Xylanase with enhanced heat resistance and use thereof

Abstract

A thermostable xylanase which is a variant of SEQ ID NO: 2 wherein the variant comprises at least one amino acid substitution at a position selected from the group consisting of 90, 105, 114 and 115 and / or comprises any set of substitutions: (a) G48C and T206C; (b) a Y123 V, a S125C, and a N171C; (c) S114C, Y1123V, S125C, and N171C, and (f) S114C, Y123V, S125C (d) Y123 V, S125C, N171C, G48C, and T206C, and a method for preparing the same; (e) S114C, Y123V, S125C, N171C, G48C, and T206C, and (f) S114C, Y123V, S125C, N171C, wherein the one or more substitutions increase the heat resistance of the xylanase relative to the parent xylanase lacking the one or more substitutions.

Description

Technical Field

[0001] The present disclosure generally relates to thermotolerant xylanases and enzyme-assisted methods using the same. background

[0002] Xylanases are important enzymes involved in the decomposition of xylanases, a non-starch polysaccharide (NSP) component of plant cell walls. Xylanases are produced by a variety of bacteria, protozoa, fungi, algae, insects, terrestrial plants, and germinating seeds. These enzymes have many industrial applications, such as clarification of wine and juice, enhanced starch separation, baking, animal feed, and pulp bleaching. Some of these industrial applications involve high temperatures that inactivate most enzymes. Therefore, there is a need for novel xylanases with higher heat resistance than those commonly found in nature.

[0003] The inclusion of non-starch polysaccharide degrading enzymes (NSPases) such as xylanases in the diets of non-ruminants has been shown to have a beneficial effect on their health by improving intestinal physiology. For example, the inclusion of NSPases in poultry feeds has been shown to improve feed digestibility in broilers. Supplementation of enzyme components in poultry feeds typically involves a high temperature process, sometimes referred to as pelleting. This process may involve temperatures reaching 95°C, which typically has a negative impact on the stability of these NSPase components, resulting in reduced activity or complete loss of one or more enzyme components. There is an urgent need to develop NSPase components that can withstand high temperature treatments such as pelleting. The addition of NSP degrading enzymes has also been shown to enhance the separation of starch in wet milling processes by releasing starch, protein and water trapped in corn fiber, which are recalcitrant to mechanical damage alone. Xylanases that tolerate the high temperatures of the wet milling process facilitate better separation and recovery of starch and increase the yield of starch-derived sugars. Sequence Listing

[0004] This application includes a sequence listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on April 28, 2023, is named "ALL.0046sequencelisting_ST26.xml" and is 4 KB in size. Overview

[0005] Enzymes with higher heat resistance than conventional enzymes are disclosed herein. In one aspect, the disclosure provides a xylanase comprising the amino acid sequence SEQ ID NO: 2, but comprising at least one amino acid substitution at positions 90, 105, 114 and / or 115; and / or any of the following substitution sets: G48C and T206C; Y123V, S125C, and N171C; S114C, Y123V, S125C, and N171C; Y123V, S125C, N171C, G48C and T206C; or S114C, Y123V, S125C, N171C, G48C, and T206C; One or more of the substitutions increase the thermotolerance of the xylanase relative to the xylanase comprising the amino acid sequence of SEQ ID NO: 2.

[0006] In another aspect, the present disclosure provides methods of using thermotolerant xylanases. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The results show that the Saccharomyces cerevisia e) Results of xylanase AZCL-arabinoxylan temperature challenge assay of supernatants of expressed Thielavia terrestris GH11 xylanase variants V22, AJ039, AJ040, AJ045 (V45), AJ102, AJ131 (V131), showing improved residual activity relative to wild type (wt), which is shown as activity relative to the same sample without heat treatment.

[0008] Figure 2 The results show that the activity of Trichoderma reesei from heterologously expressing Thielavia terrestris GH11 xylanase variants AJ045 (V45) and AJ131 (V131) is Trichoderma reesei ) were subjected to a xylanase AZCL-arabinoxylan temperature challenge assay showing improved residual activity relative to the wild type (wt), which is shown as activity relative to the same sample without heat treatment.

[0009] Figure 3 The Cp (kJ / mol / K) versus temperature for variant V45 for the first, second, and third melts is shown.

[0010] Figure 4The Cp (kJ / mol / K) versus temperature for variant V131 for the first, second, and third melts is shown. Details

[0011] NSP enzymes are a common component in feed enzyme products, which are xylan hydrolyzing active endo-β-1,4-xylanase (EC 3.2.1.8). Thielavia terrestris ) (Thite) xylanase (SEQ ID NO: 2; glycoside hydrolase family GH11) has been identified and characterized as the lead component in the thermostable NSPase disclosed herein. Trichoderma reesei ) and Saccharomyces cerevisiae ( Saccharomyces cerevisia e). The xylanase has a melting temperature of about 73°C as determined by differential scanning calorimetry (DSC), which potentially inactivates it in the pelleting process. Using rational site-directed mutagenesis and combined protein engineering, amino acid substitutions that statistically improve the residual activity of the xylanase after heat challenge were identified herein.

[0012] Industrial applications that would benefit from the thermotolerant xylanases disclosed herein include, but are not limited to, animal feed manufacturing, baking, pulp bleaching, textile bleaching, and conversion of biomass to biofuels (eg, ethanol).

[0013] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises at least one amino acid substitution at position 90, 105, 114, and / or 115.

[0014] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises any one of the following sets of substitutions: G48C and T206C; Y123V, S125C, and N171C; S114C, Y123V, S125C, and N171C; Y123V, S125C, N171C, G48C, and T206C; and S114C, Y123V, S125C, N171C, G48C, and T206C.

[0015] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the following: a. at least one amino acid substitution at position 90, 105, 114 and / or 115; and / or b. One of the following replacement sets: G48C and T206C; Y123V, S125C, and N171C; S114C, Y123V, S125C, and N171C; Y123V, S125C, N171C, G48C, and T206C; and S114C, Y123V, S125C, N171C, G48C, and T206C.

[0016] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises at least one substitution selected from the group consisting of S90T, Q105V, Q105I, S114C, S114P, and A115S.

[0017] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitution S90T. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitution Q105V. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitution Q105I. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitution S114C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitution S114P. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitution A115S.

[0018] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T and Q105V. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T and Q105I. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T and S114C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T and S114P. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T and A115S.

[0019] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105V and S114C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105V and S114P. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105V and A115S.

[0020] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105I and S114C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105I and S114P. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105I and A115S.

[0021] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S114C and A115S. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S114P and A115S.

[0022] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T, Q105V, and S114C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T, Q105V, and S114P. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the substitutions S90T, Q105V, and A115S.

[0023] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S90T, Q105I, and S114C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S90T, Q105I, and S114P. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S90T, Q105I, and A115S.

[0024] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105V, S114C, and A115S. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105V, S114P, and A115S.

[0025] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105I, S114C, and A115S. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Q105I, S114P, and A115S.

[0026] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises amino acid substitutions S90T, Q105V, S114C, and A115S.

[0027] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises amino acid substitutions S90T, Q105V, S114P, and A115S.

[0028] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises amino acid substitutions S90T, Q105I, S114C, and A115S.

[0029] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises amino acid substitutions S90T, Q105I, S114P, and A115S.

[0030] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions G48C and T206C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Y123V, S125C and N171C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S114C, Y123V, S125C and N171C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions Y123V, S125C, N171C, G48C and T206C. In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises substitutions S114C, Y123V, S125C, N171C, G48C and T206C.

[0031] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the following: (a) any substitution, alone or in combination, described in the above paragraphs; and (b) substitutions G48C and T206C.

[0032] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the following: (a) any substitution, alone or in combination, described in the above paragraphs; and (b) substitutions Y123V, S125C, and N171C.

[0033] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the following: (a) any substitution or combination of substitutions described in the above paragraphs except S114P; and (b) substitutions S114C, Y123V, S125C, and N171C.

[0034] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the following: (a) any substitution, alone or in combination, described in the above paragraphs; and (b) substitutions Y123V, S125C, N171C, G48C, and T206C.

[0035] In some embodiments, the thermotolerant xylanase comprises the amino acid sequence of SEQ ID NO: 2, but comprises the following: (a) any individual substitution or combination of substitutions described in paragraph; and (b) substitutions S114C, Y123V, S125C, N171C, G48C, and T206C.

[0036] Relative to the Thite xylanase comprising the amino acid sequence SEQ ID NO: 2, these amino acid substitutions increase the thermotolerance of the thermotolerant xylanase. "Thermostability" is defined as an increase in residual enzyme activity relative to the wild-type or parent enzyme after a thermal challenge or step involving incubation of the enzyme at a temperature greater than 60°C. The increased thermotolerance can be conferred by modifying the amino acid sequence or composition of the molecule so that it denatures at a higher temperature than the wild-type or parent enzyme. The increased thermotolerance can also be conferred by amino acid modification or substitution, which enables the enzyme to refold after denaturation or thermal inactivation.

[0037] The term "wild-type" is defined herein as a xylanase having the amino acid sequence of SEQ ID NO: 2. Wild-type xylanases, also referred to as parent or parent enzymes, can be heterologously expressed in naturally occurring microorganisms such as bacteria, yeast, and filamentous fungi.

[0038] The term "variant" is defined herein as a xylanase in which the amino acid sequence SEQ ID NO: 2 has been modified by one or more amino acid substitutions relative to SEQ ID NO: 2. The variant enzymes may be heterologously expressed in naturally occurring microorganisms such as bacteria, yeast, and filamentous fungi.

[0039] "Improved thermostability", also referred to as "improved residual activity", is defined herein as improved retention of enzyme activity after a defined high temperature challenge relative to the wild-type enzyme activity.

[0040] "Improved thermostability" is defined herein as an improved ability for structural refolding and retention of enzymatic activity after thermal denaturation or thermal inactivation relative to wild-type structural refolding and wild-type enzyme activity.

[0041] Thermotolerant xylanases disclosed herein can be identified using methods well known in the art. Such methods involve substitution of amino acid residues in the polypeptide sequence, followed by screening for variants with improved thermotolerance (see, e.g., H. Yang et al. 2015. Chem. Bio. Eng. Rev. 2: 87-94; X. F. Zhang et al. 2016. Sci. Rep. 6: 33797; H. Yu et al. 2017. Sci. Rep. 7: 41212; Z. Xu et al. 2020. Crit. Rev. Biotechnol. 40: 83-98).

[0042] Methods using thermostable xylanases The thermotolerant xylanases disclosed herein can be used in a variety of methods. In one aspect, the thermotolerant xylanases disclosed herein can be used in a method selected from the group consisting of wet milling and dry milling corn milling. The milling method can include soaking, that is, soaking the organic material in a liquid (usually water) to extract flavor substances and / or soften the organic material. The use of the thermotolerant xylanases disclosed herein allows for improved by-product recovery, accelerated soaking kinetics, manufacturing energy savings and value addition in the form of enriched mill streams, including but not limited to light steep water / saccharification tank effluent entering the fermenter. The use of the thermotolerant xylanases disclosed herein can be easily implemented in a wet milling method with minimal changes to the manufacturing plant.

[0043] In one aspect, methods using the thermotolerant xylanases disclosed herein have advantages and provide benefits over methods involving conventional wild-type enzymes. In one aspect, the methods disclosed herein include enzyme preparations wherein the spray-dried thermotolerant NSPase disclosed herein has a higher activity per unit of enzyme protein (>5,000 XU / g) than conventional wild-type enzyme protein in solid form (~3,500 XU / g).

[0044] In one aspect, the methods disclosed herein include enzyme preparations wherein the spray-dried thermostable NSPase disclosed herein has significantly higher activity and thermotolerance compared to conventional liquid-based processing. Thus, to achieve superior results over conventional products, a lower enzyme dosage of the thermostable NSPase disclosed herein is required relative to soaking or mashing solids.

[0045] The NSP enzymes disclosed herein have greater demonstrated heat tolerance relative to other treatments (e.g., Frontia® Fiberwash (a product with wild-type xylanases, provided by Novozymes A / S)) and also have improved performance under real-world soaking and fiber washing conditions relative to such treatments.

[0046] In one aspect, the methods disclosed herein do not require changes to the manufacturing plant layout, as compared to conventional methods that require, for example, installation of culture tanks and piping to store the first milled corn during enzyme treatment prior to the second milling. In addition, unlike conventional products that must be added to the fiber tank after the milling process, the thermostable NSP enzymes disclosed herein can be added directly to the steepwater tank and / or mixed with the corn entering the steepwater tank and still improve downstream separations, for example, reducing total starch in the fiber.

[0047] The above method is contrary to the view of some persons skilled in the art that the cellulolytic and xylanolytic enzymes added to the steep liquor are of no use, especially when considering the recirculation of the steepwater back into the steep liquor. When the corn is screened, it will carry only a nominal amount of active enzyme protein into the mill. Therefore, most of the enzyme activity will remain on the steep liquor side, and only maintenance dosing will be required once the target activity in the steep liquor is reached. In the presence of the thermostable NSP enzymes disclosed herein, the steep liquor also requires lower SO 2 concentration, while releasing additional fermentable C6 and C5 sugars from the corn. From an ethanol fermentation perspective, higher sugar concentrations in the steepwater will require lower energy input to achieve the desired dextrose equivalent (DE) target via evaporation and will have lower concentrations of SO 2 , thus reducing the stress on the yeast. In the infusion, the presence of C5 and C6 sugars and the reduced SO 2 Helps improve the health of the soaking microbiome - promoting the growth of naturally occurring Lactobacillus.

[0048] The presence of enzymes in the soaking water has also been shown to improve germ separation, making the germ more buoyant in the soaking water mixture, which facilitates easier separation of the germ portion during the wet milling process. When the enzyme is present in the soaking water, the soaking water has higher magnesium, potassium and phosphorus levels relative to standard milling conditions during the soaking process. This aspect is important because the soaking water is usually concentrated and mixed with corn gluten feed. The method disclosed herein provides an enriched feed material relative to feed materials produced by conventional methods that do not include the use of xylanase. Elements such as calcium and sodium are also present in the enzyme-treated soaking water at higher levels. Although also beneficial to animal health, these minerals are known to improve the activity of certain enzymes, such as alpha-amylase used in downstream fermentation, and the activity of xylanases commonly used in animal feed and added to the corn steep liquor itself. Zinc has also been identified as a promoter of xylanase activity. In the method disclosed herein, manganese and boron levels are also increased. Boron can improve the absorption of minerals by livestock, while manganese has been shown to resist fatty liver disease in cattle. These two elements are also well utilized by metalloenzymes.

[0049] In the presence of enzyme treatment, inositol levels in the soak water increased, while phytate / phytic acid levels were lower. From an animal nutrition perspective, lower levels of phytate equate to lower levels of metal ion chelators, facilitating mineral absorption by livestock. Inositol has been identified as a potential growth promoter for poultry and an essential micronutrient for aquaculture.

[0050] From a commercial operation perspective, the methods disclosed herein can reduce starch delivery in fiber by more than 20% on a wild-type basis with the addition of a 0.1% (initial) dose of enzyme protein to unsteeped corn, an increase of >20% over that achieved with conventional products.

[0051] In a wet milling unit operation, a 10% reduction in fiber-bound starch can equate to approximately >$10MM in annual cost savings and reduce the amount of corn that must be milled to meet production targets.

[0052] In addition, the steeping process produced a steepwater containing 20% ​​more C5 and C6 sugars (combined) relative to baseline conditions. This promoted the growth of Lactobacillus species but not Acetobacter ( Acetobacter ) species (lactate concentrations exceeded 3000 ppm for 24-40 h, while acetic acid remained virtually unchanged).

[0053] From the perspective of enzyme dosage cost, in factory conditions, it is preferred to use heat-resistant solid enzymes, where internal temperature and humidity can vary significantly throughout the year. The solid material in the supersack can be stored in a dark, dry place, and no refrigerated tank is required to ensure that it remains active / active over time. High activity per unit protein is preferred because a lower dosage regimen is required. This is combined with the improved separation delivered in the mill and the reduction in energy demand in the entire method, creating highly desirable cost savings for both batch soaking and continuous soaking (the latter is considered to be an inferior process relative to batch soaking). The enzyme disclosed herein is also feasible in liquid form, which can be more easily pumped and added than solid form. In the case of tank refrigeration failure or mill heating equipment failure, it is desirable that the heat-resistant enzyme disclosed herein is in liquid form. Effectively, if the temperature peak is increased to 60°C-70°C range, the heat-resistant enzyme disclosed herein remains active for the intended use without losing the entire enzyme tank.

[0054] Those skilled in the art who benefit from this disclosure will recognize that the methods disclosed herein provide cleaner separations with lower energy inputs than conventional methods. Corn processors will recognize that the methods disclosed herein allow enzymatic wet milling to be more economically viable. Currently, enzymatic wet milling is not widely practiced in the United States.

[0055] Aspects and benefits of the present invention are further described in the following examples.

[0056] Example 1 . This example describes the identification of several amino acid substitutions that improve the thermotolerance of xylanase. The yeast (Saccharomyces cerevisiae) codon-optimized Thite GH11 xylanase coding sequence (SEQ ID NO: 1) was synthesized and subcloned into the yeast expression plasmid pYES2 (Invitrogen, Carlsbad, CA) to produce pAJ021. This plasmid was used as a backbone to produce wild-type Thite GH11 xylanase and to design Thite GH11 xylanase variants. Variants were designed using rational design enhanced with protein engineering software.

[0057] Screening for thermostability was facilitated by transforming and expressing wild-type xylanase and variant xylanase in Saccharomyces cerevisiae. The construction of these variants was accomplished using two different methods. Single-site substitutions were produced for each variant position using yeast gap repair and specific left and right PCR fragments described by Raymond et al. (reference: General Method for Plasmid Construction Using Homologous Recombination, BioTechniques 26: 134-141 January 1999). Each PCR fragment was designed to incorporate specific amino acid substitutions at the 3' and 5' ends of the left and right fragments, respectively. The 3' end of the left PCR fragment was designed to contain 37 bp overlapping sequence homology with pYES2 located downstream of the GAL1 promoter, and the 3' end of the right PCR fragment was designed to contain 25 bp overlapping sequence homology with pYES2 located upstream of the CYC1 terminator. The left PCR was designed to contain a yeast consensus sequence (CACAAA) immediately upstream of the ATG methionine. The left and right PCR fragments were combined with the pYES2 vector that had been restricted with HindIII and XbaI, and then used in yeast transformation similar to Dohmen et al. (reference: An Efficient Transformation Procedure Enabling Long-term Storage of Competent Cells of Various Yeast Genera. Yeast 7: 691-692). Transformants containing repaired sequences were selected on SC-U selective plates. Combinations or multiple substitution variants were constructed using synthetic fragments produced from external synthetic DNA services. These fragments were subcloned into the pYES2 vector and transformed similarly. Colonies from transformations that exhibited uracil prototrophy were selected and grown on an Infors oscillator at 1000 RPM at 30°C in 96 deep wells (1.1 ml) containing 500 μL of SC-U culture medium containing 2% glucose. After 24 hr, the culture solution was centrifuged at 700 × g and replaced with SC-U plus 2% galactose. Cultures were similarly grown for 72 hrs and sample cultures were then harvested by centrifugation at 700 xg for 10 min. Variant sample cultures were screened using the heat challenge AZCL-arabinoxylan (Megazyme, Bray, Ireland) assay.

[0058] Example 2.

[0059] Xylanase activity in culture supernatants was determined using AZCL-arabinoxylan, an insoluble, finely divided wheat arabinoxylan substrate (Megazyme, Bray, Ireland). The substrate was prepared at a concentration of 0.2% AZCL-arabinoxylan in 50 mM sodium citrate buffer (pH 4.2) and the reaction was incubated at 50°C ( Figure 1 Further enzyme evaluation was performed under reaction conditions of pH 5.3 and incubated at 37°C ( Figure 2 For thermal stability testing, supernatant samples were placed in Figure 1 and Figure 2 The enzyme activity (hydrolysis of substrate) was measured by the release of AZCL and the detection of absorbance at 595 nm. The residual activity percentage was calculated by comparing the activity of the enzyme samples treated at elevated temperatures with the activity of the same samples not heat challenged.

[0060] When compared to the wild-type (wt) Thite GH11 xylanase, 11 variants (incorporating 9 amino acid positions) showed improved thermotolerance as measured by residual activity. Figure 1 and Table 2. Figure 1 The results of the AZCL-arabinoxylan challenge assay of supernatants from Thielavia terrestris GH11 xylanase variants V22, AJ039, AJ040, AJ045 (V45), AJ102, AJ131 (V131) expressed from S. cerevisiae are shown, showing improved residual activity relative to the wild type. The variants are shown in Table 1 below.

[0061] Table 1

[0062] Improved variants of Thielavia terrestris GH11 xylanase and corresponding designed amino acid substitutions involved in improved xylanase thermostability. Note: Amino acid substitution numbers refer to their position relative to the N-terminal amino acid of the mature protein.

[0063] Table 2 shows the results of an AZCL-arabinoxylan challenge assay performed on supernatants from S. cerevisiae heterologously expressing Thielavia terrestris GH11 xylanase (variants with improved residual activity relative to wild type). Variants V17, V22; V24, V40, V42.

[0064] Table 2

[0065] Example 3.

[0066] AJ045 (aka V45; Y123V, S125C, N171C, G48C, T206C) and AJ131 (aka V131; S114C, Y123V, S125C, N171C) expression and thermotolerance in Trichoderma reesei. Four V131 and five V45 Trichoderma reesei laboratory scale fermenters were run and culture supernatants were tested in the AZCL-arabinoxylan heat challenge activity assay. The results showed that the improved residual activity profile observed in variant V45 when expressed in Saccharomyces cerevisiae was very similar to the improvements seen when the variant was produced in Trichoderma reesei.

[0067] Example 4 Differential Scanning Calorimetry (DSC) determinations were performed on yeast expressing V22 and T. reesei, and DSC determinations were performed on yeast expressing AJ045 and AJ131, and are shown in Table 3 below.

[0068] Table 3

[0069] Figure 2 Results of AZCL-arabinoxylan challenge assays of supernatants from Trichoderma reesei heterologously expressing Thielavia terrestris GH11 xylanase variants AJ045 (V45) and AJ131 (V131) are shown, showing improved residual activity relative to wild type. Of particular interest is the thermotolerant properties of AJ045. While this variant clearly showed superior performance in the AZCL-arabinoxylan challenge assay ( Figure 1 and Figure 2 ), but this variant showed little improvement in DSC Tm when compared to the wild-type GH11 xylanase. However, the ability of AJ045 to refold after the protein has been subjected to a 10-minute melting treatment may be particularly important in the animal feed industry.

[0070] Example 5.

[0071] Differential scanning calorimetry (DSC) was used to perform refolding experiments on wild-type Thielavia terrestris GH11 xylanases, V22, V45 and V131 expressed in yeast saccharomyces cerevisiae and purified from yeast saccharomyces cerevisiae. The samples were purified in 2 steps: 1) captured by self-filling Capto Phenyl ImpRes (hydrophobic interaction chromatography) and 2) optimized by HiLoad 16 / 600Superdex 75 (size exclusion chromatography). In DSC, the purified samples were first heated to a few degrees Celsius (10-15 minute heating time) above Tm. After the melt, the sample was allowed to balance to 25°C, and then subjected to a typical DSC run ramped to 110°C at 90°C / hr. The results are shown in Table 4 below.

[0072] Table 4. First and second melting Tm and ΔH of xylanases expressed in S. cerevisiae.

[0073]

[0074] *Expressed in filamentous fungal hosts

[0075] The refolding characteristics of V45 and the elevated melting temperatures of V131 were then verified by expressing the variants in a filamentous fungus host. V45 and V131 were expressed and purified from a filamentous fungus host and tested in a refolding DSC experiment with the addition of a third melting step. The results for V45 and V131 are shown in Tables 5 and 6, respectively; their corresponding DSC thermograms are shown in Tables 5 and 6. Figure 3 and Figure 4 As shown in . The DSC thermogram shows the relationship between the heat capacity Cp (kJ / mol / K) of the first, second and third melting and the temperature T (°C). Since protein unfolding is an endothermic process, it is observed as a positive shift in the thermogram. The midpoint of the transition (peak) is Tm, and the area under the transition is the enthalpy (ΔH) of the protein unfolding process.

[0076] Table 5. Tm and ΔH of each melting point of V45

[0077] Table 6. Tm and ΔH of each melting point of V131

[0078] Example 6 . Table 7 shows the use of the thermostable NSPase Thielavia xylanase variant disclosed herein (referred to as "tNSPase" in Table 6) in corn wet milling compared to wild-type xylanase. In this example, 400 g dry corn, 466 g deionized water (DI), 0.74 g sodium metabisulfite, and 14.01 g lactic acid were mixed in a 1 L bottle. The bottle was placed in a shaking water bath set to 52°C. The corn was soaked for 40 hours. The soaking water was sampled at 0, 16, 24 and 40 hours, and the concentrations of the following compounds were measured by HPLC. Phytate, free lactic acid, dextrose, xylose, fructose, inositol, and galactose are desirable in various applications, for example, ethanol fermentation. However, free acetic acid is undesirable in many applications, for example, ethanol fermentation. The tNSPase of this example is V45 identified above. As shown in Table 7, use of the NSPase of the present invention provided better or similar results than use of wild-type xylanase, even though the tNSPase enzyme loading wt / wt% was less than that of the wild-type xylanase, i.e., 0.06% compared to 0.11%, 0.04% compared to 0.07%, and 0.02% compared to 0.04%.

[0079] Table 7

[0080]

[0081]

[0082] The methods and uses of the thermotolerant xylanases disclosed herein can provide advantages over methods using conventional wild-type enzymes. These advantages include the following: - Reduce SO added during wet milling to treat corn kernels 2 Chemical substances; Without being bound by theory, it is believed that the thermotolerant xylanases of the present disclosure assist and / or promote chemical penetration and reaction with corn kernels to a greater extent than conventional wild-type enzymes. Compared to 2000 ppm SO when conventional wild-type enzymes are used 2 Compared to traditional dosages of 500 ppm, it was found that in the presence of the thermotolerant xylanase disclosed herein, only 500 ppm was needed to substantially soften corn kernels.

[0083] - The thermotolerant xylanase of the present invention has higher activity than conventional wild-type enzymes. Therefore, the thermotolerant xylanase of the present invention can be used at a lower dosage than conventional wild-type enzymes. See Table 6 above.

[0084] - After a heat challenge or a step involving incubation of the enzyme at a temperature above 60°C, the thermotolerant xylanase has a higher residual enzyme activity relative to the wild-type or parent enzyme.

[0085] Those skilled in the art having the benefit of this disclosure will recognize that the thermotolerant Thielavia xylanase variants of the present disclosure can be used at low dosage levels to release (hemi)cellulosic material in the form of C5 and C6 sugars from dry ground corn. An additional benefit obtained will be higher ethanol yields.

[0086] One skilled in the art having the benefit of this disclosure will recognize that the thermotolerant Thielavia xylanase variants of the present disclosure may be used in continuous soaking apparatus and methods.

[0087] Those skilled in the art will recognize, using the knowledge gained from the present disclosure, that various changes may be made to the disclosed compositions and methods in obtaining the advantages and other advantages disclosed herein without departing from the scope of the present disclosure. Therefore, it should be understood that the features of the present disclosure are susceptible to modification and / or replacement. The specific embodiments shown and described herein are intended for illustrative purposes only, rather than for limitations of the present invention as set forth in the appended claims.

Claims

1. A first xylanase comprising the amino acid sequence of SEQ ID NO: 2, but comprising an amino acid substitution at a position selected from the group consisting of 90, 105, 114 and 115 of SEQ ID NO: 2 and / or any set of substitutions selected from the group consisting of: (a) G48C and T206C; (b) Y123V, S125C, and N171C; (c) S114C, Y123V, S125C, and N171C; (d) Y123V, S125C, N171C, G48C and T206C; or (e) S114C, Y123V, S125C, N171C, G48C, and T206C; Wherein the one or more amino acid substitutions increase the thermotolerance of the xylanase relative to a second xylanase comprising the amino acid sequence of SEQ ID NO:

2.

2. The first xylanase of claim 1, wherein the at least one substitution is selected from the group consisting of S90T, Q105V, Q105I, S114C, S114P and A115S.

3. The first xylanase of claim 1, wherein the amino acid substitutions comprise Y123V, S125C, N171C, G48C, and T206C.

4. A method of using the first xylanase of claim 1, wherein the method is a milling method.

5. The method of claim 4, wherein the milling process is a corn milling process.

6. The method of claim 5, wherein the corn milling method is selected from the group consisting of wet milling and dry milling.

7. A method of using the first xylanase of claim 3, wherein the method is a milling method.

8. The method of claim 7, wherein the milling process is a corn milling process.

9. The method of claim 8, wherein the corn milling method is selected from the group consisting of wet milling and dry milling.

10. The method of claim 4, wherein the method comprises granulation.

11. The method of claim 10, wherein the pelleting comprises forming granules comprising the first xylanase and an animal feed.

12. An animal feed comprising the first xylanase of claim 1.

13. The animal feed according to claim 12, wherein the animal feed is selected from the group consisting of poultry feed and aquaculture feed.

14. A method of using the first xylanase of claim 1, wherein the method comprises a thermal challenge involving subjecting the first xylanase to a temperature above 60°C.

15. A method of using the first xylanase of claim 1, wherein the method is selected from the group consisting of animal feed manufacturing, baking, pulp bleaching, textile bleaching, and conversion of biomass to biofuel.

16. The method of claim 15, wherein the method is to convert biomass into biofuel.

17. The method of claim 16, wherein the biofuel comprises ethanol.

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