nutrients, except that ME was 71 kcal / kg lower due to the energy contribution from xylanase. The phytate content was high during the starter phase and gradually decreased in the subsequent phases (phytate-P: 0.34% during the starter phase, 0.33% during the growth phase, 0.29% during the fattening phase 1, and 0.28% during the fattening phase 2). PC also contained 1.54% to 0.94% (on a feed basis) of Pi from MCP depending on the growth stage (Table 16) and 2,000 xylanase units (XU) of a commercial xylanase produced in Trichoderma reesei per kg of diet (Danisco xylanase, DuPont Animal Nutrition) in all stages. The second diet was formulated to be free of inorganic phosphate (IPF), identical to PC except that calcium (Ca) was reduced by 0.2 percentage points and added Pi was completely absent, supplemented with a novel consensus bacterial 6-phytase variant expressed in Trichoderma reesei (PhyG; DuPont Animal Nutrition) at dosage levels of 3,000 FTU / kg during days 0 to 11, 2,000 FTU / kg during days 11 to 22, and 1,000 FTU / kg during days 22 to 42. Feeds were prepared in bulk by mixing all ingredients except amino acids, vitamin and mineral premixes and enzymes. Amino acid, vitamin and mineral premixes were mixed with 10 kg of feed before adding to the mixer. Enzymes were premixed with ground corn and then added to the diet to ensure even distribution. Pelleting was performed at 80°C.

[0425] Table 16. Composition and calculated nutrient content (%, feed basis) of diets by stage treatment.

[0426]

[0427]

[0428]

[0429] 1 IPF, inorganic phosphate free

[0430] 2One kilogram of feed contains: Vitamin A: 10000IU; Vitamin D3: 4800IU; Vitamin E: 45mg; Vitamin K3: 3mg; Vitamin B1: 3mg; Vitamin B2: 9mg; Vitamin B6: 4.5mg: Vitamin B 12 : 40mg; folic acid: 1.8mg; biotin: 150mg; calcium pantothenate: 16.5mg; niacin: 65mg; Mn (as MnSO4.H2O): 90mg; Zn (as ZnO): 66mg; I (as KI): 1.2mg; Fe (as FeSO4.H2O): 54mg; Cu (as CuSO4.5H2O): 12mg; Se (as NaSeO3): 0.18mg; BHT: 25mg; calcium formate: 5mg; silicic acid, dried and precipitated: 25mg; calcium stearate: 25mg; calcium carbonate: up to 4g.

[0431] 3 The contribution of phytase from the basal diet was excluded.

[0432] Sampling and Measurements: Birds and feed were weighed on days 0, 11, 22, 35 and 42 and used to calculate average BW, average daily feed intake (ADFI) and average daily gain (ADG) expressed on a per pen basis. Pens were monitored for bird mortality daily, recorded and used to calculate mortality-corrected feed conversion ratios (FCR). In addition, for the entire period only (days 0 to 42), a weight-corrected FCR (FCRc) was calculated by correcting the FCR value with the BW difference of 3 points / 100 g to the PC.

[0433] On days 11, 22, and 42, 2 birds per pen were euthanized by cervical dislocation and the right and left tibiae collected were sampled for ash content. Tibiae were pooled by pen and frozen at -20°C prior to analysis.

[0434] Diet samples were analyzed by IRTA Mas Bové, Spain for DM, CP, ash, P and Ca. Phytase activity was measured by IRTA (Constantí, Spain) and xylanase was analyzed by DuPont Research Centre, Brabrand, Denmark.

[0435] Chemical analysis: The right tibia was thawed and the fibula, muscle and connective tissue were removed. The bone was then oven dried, defatted in ether, oven dried again and then ashed according to the method previously described by Dersjant-Li et al. (2020b). The dry matter, CP and ash of the feeds were analyzed according to AOAC methods 925.09, 942.05 and 968.06 (AOAC, 2000), respectively. Phosphorus in the feeds was analyzed by IRTA (Mas bové, Spain) according to AOAC method 965.17 (AOAC, 1990). Calcium in the feeds was analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Agilent Technologies model 7700X) at the Office Laboratory Castellbisbal (Barcelona, ​​Spain) according to the method described by Pacquette et al. (2018). Phytase was determined by ISO method 30024:2009 (Animal feed - Determination of phytase activity), where one phytase unit (FTU) is defined as the amount of enzyme that releases 1 μmol of inorganic phosphate from phytate per minute at pH 5.5 and 37°C. Xylanase activity was determined according to the method of Romero et al. (2014), where one XU is defined as the amount of enzyme that releases 0.48 μmol of reducing sugars such as xylose from wheat arabinoxylan per minute at pH 4.2 and 50°C. All feed analyses were performed in duplicate.

[0436] Statistical analysis: All data are presented with the pen as the experimental unit. All data were analyzed by 2 × 2 factorial analysis of variance (ANOVA) to evaluate the main effects of treatment and sex and their interactions on performance and ash mass. In cases where significant interactions between factors were identified, means were separated using Tukey's test. Analyses were performed using the Fit Model platform of JMP 14.0 (JMP, 2019; SAS Institute Inc., Cary, NC). Differences were considered significant when PeP < 0.10 and were considered a trend.

[0437] result

[0438] The levels of nutrients and enzyme activities analyzed in the dietary treatments are presented in Table 17.

[0439] Table 17. Nutrient content (% original) and enzyme activity of the analyzed treatment diets by phase

[0440]

[0441] 1 IPF, inorganic phosphate free

[0442] 2 Based on the phytic acid-P levels analyzed in the ingredients

[0443] Growth performance and mortality: ADG, ADFI and FCR of birds receiving the IPF diet were comparable or improved compared to PC during all individual growth stages (Table 18): During days 0 to 11, mean ADG, ADFI and FCR in treatment IPF were comparable to the corresponding values ​​produced by PC; during days 11 to 22, ADG and ADFI increased, and FCR decreased in treatment IPF compared to PC (P < 0.001 in all cases); during days 22 to 35, ADG increased in treatment IPF (P < 0.01), while ADFI and FCR did not differ from PC; during days 35 to 42, ADG increased in treatment IPF relative to PC (P < 0.05), and FCR tended to decrease compared to PC (P < 0.10) (Table 18). The positive growth performance effect of treatment IPF relative to PC was greatest during days 11 to 22 (ADG +6.02%, ADFI +4.57%, FCR -2 points, P < 0.001 relative to PC, Table 18).

[0444] Table 18. Effects of diet and sex on broiler growth performance measures by stage; factor analysis of main and interaction effects 1 .

[0445]

[0446] 1 Factor analysis was a 2 x 2 factor analysis of variance (ANOVA) with 2 diets and 2 sexes.

[0447] All growth performance parameters (ADG, ADFI and FCR) were corrected for mortality.

[0448] 2 IPF, inorganic phosphate free.

[0449] On a cumulative basis, treatment with IPF improved BW, ADG, ADFI, and FCR during each of days 0 to 22 and 0 to 35 compared to PC (P < 0.05 in all cases) (Table 19). For the entire period (days 0 to 42), IPF improved BW by 3.97% (P < 0.01), ADG by 4.10% (P < 0.01), ADFI by 1.94% (P = 0.05), and FCRc by 6 points (P < 0.001) at day 42 relative to PC (Table 19).

[0450] Table 19. Cumulative effects of diet and sex on growth performance measures in broiler chickens; factor analysis of main and interaction effects 1 .

[0451]

[0452] 1 Factor analysis was a 2 x 2 factorial analysis of variance (ANOVA) with 2 diets and 2 sexes. All growth performance parameters (ADG, ADFI and FCR) were corrected for mortality.

[0453] 2 FCRc: Body weight corrected FCR, calculated by correcting the FCR value by the BW difference of 3 points / 100 g and PC.

[0454] 3 EPEF, European Performance Efficiency Factor (Aviagen, 2018) = ADG(g) x (100 - mortality (%)) / (10 x FCR).

[0455] 4 Grower Targets: Ross 308 / Ross 308 FF Broilers: Performance Targets, Aviagen, 2019.

[0456] 5 IPF: Inorganic phosphate free.

[0457] Regardless of treatment, male birds consistently showed higher BW, ADG, ADFI and lower FCR (P < 0.05) than female birds during most individual growth phases (Table 18) and cumulative growth phases (Table 19). Exceptions to this general trend include: Days 0 to 11, during which there were no differences in growth performance outcome measures between male and female birds; and Days 35 to 42 and overall (Days 0 to 42), during which FCR was equivalent in males and females, but BW, ADG and ADFI were higher in males. The greatest gender differences were seen during Days 22 to 35 (ADG: +18.54% (P < 0.001); ADFI: +16.85% (P < 0.001), FCR: -2.2 points (P = 0.05) for males versus females).

[0458] There were interactions between sex and dietary treatment for FCR during days 11 to 22 (P < 0.05), days 0 to 22 (P < 0.01), and days 0 to 42 (P < 0.05), and for FCRc during the entire period (days 0 to 42) (P < 0.001). The nature of these interactions was such that the beneficial effects of the IPF diet in reducing FCR (relative to PC) were greater in males than in females (effect size differences of 1.9 FCR points during days 0 to 22 and 4.0 FCR points during days 0 to 42) ( Figure 1A and Figure 1B ).

[0459] Mortality across treatments and growth stages was equal to or less than 3%. There were no differences in mortality levels by sex or treatment at any stage (Tables 18 and 19).

[0460] The consistency of bird BW between birds in the pen by diet and sex (based on individual bird weights) is presented in Table 20. The CV of body weight was consistently 12% or less on each of Days 11, 22, and 42. There were no main or interaction effects of treatment diet or sex on the BW CV values, except that there was a trend for lower CV of BW for the IPF treatment relative to PC on Day 22 (P<0.10), and the CV of BW was higher in males than in females on Day 42 (P<0.05) (Table 20).

[0461] Table 20. Consistency (coefficient of variation (CV)) of BW in birds by diet and sex 1 .

[0462]

[0463] 1 Birds were weighed individually and the coefficient of variation was calculated on a per pen basis.

[0464] 2 IPF, inorganic phosphate free

[0465] CV, coefficient of variation = standard deviation / mean

[0466] Tibia ash content and breaking strength: Tibia ash content at all time points (days 11, 22, and 42) remained comparable in the IPF treatment to PC and did not differ between males and females (Table 21).

[0467] Table 21 Effect of PhyG in combination with xylanase on tibia ash (%DM) at days 11, 22 and 42; factor analysis of main and interaction effects 1 .

[0468]

[0469] 1 The factor analysis was a 2 x 2 analysis of variance (ANOVA) with 2 diets and 2 sexes.

[0470] IPF, inorganic phosphate free.

[0471] In summary, during all growth stages, the addition of PhyG to a Pi-free, Ca-reduced diet (treatment IPF) at a specified dosage regimen produced growth performance metrics comparable to (or improved over) PC. Bone mineralization (measured by tibia ash content) also remained comparable to PC. These results complement those of the previous example, in which the combination of PhyG and xylanase similarly maintained or improved growth performance during all stages of the Pi-free diet compared to a positive control without exogenous enzymes, and provide further evidence of the efficacy of PhyG in the Pi-free diet. In addition, the results provide stronger evidence that the beneficial effects of PhyG and xylanase are separate in the test setting.

[0472] BW and FCR on days 35 and 42 were improved in birds treated with IPF compared to PC and consistently met or exceeded breeder recommendations. In the IPF treatment, BW was 103.1% (day 35) and 100.1% (day 42) of breeder targets, and FCR was 107.6% (day 35) or 105.6% (day 42) of breeder targets. In addition, the calculated European Production Efficiency Factor (EPEF; Aviagen, 2019, Ross 308 / Ross308FF Broilers: Performance Targets, accessed September 3, 2020) (a measure of production efficiency based on ADG from days 0 to 42), mortality, and FCR were higher (+4.9%) in birds treated with IPF compared to PC. Based on the feed intake measured at each stage and the feed costs (including enzyme costs) calculated based on the market prices of feed ingredients in 2020, the total feed cost per kg body weight gain (BWG) for each diet was estimated. The calculated cost for the PC diet was 0.416€ / kg BWG and the calculated cost for the IPF diet was 0.405€ / kg BWG. On this basis, the diets supplemented with PhyG provided producers with significant (P<0.001) feed cost savings.

[0473] Without being bound by theory, the improvements in ADG and FCR observed in birds in the IPF treatment over the levels of PC during days 11 to 22, 0 to 22, 0 to 35, and 0 to 42, as well as the improvements in BW on days 21, 35, and 42, while tibia ash remained comparable to PC, suggest an "extra-phosphorus" impact of PhyG in this study, i.e., a beneficial effect on the digestion and / or retention of nutrients other than P, which positively affected growth performance. Marchal et al. (Poultry Science Association 109th Annual Meeting. 2020 (Abstract 153)) observed similar effects on BW and FCR with an IPF diet containing PhyG during days 0 to 21, with PhyG dosed at 3,000 FTU / kg during the starter phase, 2,000 FTU / kg during the grower phase, and 1,000 FTU / kg during the finisher phase (data from two trials analyzed together). Dersjant-Li et al. (Poultry Science Association 109 th The additional phosphorus effect of PhyG at 1,000 FTU / kg in a Pi-free broiler diet was further confirmed by a study in the Poultry Science Association Annual Meeting. 2020 (Abstract 149), which resulted in improved AIDs of DM, N, GE, and AA in 15-day-old male broilers (Dersjant-Li et al., Poultry Science Association 109 th Annual Meeting. [Poultry Science Association 109th Annual Meeting] 2020 (Abstract 149). Without being bound by theory, it is possible that the high dose levels of PhyG used in this study during the starter phase (3,000 FTU / kg) and grower phase (2,000 FTU / kg), when bird growth rates are high, combined with sufficient available substrate provided by the phytate content of these diets, enabled the birds to derive greater benefit from the presence of phytase during these early growth phases.

[0474] In conclusion, the addition of the novel consensus bacterial 6-phytase variant PhyG at phase-decreasing dosage levels to compound diets containing xylanases without supplemented inorganic P maintained or improved broiler growth performance comparable to that of a nutritionally adequate control diet during all growth phases. Tibia ash also remained comparable to the control diet. These findings add to the existing evidence on the efficacy of PhyG as a complete replacement for Pi in broiler diets containing xylanases and confirm the suitability of the phase-adopted graded PhyG dosage levels and dietary phytate-P content in the dietary settings tested.

[0475] Example 4: Evaluation of novel consensus bacterial 6-phytase variants in diets with or without xylanase Effects of PhyG on growth performance and bone mineralization in broiler chickens

[0476] This example shows the effect of a novel consensus bacterial 6-phytase variant (PHY-13885, "PhyG") on growth performance and bone mineralization in broiler chickens evaluated in diets with or without xylanase.

[0477] Materials and methods

[0478] Birds and Housing: All animal care procedures were approved by the Institutional Animal Ethics Committee of Schothorst Feed Research.

[0479] A total of 26,240 Ross 308 one-day-old broilers (mixed males and females) were obtained from a commercial hatchery and assigned to four dietary treatments in 32 pens at 820 birds / pen and 8 pens / treatment. The pens contained fresh wood shavings and were located in broiler houses where the ambient temperature was initially maintained at 34.5°C, reduced to 20°C at 37 days of age, under a 24:0 h light-dark cycle for the first 24 h, followed by 22 L:2D h. From day 3 onwards, the light-dark cycle was 8L:4D:10L:2D. Birds had ad libitum access to water and diet during the trial.

[0480] Dietary treatments: Four dietary treatments were tested in a 2 × 2 factorial arrangement with 2 levels of xylanase and 2 levels of phytase, including: 1) a positive control (PC1) based on mixed cereal and oilseed meals containing inorganic phosphate (Pi) from monocalcium phosphate (MCP), 2) a PC1 supplemented with 2,000 XU / kg xylanase and reduced by 75 kcal / kg PC1 (PC2), 3) and 4) of ME were reformulated to be free of inorganic phosphate (IPF) and reduced in Ca (-0.15 percentage points), supplemented with 3,000 FTU / kg (1-10d, 0.33% phytate-P) during the starter phase, 2000 FTU / kg (10-22d, 0.31% phytate-P) during the grower phase and 1000 FTU / kg (22-37d, 0.26% phytate-P) of PhyG (IPF1 and IPF2) during the finisher phase. The detailed composition of the diets is given in Table 22. The starter diet was fed in the form of crumbs, while the grower and finisher diets were fed in the form of standard pellets (3.0 mm).

[0481] Table 22. Feed formulation and calculated nutrients of the test diet (as is)

[0482]

[0483]

[0484]

[0485] * Vitamin and mineral premix supplied per kg of diet (based on 0.5% dose):

[0486] Vitamin A 10,000IU; Vitamin D3 3,333IU; Vitamin E 50mg; Vitamin K3 2.5mg; Vitamin B1 2.5mg; Vitamin B2 7.5mg; Vitamin B6 5mg; Vitamin B12 25mg; Niacin 50mg; D-pantothenic acid 15mg; Choline chloride 500mg; Folic acid 1.5mg; Biotin 0.25mg; Fe 50mg (as FeSO4.H2O); Cu 12.5mg (as CuSO4.5H2O); Mn 75mg (as MnO); Zn 70mg (as ZnSO4.H2O); I 2mg (as Ca(IO3)2); Se 0.25mg (as Na2SeO3); Antioxidants (Luctanox EF 5mg; BHT 2.01mg, propyl gallate 0.17mg).

[0487] ** Premix Sacox contains 12,000 mg of salinomycin sodium (Huvepharma NV) to deliver 70 ppm at an inclusion rate of 0.582% in the finished feed.

[0488] Sampling and measurements: Approximately 180 birds were randomly selected upon arrival at the experimental farm (day 0) and body weight (BW) was measured. On days 10 and 22, the BW of each pen was monitored by an automatic weighing scale. On day 37, all birds were weighed manually per pen because the automatic weighing platform was inaccurate for measuring the BW of birds in the late growth and fattening stages. At the end of each period, feed intake was recorded on a per-pen basis and used to calculate FCR, corrected for mortality. On days 21 and 36, left tibiae of 4 male birds per pen were extracted and pooled for determination of defatted tibia ash content. Adjacent tissues were removed by autoclaving the bones; the tibiae were initially dried at 40°C and then at 70°C overnight. Fat was extracted using 100% petroleum ether according to a modified method of Watson et al. (2006). The fat-extracted tibiae were dried at 103°C for 4 hours and ashed in a muffle furnace at 700°C for 24 hours to determine the ash content (data to be reported). The tibiae and humeri were analyzed for breaking strength according to a three-point bending setup. Four hours before analysis, the bones were taken out of the refrigerator and gradually adjusted to 20°C. The width and thickness of each bone were measured. The bones were then positioned on two brackets. For the tibiae collected at D21, the distance between the brackets was 40 mm, for the tibiae collected at D36, the distance between the brackets was 45 mm, and for the humeri collected at D36, the distance between the brackets was 40 mm. An increasing force was applied to the middle of the tibia until it finally broke. The flexural strength was measured in pressure (unit: MPa). The pressure was calculated as (3*force applied at break*bracket length) / (2*width*thickness 2 )). Force is expressed in Newton; bracket length, width and thickness are expressed in mm. Measurement is carried out according to ISO 178 (Determination of flexural properties of rigid plastics).

[0489] Chemical Analysis: Representative samples of all treatment diets were analyzed for dry matter (DM), crude protein (CP), crude fat, crude fiber, Ca, P, phytate-P, and phytase. Nutritional analyses were performed by Schothorst Feed Research (Lelystad, The Netherlands).

[0490] For all analyses, samples were analyzed in duplicate. Nutrients were analyzed according to the following methods: moisture, NEN-ISO 6496 (1999); crude protein, NEN-EN-ISO 16634 (2008); crude fat, NEN-ISO 6492 (1999); crude fiber, NEN-ISO 6865 (2001); starch, NEN-ISO 15914 (2005); phosphorus, NEN-ISO 6491 (1999); calcium, NEN-EN-ISO 6869 (2001); and ash, NEN-ISO 5984 (2003). Phytate phosphorus (PP [inositol hexaphosphate (IP6)]) concentration in the diet and phytase activity in the diet were determined by DuPont Laboratories (Brabrand, Denmark) using the method described by Yu et al. (2012). One phytase unit (FTU) is defined as the amount of enzyme that releases 1 μmol of inorganic orthophosphate per minute from a sodium phytate substrate at pH 5.5 and 37°C (AOAC, 2000).

[0491] Statistical analysis: Data were analyzed based on the pen as the experimental unit and analyzed using JMP14. Data were analyzed by two-way ANOVA with 2 levels of xylanase and 2 levels of phytase arranged as 2×2 factors and block as a random effect. The following model was used: Yij=μ+Blocki+Phytasej+Xylanasek+(phytase×xylanase)jk+eijk,

[0492] Where: Yij = dependent variable; μ = grand mean; Blocki = replicate effect (i = 1...8); Phytasej = PhyG effect (j = 1, 2); Xylanasek = xylanase effect (k = 1, 2), (phytase × xylanase)jk = effect of interaction between PhyG and xylanase, and eijk = residual error.

[0493] Differences between treatments were identified based on the Tukey test, with P < 0.05 and P < 0.1 considered to indicate a significant trend.

[0494] result

[0495] There were no phytase × xylanase interactions for any growth performance and bone parameters (Tables 23 and 24). IPF treatment maintained final body weight at each stage and overall from 0 to 37 days compared to the corresponding control group. Factor analysis showed that phytase reduced feed intake from 10 to 22 days and 22 to 37 days and cumulative feed intake from 0 to 22 days and 0 to 37 days (P<0.05). Similarly, water intake from 10 to 22 days and 22 to 37 days and cumulative water intake from 0 to 37 days was reduced by phytase supplementation (P<0.05). Feed conversion ratio from 22 to 37 days and cumulative feed conversion ratio from 0 to 37 days were improved by phytase supplementation (P<0.05). Cumulative feed cost per kg body weight gain was reduced by phytase and xylanase supplementation from 0 to 37 days of cumulative age (P<0.05).

[0496] Table 23. Effects of complete replacement of inorganic P with phytase and xylanase on dietary phase-specific and cumulative growth performance of 0-37 days of age

[0497]

[0498]

[0499]

[0500]

[0501] Table 24. Effect of complete replacement of inorganic P with phytase and xylanase on bone ash (% DM) and bone strength (MPa)

[0502]

[0503]

[0504]

[0505] IPF treatment maintained bone breaking strength at all sampling points. On day 21, tibia breaking strength was increased by phytase supplementation (P=0.011).

[0506] In diets with and without phytase, xylanase supplementation was able to compensate for the 75 kcal ME reduction compared to the control and maintain performance.

[0507] The data demonstrate that phytase alone or in combination with xylanase can completely replace inorganic P in broiler chickens from day 1, and maintain or improve broiler performance and bone breaking strength in a commercial setting. Sequence Listing <110> DuPont Nutrition Biosciences APS <120> Diet formulations <130> NB41175 <160> 37 <170> PatentIn Version 3.5 <210> 1 <211> 413 <212> PRT <213> Artificial sequence <220> <223> PHY-13594 <400> 1 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 2 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-10931 <400> 2 Ser Glu Thr Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Asn Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu 100 105 110 Thr Ile His His Gln Ser Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln Tyr 145 150 155 160 Tyr Ala Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu His Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 3 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-10957 <400> 3 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Trp Phe Gln Gln Gln Gly Ile Leu Ser Lys Glu Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Pro Leu 100 105 110 Thr Ile His His Gln Ser Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Lys Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro 405 410 <210> 4 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-11569 <400> 4 Ser Glu Thr Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asn Ile Lys Val Val Asp Pro Met Phe His 115 120 125 Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Ala Pro Glu Leu Ala Leu Met Ser Ala Val Leu Asn Phe Pro Ala 165 170 175 Ser Pro Tyr Cys Gln Gln His Ser Val Asp Lys Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Ser Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 5 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-11658 <400> 5 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Leu Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Trp Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Ala Asp Val Gly Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Arg Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 6 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-11673 <400> 6 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Glu Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Pro Leu 100 105 110 Thr Ile His His Gln Ser Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Thr Gly Leu Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp Lys 145 150 155 160 Tyr Ala Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Lys Leu Ser Ile Gly Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Val 385 390 395 400 Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 7 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-11680 <400> 7 Ser Glu Thr Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His 145 150 155 160 Tyr Arg Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Thr Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Ile Ile Thr Asp Asp Gly Asn Lys Val 195 200 205 Ala Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Lys Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro 405 410 <210> 8 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-11895 <400> 8 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Leu Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Trp Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Lys Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Ala Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Thr Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Thr Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 9 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-11932 <400> 9 Ser Glu Thr Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Ala Glu Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His 145 150 155 160 Tyr Arg Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Ala Arg His Ser Gly Asp Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Met Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 10 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-12058 <400> 10 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Val Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Lys Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Pro Leu 100 105 110 Thr Ile His His Gln Ser Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Ala Gln His Ser Gly Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Ile Pro 405 410 <210> 11 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-12663 <400> 11 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Val Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Trp Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu 100 105 110 Thr Ile His His Gln Lys Asn Ile Lys Val Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Asp Glu Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Arg Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro 405 410 <210> 12 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-12784 <400> 12 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Leu Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Asn Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Ile Leu Ser Lys Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Met Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln Tyr 145 150 155 160 Tyr Ala Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Thr Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Thr Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Thr Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro 405 410 <210> 13 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13177 <400> 13 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Val Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Asn Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Ala Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Arg Cys Asp Leu 100 105 110 Thr Ile His His Gln Lys Asp Ile Lys Gln Val Asp Pro Met Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Ala Gln His Ser Gly Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Arg Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 14 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13371 <400> 14 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Gly Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asn Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ala Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Ile Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 15 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13460 <400> 15 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Ala Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Val His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asn Lys Ser Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Met Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Met Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 16 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13513 <400> 16 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Gly Pro Ile Glu Thr Leu Asn Gln Arg 145 150 155 160 Tyr Gln Ala Glu Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 脯氨酸-亮氨酸-亮氨酸-谷氨酰胺-苏氨酸-异亮氨酸-缬氨酸-天冬酰胺-丙氨酸-亮氨酸-天冬酰胺-丝氨酸-天冬酰胺-苏氨酸-苏氨酸-丙氨酸 275 280 285 精氨酸-谷氨酸-亮氨酸-脯氨酸-天冬氨酸-异亮氨酸-丝氨酸-脯氨酸-天冬氨酸-缬氨酸-赖氨酸-异亮氨酸-亮氨酸-苯丙氨酸-亮氨酸-丙氨酸 290 295 300 甘氨酸-组氨酸-天冬氨酸-苏氨酸-天冬酰胺-异亮氨酸-丙氨酸-天冬酰胺-异亮氨酸-甘氨酸-甘氨酸-甲硫氨酸-亮氨酸-甘氨酸-甲硫氨酸-苏氨酸 305 310 315 320 色氨酸-苏氨酸-亮氨酸-脯氨酸-甘氨酸-谷氨酰胺-脯氨酸-天冬氨酸-天冬酰胺-苏氨酸-脯氨酸-脯氨酸-甘氨酸-甘氨酸-丙氨酸-亮氨酸 325 330 335 亮氨酸-苯丙氨酸-谷氨酸-亮氨酸-色氨酸-精氨酸-天冬氨酸-赖氨酸-天冬氨酸-甘氨酸-苏氨酸-谷氨酰胺-酪氨酸-缬氨酸-丝氨酸-缬氨酸 340 345 350 组氨酸-甲硫氨酸-缬氨酸-酪氨酸-谷氨酰胺-苏氨酸-亮氨酸-丙氨酸-谷氨酰胺-亮氨酸-精氨酸-天冬酰胺-甲硫氨酸-苏氨酸-脯氨酸-亮氨酸 355 360 365 苏氨酸-亮氨酸-赖氨酸-谷氨酸-脯氨酸-丙氨酸-甘氨酸-丝氨酸-缬氨酸-脯氨酸-亮氨酸-赖氨酸-异亮氨酸-脯氨酸-甘氨酸-半胱氨酸 370 375 380 天冬氨酸-天冬氨酸-谷氨酰胺-苏氨酸-丙氨酸-谷氨酸-甘氨酸-酪氨酸-半胱氨酸-脯氨酸-亮氨酸-天冬氨酸-苏氨酸-苯丙氨酸-苏氨酸-精氨酸 385 390 395 400 亮氨酸-缬氨酸-天冬酰胺-谷氨酰胺-缬氨酸-缬氨酸-谷氨酸-脯氨酸-丙氨酸-半胱氨酸-谷氨酰胺-亮氨酸-脯氨酸 405 410 <210> 17 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13637 <400> 17 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln His His Ser Gly Asp Gln Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Val Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 18 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13705 <400> 18 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Arg Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Arg Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 19 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13713 <400> 19 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro 65 70 75 80 Thr Pro Asp Ala Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile His Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Met Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 20 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> PHY-13747 <400> 20 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Asp Leu 100 105 110 Ala Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Met Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Glu Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Met Gln Thr Ile Ser Asn Ala Leu Asn Pro Asn Ala Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile 405 410 <210> 21 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> PHY-13779 <400> 21 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro His Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Glu Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile 405 410 <210> 22 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13789 <400> 22 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro 65 70 75 80 Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala 180 185 190 Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 23 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13798 <400> 23 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Ala Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asn Ile Ser Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala 180 185 190 Gln Met Met Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asn Pro Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 24 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13868 <400> 24 Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Glu Lys Val Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Ser Gln Thr Tyr Glu 130 135 140 Ala Val Glu Lys Gln Ala Gly Gly Pro Ile Glu Thr Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Arg His Ser Gly Asp Lys Thr Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Glu Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 25 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13883 <400> 25 Ser Glu Ala Ala Pro Ser Gly Tyr Gln Leu Glu Lys Val Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asn Ile Ser Gln Ala Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 26 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13885 <400> 26 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 27 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-13936 <400> 27 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Pro Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Ala Ile 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Glu Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 28 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> PHY-14004 <400> 28 Ala Glu Glu Ala Asn Gly Met Lys Leu Gln Lys Ala Val Ile Leu Ser 1 5 10 15 Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg Asp 20 25 30 Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr Ile 35 40 45 Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr Arg 50 55 60 Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro Thr 65 70 75 80 Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg Lys 85 90 95 Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu Thr 100 105 110 Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His Pro 115 120 125 Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln Ala 130 135 140 Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His Tyr 145 150 155 160 Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys Ser 165 170 175 Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala Asn 180 185 190 Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val Gln 195 200 205 Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe Leu 210 215 220 Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile His 225 230 235 240 Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln Phe 245 250 255 Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr Pro 260 265 270 Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala Arg 275 280 285 Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala Gly 290 295 300 His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr Trp 305 310 315 320 Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu 325 330 335 Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val His 340 345 350 Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr 355 360 365 Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp 370 375 380 Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu 385 390 395 400 Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 29 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-14215 <400> 29 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu 100 105 110 Ala Ile His His Gln Gln Asn Ile Thr Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Arg His Ser Gly Asp Lys Thr Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Lys Val 195 200 205 Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Ser Gln Trp Lys Ser Leu Leu Asn Leu His Asn Ala His 245 250 255 Phe Asn Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Glu Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 30 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-14256 <400> 30 Ser Asp Thr Ala Pro Ala Gly Tyr Gln Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Ala Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr 245 250 255 Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 31 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> PHY-14277 <400> 31 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Pro Lys Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Arg Arg His Ser Gly Asp Lys Thr Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile 405 410 <210> 32 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> PHY-14473 <400> 32 Ala Glu Glu Gln Asn Gly Met Lys Leu Gln Lys Ala Val Ile Leu Ser 1 5 10 15 Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg Asp 20 25 30 Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr Ile 35 40 45 Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr Arg 50 55 60 Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro Thr 65 70 75 80 Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg Lys 85 90 95 Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Ala Ile Thr 100 105 110 Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His Pro 115 120 125 Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln Ala 130 135 140 Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His Tyr 145 150 155 160 Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys Ser 165 170 175 Pro Tyr Cys Arg His Gln Ser Gly Asp Lys Thr Cys Asp Phe Ala Asn 180 185 190 Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val Gln 195 200 205 Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe Leu 210 215 220 Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile His 225 230 235 240 Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln Phe 245 250 255 Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr Pro 260 265 270 Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu Ser 275 280 285 Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala Gly 290 295 300 His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr Trp 305 310 315 320 Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu 325 330 335 Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val Lys 340 345 350 Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr 355 360 365 Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp 370 375 380 Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu 385 390 395 400 Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 33 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-14614 <400> 33 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu 100 105 110 Thr Ile His His Gln Gln Asn Ile Thr Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Gln His Ser Val Asp Lys Pro Cys Asp Phe Ala 180 185 190 Asn Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu 275 280 285 Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 34 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-14804 <400> 34 Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Pro Arg Asp Asn Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Glu Leu Asn Gln His 145 150 155 160 Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 35 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-14945 <400> 35 Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr 50 55 60 Arg Glu Arg Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro 65 70 75 80 Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Ala Ile 100 105 110 Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His 115 120 125 Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys 165 170 175 Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val 195 200 205 Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His 245 250 255 Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro 405 410 <210> 36 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> PHY-15459 <400> 36 Ala Glu Pro Gln Asn Gly Met Lys Leu Asp Lys Ala Val Ile Leu Ser 1 5 10 15 Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg Asp 20 25 30 Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr Ile 35 40 45 Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr Arg 50 55 60 Gln Tyr Phe Gln Gln Gln Gly Ile Leu Ser Lys Asp Arg Cys Pro Arg 65 70 75 80 Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg Lys 85 90 95 Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu Thr 100 105 110 Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His Pro 115 120 125 Val Lys Gly Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln Ala 130 135 140 Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His Tyr 145 150 155 160 Arg Pro Glu Leu Ala Leu Met Ser Ala Val Leu Asn Phe Pro Ala Ser 165 170 175 Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala Gln 180 185 190 Met Met Pro Ser Lys Leu Tyr Ile Thr Asp Asp Gly Asn Glu Val Gln 195 200 205 Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe Leu 210 215 220 Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile His 225 230 235 240 Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Tyr Phe 245 250 255 Asp Leu Met Tyr Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr Pro 260 265 270 Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala Arg 275 280 285 Glu Leu Pro Asp Ile Ser Pro Asp Asn Arg Ile Leu Phe Leu Ala Gly 290 295 300 His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp Trp 305 310 315 320 Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu 325 330 335 Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val His 340 345 350 Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr 355 360 365 Leu Gln Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Ser Cys Asp 370 375 380 Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Lys Leu 385 390 395 400 Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro 405 410 <210> 37 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PHY-16513 <400> 37 Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu 1 5 10 15 Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg 20 25 30 Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr 35 40 45 Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr 50 55 60 Arg Gln Tyr Phe Gln Met Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro 65 70 75 80 Asn Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg 85 90 95 Lys Thr Gly Glu Ala Phe Leu Arg Gly Leu Ala Pro Glu Cys Asp Leu 100 105 110 Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His 115 120 125 Pro Val Lys Gly Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln 130 135 140 Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His 145 150 155 160 Tyr Arg Pro Glu Leu Ala Leu Met Ser Ala Val Leu Asn Phe Pro Ala 165 170 175 Ser Pro Tyr Cys Gln Gln His Ser Val Asp Lys Pro Cys Asp Phe Ala 180 185 190 Asn Ala Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val 195 200 205 Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe 210 215 220 Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile 225 230 235 240 His Asn Glu Ser Gln Trp Lys Ser Leu Leu Asn Leu His Asn Ala His 245 250 255 Phe Asn Leu Met His Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr 260 265 270 Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala 275 280 285 Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala 290 295 300 Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp 305 310 315 320 Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu 325 330 335 Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val 340 345 350 Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu 355 360 365 Thr Leu Asn Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Ser Cys 370 375 380 Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Val 385 390 395 400 Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro 405 410

Claims

1. A poultry diet comprising (i) an engineered phytase polypeptide; and (ii) no or substantially no inorganic phosphorus, wherein the phytase polypeptide consists of the amino acid sequence of SEQ ID NO:

26.

2. The diet of claim 1 further comprising one or more of: (a) a direct-fed microbial comprising at least one bacterial strain, (b) at least one other enzyme, and / or (c) an essential oil.

3. The diet of claim 1 or 2, wherein the engineered phytase polypeptide is present in an amount of at least 0.1 g / ton of feed.

4. A diet as claimed in any one of claims 1 to 3 comprising 0.62% to 0.72% calcium in a starter diet, 0.54% to 0.64% in a grower diet, and / or 0.42% to 0.55% calcium in a finisher diet.

5. A diet as claimed in claim 4, wherein the finishing diet comprises 0.46% to 0.55% or 0.42% to 0.50% calcium.

6. A diet according to any one of claims 1 to 5 comprising 1.18% to 1.22% amino acids in a starter diet, 1.06% to 1.10% in a grower diet, and / or 0.88% to 1% in a finisher diet.

7. A diet according to claim 6, wherein the finishing diet comprises 0.96% to 1.0% or 0.88% to 0.92% amino acids.

8. A diet as claimed in claim 6 or 7, wherein the amino acids include digestible lysine.

9. The diet of any one of claims 1 to 8 comprising 2824 to 2950 kcal / kg of metabolizable energy in a starter diet, 2924 to 3050 kcal / kg in a grower diet, and / or 2970 to 3120 kcal / kg in a finisher diet.

10. The diet of claim 9, wherein the finishing diet comprises 2970 to 3100 kcal / kg or 2994 to 3120 kcal / kg of metabolizable energy.

11. The diet of any one of claims 1 to 10 comprising 0.13% to 0.17% sodium.

12. The diet of any one of claims 1-11, wherein the diet contains a phytate source comprising one or more of corn, wheat, soybean meal, rapeseed, rice and / or wheat bran.

13. The diet of any one of claims 1-12, wherein the diet further comprises oat hulls.

14. The diet of any one of claims 1-13, wherein the diet lacks meat and / or bone meal.

15. The diet of any one of claims 1-14, further comprising one or more additional feed enzymes selected from the group consisting of xylanases, proteases, amylases and glucoamylases.

16. The diet of any one of claims 1-15, wherein the diet is a starter diet.

17. The diet of any one of claims 1-15, wherein the diet is a growing diet.

18. The diet of any one of claims 1-15, wherein the diet is a finishing diet.

19. The diet of any one of claims 1-18, wherein the poultry is a broiler or laying hen.

20. Use of a diet as claimed in any one of claims 1 to 19 in the preparation of a food or feed for improving animal performance in one or more parameters by administration to an animal in an effective amount.

21. The use of claim 20, wherein the one or more parameters are selected from the group consisting of: increased feed efficiency, increased weight gain, decreased feed conversion ratio, improved digestibility of nutrients or energy in feed, and improved nitrogen retention.

22. The use of claim 20 or claim 21, wherein the animal is poultry.

23. The use according to claim 22, wherein the poultry is selected from the group consisting of turkeys, ducks, chickens, geese, pheasants, quails and emus.

24. The use according to claim 23, wherein the chicken is selected from the group consisting of laying hens and broiler chickens.

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