Xylanase mutants and their applications

Abstract

The present invention provides a xylanase mutant. The xylanase mutant is obtained by mutating the xylanase (xynCDBFV) gene (Genebank: KP691331.1) of the parent rumen fungus Neocallimastix patriciarum, and substituting the amino acid sequence of the parent xylanase shown in SEQ ID NO: 1. Compared with the parent xylanase shown in SEQ ID NO: 1, the xylanase mutant of the present invention has significantly improved acid stability and low pH specific enzyme activity, which is conducive to the industrial application of the enzyme.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to an acid-resistant xylanase mutant, its encoding gene and application. Background Art

[0002] Xylanases (EC 3.2.1.8) are a complex enzyme system that degrades xylans in hemicellulose into xylo-oligosaccharides and xylose. The primary enzyme is β-1,4-endo-xylanase. Xylanases are widely used in the feed, food, and paper industries. As feed enzymes, xylanases can effectively degrade xylans in feed, degrade anti-nutrients, reduce feed viscosity, and improve feed utilization efficiency. During animal feed digestion, feed is first pre-processed in the stomach before being digested and absorbed in the intestines. The stomach is an acidic environment with a pH of 2.5-3.5. Ordinary xylanases are easily inactivated, resulting in the enzyme preparation being unable to perform its enzymatic activity by the time it reaches the intestines. Therefore, xylanases need to possess good acid resistance to better perform their enzymatic activity.

[0003] Currently, most commercially available xylanases are neutral xylanases, which have high enzymatic activity at pH 5.0-7.0. However, their activity is low at low pH (pH 2.5-3.5), and they are not acid-resistant and easily inactivated, limiting their application. Acid xylanases are a class of xylanases that can maintain high enzymatic activity and stability under acidic conditions. However, natural acid xylanases are relatively rare and have low enzymatic activity, resulting in long R&D cycles and very high costs for industrial applications. Currently, there is an acid xylanase, XYL10A, but its enzymatic activity near its optimum pH of 2.4 is significantly different from its peak enzymatic activity, indicating that this xylanase only has high enzymatic activity within a very narrow pH range, limiting its application.

[0004] During feed production, enzyme preparations are mixed with feed and then pelletized at high temperatures. During this process, the enzyme is easily denatured and inactivated. Therefore, the development of heat-resistant acid xylanase is beneficial for its application in the feed industry. Currently, the activity of most xylanases on the market drops to 20%-30% or even lower when the reaction temperature is 70℃-80℃.

[0005] Therefore, screening out acid-resistant and heat-resistant xylanases is of great significance for reducing the production cost of current feed xylanases and improving their utilization efficiency. At the same time, it is also of great significance for industries such as food processing and medicine that are suitable for use under acidic conditions. Summary of the Invention

[0006] Based on this, the present invention is necessary to provide a xylanase mutant and its industrial application. Compared with existing xylanases, the xylanase mutant has higher acid resistance and low pH enzyme activity, making it suitable for application in industries such as food, feed and medicine.

[0007] The specific technical solutions are as follows:

[0008] A xylanase mutant, wherein the amino acid sequence of the xylanase mutant is subjected to one or more of the following mutations relative to the parent xylanase shown in SEQ ID NO: 1: K15E, V16I, K83E, F120S, N128S, D171R, G201S and D225G.

[0009] A nucleic acid molecule comprising one or more of the nucleotide fragments shown in (a) and (b) below:

[0010] (a) a nucleotide fragment encoding any one of the above xylanase mutants;

[0011] (b) consists of a nucleotide fragment that is completely complementary to a).

[0012] A recombinant expression vector comprises the nucleic acid molecule.

[0013] A host cell comprises the nucleic acid molecule or the recombinant expression vector.

[0014] A method for preparing an acid-resistant xylanase mutant, comprising the following steps:

[0015] Cultivating the above-mentioned host cell;

[0016] The host cell is induced to express the xylanase mutant.

[0017] The xylanase mutant is used as a xylan degrading enzyme; and / or in the preparation and / or processing of food, feed, nutritional additives, textiles, detergents, paper and medicines.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The xylanase mutant provided by the present invention has significantly improved acid stability and low pH specific enzyme activity compared with the parent xylanase shown in SEQ ID NO: 1, which is beneficial to the industrial application of the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The optimal reaction pH of the mutant obtained by the present invention and the parent xylanase is shown;

[0021] Figure 2The results show that the mutants obtained in the present invention have thermal stability compared with the parent xylanase. DETAILED DESCRIPTION

[0022] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0024] One embodiment of the present invention provides a xylanase mutant, obtained by multiple mutagenesis and high-throughput screening of the xylanase (xynCDBFV) gene (Genebank: KP691331.1) from the rumen fungus Neocallimastix patriciarum, with improved acid stability and low-pH specific enzyme activity. The amino acid sequence of the xylanase mutant has one or more of the following mutations relative to the parent xylanase set forth in SEQ ID NO: 1: K15E, V16I, K83E, F120S, N128S, D171R, G201S, and D225G. The xylanase mutant has xylanase activity, including but not limited to hydrolase activity, such as the ability to hydrolyze glycosidic bonds present in xylan, such as catalyzing the hydrolysis of internal β-1,4-xylosidic bonds; and has higher enzymatic activity at pH 3.0 than the xylanase set forth in SEQ ID NO: 1.

[0025] In a specific example, the amino acid sequence of the xylanase mutant undergoes one or two of the following mutations relative to the parent xylanase set forth in SEQ ID NO: 1: K15E, V16I, K83E, F120S, N128S, D171R, G201S, and D225G. It should be noted that the "K15E" mutation indicates that the amino acid at position 15 of the amino acids set forth in SEQ ID NO: 1 is mutated from K (lysine) to E (glutamic acid). The "F120S and K15E" two-point mutation indicates that the amino acid at position 15 of the amino acids set forth in SEQ ID NO: 1 is mutated from K (lysine) to E (glutamic acid), and the amino acid at position 120 is mutated from F (phenylalanine) to S (serine). Other similar treatments are similarly applied.

[0026] In a preferred embodiment, the xylanase mutant of the present invention comprises the following mutations relative to the parent xylanase set forth in SEQ ID NO: 1: K15E; V16I; K83E; F120S; N128S; D171R; or G201S. The xylanase mutants comprising these single-point mutations exhibit significantly better acid stability and low pH specific enzyme activity than the parent xylanase.

[0027] In a preferred embodiment, relative to the parent xylanase shown in SEQ ID NO: 1, the xylanase mutant of the present invention comprises the following mutations: K15E+V16I; or K15E+K83E; or K15E+F120S; or K15E+N128S; or K15E+D171R; or K15E+G201S; or K15E+D225G; or V16I+K83E; or V16I+F120S; or V16I+N128S; or V16I+D171R; or V16I+G201S; or K15E+D225G; or K15E+D171R; or V16I+K83E; or V16I+F120S; or V16I+N128S; or V16I+D171R; or V16I+G201S; or V16I+D225G; or K15E+D171R; or V16I+G201S; or V16I+D225G; or K15E+D171R; or V16I+K83E; or V16I+ 3E+F120S; or K83E+N128S; or K83E+D171R; or K83E+G201S; or K83E+D225G; or F120S+N128S; or F120S+D171R; or F120S+G201S; or F120S+D225G; or N128S+D171R; or N128S+G201S; or N128S+D225G; or D171R+G201S; or D171R+D225G; or G201S+D225G. It should be noted that "K15E+V16I" indicates that the amino acids at positions 15 and 16 are mutated compared to the amino acids shown in SEQ ID NO: 1. The same applies to other similar treatments. The acid resistance stability and low pH specific enzyme activity of the xylanase mutant with two point mutations are significantly better than those of the parent xylanase.

[0028] In a more preferred embodiment, relative to the parent xylanase set forth in SEQ ID NO: 1, the xylanase mutant of the present invention comprises the following mutations: K15E+F120S; or V16I+F120S; or K83E+F120S; or F120S+N128S; or F120S+D171R; or F120S+G201S; or F120S+D225G. The xylanase mutants of the present invention comprising the above two point mutations have improved acid stability and low pH specific enzymatic activity.

[0029] In a specific example, the amino acid sequence of the xylanase mutant includes at least one of the following sequences: 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: 15 and SEQ ID NO: 16.

[0030] In one specific example, the xylanase mutant is derived from the rumen fungus Neocallimastix patriciarum.

[0031] One embodiment of the present invention further provides a nucleic acid molecule or a recombinant expression vector or a host cell.

[0032] The nucleic acid molecule comprises at least one of the nucleotide sequences shown in (a) and (b) below:

[0033] a) a nucleotide sequence encoding any one of the above xylanase mutants;

[0034] b) consists of a nucleotide sequence completely complementary to a).

[0035] In a specific example, the nucleotide sequence of the xylanase mutant according to the present invention is shown in 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, and SEQ ID NO: 31. The recombinant expression vector comprises the above-mentioned nucleic acid molecule.

[0036] In a specific example, the vector can be a plasmid, a virus, a bacteriophage, etc. Other vectors can also be selected according to the needs of those skilled in the art.

[0037] The host cell contains the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector.

[0038] In a specific example, the host cell can be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell, or a mammalian cell. Preferably, the host cell is a fungal cell, more preferably, a yeast cell or a filamentous fungal cell, and more preferably, a Pichia pastoris.

[0039] One embodiment of the present invention further provides a method for preparing an acid-resistant xylanase mutant or a polypeptide having xylanase activity, comprising the following steps:

[0040] a) constructing a recombinant expression vector comprising a nucleic acid molecule encoding a xylanase mutant;

[0041] b) introducing the recombinant expression vector into a host cell to obtain a recombinant host cell;

[0042] c) inducing the above recombinant host cell to express the xylanase mutant or the polypeptide having xylanase activity.

[0043] In a specific example, the method may further include recovering and purifying the xylanase mutant.

[0044] Furthermore, one embodiment of the present invention also provides a method for producing the above-mentioned xylanase mutant, comprising the following steps:

[0045] Cultivating the recombinant host cell under suitable conditions to produce a xylanase mutant having xylanase activity; and

[0046] The xylanase mutant is recovered and purified to obtain the produced xylanase mutant.

[0047] Xylanase proteins can be expressed in a variety of expression systems, and appropriate downstream processing and purification steps must be selected accordingly. In one embodiment of the present invention, xylanases can be expressed in bacterial hosts, and the protein secreted into the periplasm or extracellular space. Cultures of the expressing organisms can be prepared in appropriate volumes according to standard fermentation methods. In a preferred embodiment, cells are grown in a fermentor, and growth conditions such as pH, temperature, oxygen, and / or nutrient supply are optionally controlled.

[0048] In some embodiments, the expressed or produced mutant xylanase is secreted into the nutrient medium and can be recovered directly from the medium. If the expressed or produced mutant xylanase is not secreted, it can be recovered from cell lysates.

[0049] In some embodiments, the first step of purification involves separating the cells from the supernatant using one or more of the following techniques: sedimentation, microfiltration, centrifugation, or flocculation. In preferred embodiments, microfiltration is the preferred method. If expressed intracellularly, the cells are treated to release the protein from the intracellular space. These treatments may include, for example, pressurization, enzymatic catalysis, osmotic shock, freezing, sonication, or other treatments, thereby producing a cell extract that may or may not undergo further purification.

[0050] In some specific examples, the xylanase is secreted into the supernatant after induction culture, and the protein in the supernatant or concentrated supernatant is further purified by one or more of the following methods: extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatography methods, including but not limited to ion exchange, hydrophobic interaction, hydroxyapatite, particle size fractionation by gel filtration, phosphocellulose or lectin chromatography and affinity chromatography, or any combination thereof. In some preferred methods, the affinity-tagged protein is purified by metal chelate affinity chromatography to obtain a high-purity target protein. In other preferred examples, the high-purity target protein is obtained by HPLC purification.

[0051] In some embodiments, the production method may further comprise processing the produced xylanase mutant.

[0052] In one specific example, the fermentation cell suspension comprising the expressed xylanase is dried as a whole using methods including, but not limited to, fluid bed drying, conveyor drying, spray drying, or drum drying, or any combination thereof.

[0053] The xylanase mutants of the present invention are thermostable and acid-stable, and can retain xylanase activity under certain conditions. Optionally, the conditions include 25°C to 95°C, optionally 37°C to 90°C, optionally 50°C to 80°C, optionally 70°C to 95°C, and optionally 80°C to 95°C. Optionally, the conditions include pH 2.5 to pH 12, optionally pH 2.5 to pH 6.0, and optionally pH 3.0 to pH 5.5. In one specific example, after treatment at 90°C for 5 minutes, the mutants still retain xylanase activity at pH 5.5. Compared to the parent xylanase set forth in SEQ ID NO: 1, the xylanase mutants of the present invention exhibit significantly improved acid stability and low-pH specific enzyme activity under low pH conditions, facilitating the industrial application of the enzyme. Low pH conditions include pH 2.5 to 3.5, and optionally pH 3.0.

[0054] Therefore, one embodiment of the present invention further provides the use of the xylanase mutant in degrading xylan, and / or in food, feed, nutritional additives, textiles, detergents, papermaking, agriculture, and medicine. Specifically, the xylanase mutant can be used in the preparation and / or processing of food, feed, nutritional additives, textiles, detergents, and the like. Specifically, the xylanase mutant can be used in pharmaceutical compositions and dietary supplements, and can be particularly used in baking, animal feed, and beverages.

[0055] In one embodiment, the activity of the xylanase mutant comprises catalyzing the hydrolysis of internal β-1,4-xylosidic linkages. In one embodiment, the activity of the xylanase mutant comprises endo-β-1,4-xylanase activity.

[0056] In one embodiment, the activity of the xylanase mutant comprises hydrolyzing xylan to produce smaller molecular weight xylose and xylose oligomers. In one embodiment, the xylan comprises arabinoxylan, such as water-soluble arabinoxylan. The water-soluble arabinoxylan can be formed into dough or bread products.

[0057] In one embodiment, the activity of the xylanase mutant includes catalyzing the hydrolysis of xylan in a cell, such as a plant cell or a microbial cell.

[0058] In one embodiment, the activity of the xylanase mutant comprises hydrolyzing polysaccharides containing 1,4-β-glycosidically linked D-xylopyranose. In one embodiment, the activity of the xylanase mutant comprises hydrolyzing hemicellulose, specifically, hydrolyzing hemicellulose in wood or pulp or paper products.

[0059] In one embodiment, the activity of the xylanase mutant comprises catalyzing the hydrolysis of xylans in a beverage, feed, or food. The feed or food comprises cereal-based animal feed, wort or beer, milk or milk products, fruit, or vegetables.

[0060] Furthermore, the present invention also provides food, feed, beverage, or beverage precursor comprising the xylanase mutant. The food may be dough or bread products. The beverage or beverage precursor may be beer or wort.

[0061] Furthermore, the present invention also provides a method for improving dough, comprising contacting dough or a bread product with at least one xylanase mutant of the present invention under conditions sufficient to improve the dough.

[0062] Furthermore, the present invention provides a method for producing a beverage, comprising applying at least one xylanase mutant of the present invention to a beverage or a beverage precursor under conditions sufficient to reduce the viscosity of the beverage.

[0063] In one embodiment, the xylanase mutants of the present invention can also be used to treat fibers and textiles, where they can be combined with other textile treatments, such as scrubbing and bleaching. Scrubbing is the process of removing non-cellulosic materials from cotton fibers, such as the cuticle (primarily composed of waxes) and the original cell wall (primarily composed of colloids, proteins, and xylose glucose). Proper wax removal is essential for achieving high wettability. This is necessary for dyeing. Removal of the primary cell wall by the process of the present invention improves wax removal, ensuring more uniform dyeing.

[0064] In a specific example, the xylanase mutants of the present invention can be made into powdered and liquid detergents, not limited to detergents for use under alkaline conditions. If necessary, the functional components in the detergent can be separated by isolation means and released at different time periods to exert their own effects. These detergent compositions also include other enzymes such as cellulases, lipases, oxidases, laccases, amylases, glucoamylases, pectinases, etc. The detergent compositions of the present invention can, for example, be formulated as detergent compositions for manual or machine laundry containing the polypeptides of the present invention. Laundry additives for pre-treating dyed fabrics can contain the xylanase mutants of the present invention. Fabric softener compositions can contain the xylanase mutants of the present invention. Alternatively, the xylanase mutants of the present invention can be made into detergent compositions for use in conventional household hard surface cleaning work.

[0065] In one embodiment, the xylanases of the invention can be used to formulate a variety of cleaning compositions. A variety of known compounds are suitable surfactants including nonionic, anionic, cationic, or zwitterionic detergents can be used.

[0066] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made according to experimental manuals or conventional conditions in the art, or according to conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0067] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0068] The following are the main experimental materials, reagents and methods used in the examples:

[0069] 1. Strains and vectors: Strains containing the xylanase xynCDBFV gene and expression plasmid, Escherichia coli strain Top10, Pichia pastoris X33, vector pPICZαA, vector pGAPzαA, and antibiotic Zeocin were purchased from Invitrogen.

[0070] 2. Enzymes and kits: Ultra-fidelity 2× Master Mix PCR polymerase and restriction endonucleases were purchased from New England Biolabs, and plasmid extraction and purification kits were purchased from Shanghai Bioengineering Corporation.

[0071] 3. Culture medium

[0072] E. coli culture medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB+Amp medium was LB medium supplemented with ampicillin at a final concentration of 100 μg / mL, and LB+Zeo medium was LB medium supplemented with Zeocin at a final concentration of 25 μg / mL.

[0073] Yeast culture medium was YPD medium (1% yeast extract, 2% peptone, 2% glucose). Yeast screening medium was YPD+Zeo medium (YPD+Zeo medium is YPD medium with Zeocin added to a final concentration of 100 μg / mL).

[0074] yeast induction medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v / v)) and BMMY (same composition as BMGY except 0.5% methanol instead of glycerol);

[0075] Recombinant yeast fermentation minimal salt medium: 5% diammonium phosphate, 0.5% potassium dihydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 1.95% potassium sulfate, 0.1% calcium sulfate, 0.03% defoamer. After autoclaving, add 4.35 mL of PTM1 (trace salt solution) per liter: copper sulfate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, and biotin 0.02%.

[0076] 4. Chemical reagents:

[0077] Xylanase standard and xylan were purchased from Sigma, and other reagents were purchased from Guangzhou Chemical Reagent Factory;

[0078] 5. Xylanase activity determination method

[0079] Xylanase activity is determined according to the national standard "Determination of Xylanase Activity in Feed Additives - Spectrophotometric Method (GB / T23874-2009)." Xylanase can degrade xylan into oligosaccharides and monosaccharides. Reducing oligosaccharides and monosaccharides react with 3,5-dinitrosalicylic acid (DNS) reagent in a boiling water bath to develop a color. The color depth of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis, and the amount of reducing sugars produced is proportional to the activity of the xylanase in the reaction solution. Therefore, by spectrophotometrically measuring the color intensity of the reaction solution, the activity of the xylanase in the reaction solution can be calculated. Different pH levels can be adjusted to detect enzyme activity according to experimental requirements.

[0080] At 37°C and pH 5.5, the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a 5 mg / mL xylan solution per minute is one enzyme activity unit (U).

[0081] Example 1 Synthesis of xylanase xynCDBFV gene and vector construction

[0082] The amino acid sequence of the xylanase (xynCDBFV) gene of the rumen fungus Neocallimastix patriciarum (Genebank: KP691331.1) is shown in SEQ ID NO: 1.

[0083] EcoRI and XbaI restriction sites were introduced at the 5' and 3' ends of the xylanase xynCDBFV gene and ligated to the Puc57-amp vector. xynCDBFV-Puc57 was inoculated into LBA medium and cultured for 24 hours. The plasmid was extracted and digested with EcoRI and XbaI. The target gene fragment was recovered by gel cutting. The product was purified and recovered and ligated to the expression vector pPICzαA to obtain the expression vector pPICzαA-xynCDBFV.

[0084] Example 2 Error-prone PCR random mutagenesis

[0085] Using the above-mentioned pPICzαA-xynCDBFV as a template, an error-prone PCR method was used to randomly introduce mutations into the xylanase xynCDBFV gene.

[0086] PCR amplification was performed using the following primers:

[0087] Xyn-F 5'-gaaaagagaggctgaagctgaattc-3', SEQ ID NO: 32;

[0088] Xyn-R 5'-tgagatgagtttttgttctagacta-3', SEQ ID NO: 33.

[0089] The amplified product was digested with EcoRI and XbaI, and the PCR amplification results were checked by agarose electrophoresis. The PCR amplified product of interest was purified and recovered. The template was digested with the restriction endonuclease DpnI and ligated into the expression vector pPICzαA, which had been digested with EcoRI and XbaI. The digested product was transformed into competent E. coli Top10 cells using a chemical transformation heat shock method. Recombinant transformants were verified by PCR in the culture medium, and the plasmids of the confirmed transformants were extracted. The mutant plasmid was linearized with the PmeI endonuclease, the linearized plasmid fragment was purified, and transformed into competent Pichia pastoris X33 cells by electroporation. Recombinant transformants were obtained using YPD+Zeo medium.

[0090] Example 3 High-throughput screening of high-specific-activity mutant strains

[0091] Using a toothpick, the yeast recombinant transformants obtained in Example 2 were individually transferred to a 24-well plate. 1 mL of BMMY medium was added to each well and cultured at 30°C, 220 rpm, for approximately 24 hours. The supernatant was then centrifuged and removed. 1.6 mL of BMMY medium was then added to each well for induction. After 24 hours of culture, the supernatant was removed by centrifugation. 200 μL of each supernatant was transferred to a 96-well plate for xylanase activity assay and specific activity calculation. High-throughput screening was performed to identify yeast recombinant transformants with enhanced specific activity.

[0092] Example 4 High-throughput screening of xylanase mutants with improved acid resistance

[0093] Yeast recombinant transformants obtained in Example 3 with xylanase activity close to or higher than that of the parent strain were picked individually with toothpicks into a 24-well plate. 1 mL of BMMY medium was added to each well and cultured at 30°C, 220 rpm for approximately 24 hours. The supernatant was then centrifuged and removed. 1.6 mL of BMMY medium was then added to each well for induction culture. After 24 hours of culture, the supernatant was centrifuged and 200 μL of each supernatant was transferred to a 96-well plate for xylanase activity and acid stability assays. Through multiple rounds of screening and comparison, xylanase mutants with significantly improved acid resistance compared to the parent strain were identified.

[0094] Example 5 Combination Mutation and Screening

[0095] In Example 3 and Example 4, based on the low pH specific activity or acid-resistant forward mutation sites, double-site or multi-site combination mutations were performed, and the parent xylanase and the xylanase mutant were purified respectively by affinity chromatography purification. The high-throughput method in Implementation Case 3 and Implementation Case 4 was continued to be used for screening, and the corresponding pH 3.0 enzyme activity and acid resistance under pH 3.0 conditions were measured, and the specific activity was calculated.

[0096] The present invention screened xylanase mutants with single point mutations of K15E, V16I, K83E, F120S, N128S, D171R, G201S, and D225G, and any combination of any two of these mutations, derived from the same parent (e.g., SEQ ID NO: 1). Studies have shown that at pH 3.0, the xylanase mutants with the single point mutations and any combination of any two of these mutations exhibit varying degrees of improvement in specific activity and acid stability relative to the parent xylanase xynCDBFV.

[0097] As shown in Table 1 below, the relative specific activity of the mutants was calculated by dividing the mutant specific activity by the specific activity of the parent xylanase. At pH 3.0, xylanase mutants harboring single point mutations (K15E, V16I, K83E, F120S, N128S, D171R, G201S, or D225G) (SEQ ID NOs: 2 to 9) exhibited significantly improved specific activity relative to the parent xylanase xynCDBFV. Xylanase mutants harboring any combination of two of these point mutations exhibited similar properties. Xylanase mutants harboring two-point mutation combinations exhibiting even superior properties (SEQ ID NOs: 10 to 16) are also listed in Table 1.

[0098] The amino acid sequence of the parent xylanase is as follows:

[0099] QSFCSSASHSGQSVKVTGNKVGTIGGVGYELWADSGNNSATFYSDGSSFSCTFQNAGDY

[0100] LCRSGLSFSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSP

[0101] FPPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHF

[0102] DQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD, SEQ ID NO: 1.

[0103] Table 1 Relative specific activities of parent xylanase xynCDBFV and mutants at pH 3.0

[0104]

[0105]

[0106] When treated at pH 3.0 for 2 hours, the xylanase mutants with single point mutations and any combination of two point mutations screened by the present invention can retain higher enzyme activity and significantly improve acid resistance stability compared to the parent xylanase xynCDBFV.

[0107] As shown in Table 2 below, at pH 3.0, xylanase mutants containing single point mutations K15E, V16I, K83E, F120S, N128S, D171R, G201S, or D225G (SEQ ID NOs: 2 to 9) retained higher enzyme activity and significantly improved acid stability compared to the parent xylanase xynCDBFV. Xylanase mutants containing any combination of two of these point mutations also exhibited similar properties. Xylanase mutants containing two point mutation combinations exhibiting even better properties (SEQ ID NOs: 10 to 16) are also listed in Table 2.

[0108] Table 2 Comparison of acid resistance of parent xylanase xynCDBFV and mutants

[0109]

[0110] Example 6 Optimal reaction pH of parent xylanase xynCDBFV and its mutants

[0111] At a temperature of 37°C, the enzyme activity of xylanase was measured at pH 3.0, pH 4.0, pH 5.0, and pH 5.5. The results are as follows: Figure 1 As shown. The enzyme activity of xylanase measured at pH 5.5 was used as a control to calculate the relative enzyme activity of each enzyme under different pH conditions. Figure 1 It can be seen that the relative enzyme activity of the xylanase mutant under different pH conditions is basically consistent with that of the parent xylanase xynCDBFV, the optimal reaction pH is pH 5.5, and the enzyme activity at pH 3.0 is significantly higher than that of the parent.

[0112] Example 7 Thermostability of the parent xylanase xynCDBFV and its mutants

[0113] Feed pelleting requires high temperature conditions. To investigate the thermal stability of xylanase xynCDBFV and its mutant at high temperatures, the thermostability of these two enzymes was tested at 90°C. The fermentation supernatant was treated at 90°C for 5 minutes, and the enzyme activity was measured at 37°C and pH 5.5. The unheated fermentation supernatant was used as a control, and the relative enzyme activity was 100%. The results are shown in Figure 2. Figure 2 As shown, after the parent xylanase xynCDBFV and the mutant were treated at 90°C for 5 min, 70% of the enzyme activity was retained, and the thermal stability of the mutant did not change.

[0114] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A xylanase mutant, characterized in that The amino acid sequence of the xylanase mutant is shown in SEQ ID NO:

16.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleotide fragment encoding the xylanase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule of claim 2.

4. A host cell, characterized in that The invention relates to a yeast cell or a filamentous fungal cell comprising the nucleic acid molecule according to claim 2 or the recombinant expression vector according to claim 3.

5. The host cell according to claim 4, wherein The yeast cells are Pichia pastoris cells.

6. A method for preparing an acid-resistant xylanase mutant, characterized in that: The method comprises the following steps: Cultivating the host cell according to claim 4 or 5; The host cell is induced to express the xylanase mutant.

7. Use of the xylanase mutant according to claim 1 as a xylan-degrading enzyme.

8. Use of the xylanase mutant according to claim 1 in the preparation and / or processing of food, feed, nutritional additives, textiles, detergents and paper.

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