Xylanase mutant with improved acid resistance and enzyme activity as well as coding gene and application of xylanase mutant

Abstract

The invention discloses a xylanase mutant with improved acid resistance and enzyme activity as well as a coding gene and application of the xylanase mutant. According to the invention, on the basis of a xylanase amino acid sequence derived from Neocallimastix patriciarum, xylanase mutants Xyn-M1, Xyn-M2, Xyn-M3 and Xyn-M4 with significantly improved acid resistance and mutants Xyn-M5, Xyn-M6 and Xyn-M7 with improved enzyme activity are obtained in a manner of combining amino acid site-saturation mutation and random mutation. The xylanase mutant provided by the invention can significantly reduce the feed conversion ratio of bred animals and improve the growth performance index of the bred animals in livestock and poultry and aquatic animal breeding, and has wide application prospects in the industries of feed, food and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a xylanase mutant with improved acid resistance and enzyme activity, a coding gene and application thereof. Background Art

[0002] In the breeding industry, xylan in feed cannot be effectively degraded in the animal digestive system, and it will also affect the absorption and utilization of other nutrients, reducing the conversion and utilization efficiency of feed. Xylanase (xylanase) is a key enzyme in the degradation of xylan by randomly hydrolyzing the 1,4-β-D-glycosidic bonds of the xylan main chain to produce xylose or oligoxylose. Adding xylanase to the feed to convert the anti-nutritional factor xylan is very economical and effective to improve the feed conversion rate.

[0003] At present, the optimum temperature of most xylanases on the market is between 40 and 70°C, and the optimum pH is between 5.0 and 7.0. The xylanase products sold are mainly produced by Pichia pastoris and Trichoderma reesei. The acid xylanase from Trichoderma can tolerate pepsin degradation to a certain extent, but the residual enzyme activity is less than 10% after heating at 80 degrees for 3 minutes. The neutral xylanase produced by yeast has high temperature resistance and enzyme activity, and the optimum pH is generally 5.5. However, under pH 3.0, the enzyme activity is only 4% of the optimum enzyme activity, and it is not resistant to pepsin degradation. It has low activity in the animal stomach environment and is easily inactivated when passing through the digestive tract. One way to solve this problem is to use coating agents and carriers to improve the acid resistance and stability of the enzyme, which will not only increase the production cost of the enzyme preparation, but also seriously affect the bioavailability of the enzyme preparation. Summary of the invention

[0004] In view of the above technical difficulties existing in the prior art, the object of the present invention is to provide a xylanase mutant with improved acid resistance and enzyme activity, and its encoding gene and application. The xylanase mutant provided by the present invention is obtained by screening by combining site-directed saturation mutagenesis and random mutagenesis, and has stronger acid resistance and better stress resistance, thereby improving its application effect as a feed additive in livestock and poultry and aquaculture.

[0005] In order to achieve the above purpose, the following technical solutions are adopted:

[0006] The present invention provides a xylanase mutant Xyn-M1 with improved acid resistance. The amino acid sequence of the mutant Xyn-M1 is shown in SEQ ID NO:4, and the mutant Xyn-M1 is obtained by changing the leucine at position 31 of the xylanase shown in SEQ ID NO:2 to methionine.

[0007] The invention provides a xylanase mutant Xyn-M2 with improved acid resistance. The amino acid sequence of the mutant Xyn-M2 is shown in SEQ ID NO:6, and the mutant Xyn-M2 is obtained by changing the serine at position 133 of the mutant Xyn-M1 to valine and the lysine at position 193 to tyrosine.

[0008] The present invention provides a xylanase mutant Xyn-M3 with improved acid resistance. The amino acid sequence of the mutant Xyn-M3 is shown in SEQ ID NO:8, and the mutant Xyn-M3 is obtained by changing the serine at position 133 of the mutant Xyn-M1 to aspartic acid and the lysine at position 193 to tyrosine.

[0009] The invention provides a xylanase mutant Xyn-M4 with improved acid resistance. The amino acid sequence of the mutant Xyn-M4 is shown in SEQ ID NO: 10, and the mutant Xyn-M4 is obtained by changing the 58th tyrosine of the mutant Xyn-M1 to threonine and the 105th serine to asparagine.

[0010] The present invention provides a xylanase mutant Xyn-M5 with improved enzyme activity. The amino acid sequence of the mutant Xyn-M5 is shown in SEQ ID NO: 12, and the mutant Xyn-M5 is obtained by changing the threonine at position 71 of the mutant Xyn-M1 to tyrosine and the glutamine at position 168 to proline.

[0011] The present invention provides a xylanase mutant Xyn-M6 with improved enzyme activity. The amino acid sequence of the mutant Xyn-M6 is shown in SEQ ID NO: 14, and the mutant Xyn-M6 is obtained by changing the glutamic acid at position 145 of the mutant Xyn-M1 to alanine and the glutamine at position 168 to proline.

[0012] The present invention provides a xylanase mutant Xyn-M7 with improved enzyme activity. The amino acid sequence of the mutant Xyn-M7 is shown in SEQ ID NO: 16, and the mutant Xyn-M7 is obtained by changing the glutamic acid at position 145 of the mutant Xyn-M1 to glutamine, the threonine at position 149 to valine, and the methionine at position 189 to leucine.

[0013] The invention also provides a gene encoding the xylanase mutant.

[0014] The invention also provides the use of the xylanase mutant in the preparation of xylooligosaccharides and xylose.

[0015] The invention also provides application of the xylanase mutant in preparing animal feed additives.

[0016] Further: the animals include pigs, chickens, ducks, fish, shrimps and crabs.

[0017] Furthermore: the xylanase mutant can increase the daily weight gain of animals and reduce the feed-to-meat ratio.

[0018] Furthermore: the xylanase mutant can improve the weight gain rate and specific growth rate of animals.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0020] The present invention adopts a method combining analysis and random mutation, starting from the gene and structure of xylanase, by introducing random mutations into the key sites of the target gene, constructing a mutant library, thereby screening the required forward mutants, further improving its acid resistance and enzyme activity under acidic conditions, making it more suitable for the animal intestinal environment. The xylanase mutants Xyn-M1, Xyn-M2, Xyn-M3, and Xyn-M4 screened by the present invention improve the enzyme activity under acidic conditions, are more suitable for the animal gastrointestinal environment, and improve the digestibility and utilization rate of feed raw materials. The enzyme activity of Xyn-M5, Xyn-M6, and Xyn-M7 is significantly improved compared with the original gene, and its stress resistance is better, and it has good heat resistance. In livestock and poultry and aquatic animal breeding, it can significantly reduce the feed-to-meat ratio of breeding animals and improve the growth performance index of breeding animals. Therefore, the xylanase mutants produced by the present invention can be widely used in industries such as animal breeding, feed, and food, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the relative enzyme activity of the xylanase and its mutants at pH 3.0.

[0022] Figure 2 is the relative enzyme activity of the xylanase and its mutants.

[0023] Figure 3 is the enzyme activity of the xylanase mutant in a 30 L fermenter.

[0024] Figure 4 The invention relates to application of the xylanase and its mutants in piglet breeding. DETAILED DESCRIPTION

[0025] The following embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims. The protection scope of the present invention and the scope of claims are not limited to the cases provided.

[0026] Unless otherwise specified, the reagents and biological materials used in the following specific examples can be obtained from commercial sources.

[0027] 1. Strains and vectors

[0028] Pichia pastoris GS115, plasmid pPIC9K, Escherichia coli DH5α, Escherichia coli BL21, and plasmid pET 21a(+) were purchased from Invitrogen; the optimized gene was synthesized by Shanghai Jierui Biotechnology Co., Ltd.; sequencing analysis and primer ordering were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0029] 2. Reagents and culture media

[0030] Plasmid extraction kit, fragment purification and recovery kit, restriction endonuclease, etc. were purchased from Bao Biotechnology (Dalian) Co., Ltd.; GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; protein marker: Blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0031] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

[0032] MD medium: 1.34% YNB, 0.4 mg / L biotin, 2% glucose;

[0033] YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0034] BMGY medium: 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% glycerol;

[0035] BMMY medium: 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% methanol;

[0036] BSM medium: 26.7 mL 85% phosphoric acid, 0.93 g calcium sulfate dihydrate, 14.9 g magnesium sulfate dihydrate, 4.13 g potassium hydroxide, 18.2 g potassium sulfate, 40 g glycerol, 4.0 mlL PMT1.

[0037] When the above culture medium is solid, add 2% agar powder.

[0038] 3. Experimental methods

[0039] Strain culture conditions: E. coli culture at 37°C, yeast culture at 30°C. The shaking speed during liquid culture was 200 rpm.

[0040] Pichia pastoris GS115 transformation method:

[0041] The activated Pichia pastoris GS115 was inoculated into 20 mL YPD liquid medium, cultured at 30 ° C in a shake flask until OD600 was 1.2-1.5, and then centrifuged at low temperature to collect the cells. The cells were washed with 20 mL ice-cold sterile water and 5 mL ice-cold sorbitol solution with a concentration of 1 mol / L, and finally resuspended with 1 mL sorbitol solution to obtain a yeast competent suspension. 100 μL of yeast competent cells and 10 μL of linearized vector were mixed and transferred to a pre-cooled electroporation cup for electroporation transformation. The conditions for electroporation transformation were 1.5 kV, 6 msec. After electroporation, 1 mL of sorbitol solution was added and transferred to a 1.5 mL centrifuge tube for incubation at 30 ° C for 1 h. Centrifuged at 5000 rpm for 5 min, the collected cells were spread on the MD screening plate and inverted for culture until a positive single clone grew.

[0042] The xylanase activity test was carried out in accordance with the national standard of the People's Republic of China "GB / T 23874-2009"; the xylanase activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar in 5.0 mg / mL xylan solution per minute at a temperature of 37°C and a pH value of 5.5, which is one xylanase activity unit, expressed as U.

[0043] Example 1: Obtaining a site-directed saturation mutant of xylanase

[0044] This example refers to the amino acid sequence of xylanase (as shown in SEQ ID NO: 2, GenBank: AKN90969.1), and the corresponding nucleotide sequence is synthesized through gene optimization, as shown in SEQ ID NO: 1. After online software analysis, 31 sites are selected for saturation mutation, and the saturation mutation primers are designed as follows:

[0045] Table 1 Saturation mutagenesis primers

[0046]

[0047] Primer XynF / 31-R was used to amplify the upstream gene fragment, and primer XynR / A31dnF was used to amplify the downstream gene fragment, and then the full-length gene was amplified by overlapping PCR (primer XynF / XynR). The amplified full-length gene was double-digested with EcoR I and Not I, purified and recovered, and connected to the pET-21a(+) vector, transformed into E. coli BL21-DE3, and positive clones were screened on ampicillin-resistant LB plates to obtain pET-Xynx. The synthesized original gene was connected to the pET-21a(+) vector and transformed into E. coli BL21-DE3 in the same way to obtain pET-Xyn0.

[0048] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min (30 cycles), post-extension at 72°C for 5 min, and storage at 15°C.

[0049] Overlap PCR reaction conditions: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 68°C for 1 min 30 s (two steps, 30 cycles), and post-extension at 72°C for 5 min, and storage at 15°C.

[0050] The screened single colonies were inoculated into a 96-well deep-well plate. Two single colonies expressing Xyn0 were inoculated into each plate as controls. 300uL LB liquid culture medium (containing 100μg / mL ampicillin) was placed in each well, and after shaking and culturing at 37°C 200rpm for 4 hours, 50uL of bacterial solution was transferred to a new 96-well plate for seed preservation. 200uL LB-Amp culture medium containing IPTG was added to the remaining bacterial solution on the plate, so that the final concentration of IPTG was 1mM and the final concentration of ampicillin was 100μg / mL. The expression of xylanase was induced by shaking and culturing at 37°C 200rpm for 10h.

[0051] The induced bacterial solution was repeatedly frozen and thawed to break it, and the broken cell solution was centrifuged to take the supernatant, and the enzyme activity of xylanase and the enzyme activity at pH 3.0 were detected. The mutant gene with higher enzyme activity than the control was sequenced. The mutant L31M with improved enzyme activity and acid resistance using Xyn0 as the starting template was screened and named Xyn-M1. The amino acid sequence is shown in SEQ ID NO: 4, and the nucleotide sequence encoded by it is shown in SEQ ID NO: 3.

[0052] SEQ ID NO: 3 GAATTCCAAAGTTTCTGTAGTTCAGCTTCTCACTCTGGACAAAGTGTAAAGGTAACCGGCAACAAGGTTGGAACTATTGGTGGTGTTGGTTACGAAATGTGGGCTGATAGTGGTAATAACAGTGCTACTTTCTATTCTGATGGTTCCTTCTCATGTACTTTCCAAAATGCTGGGGATTACTTATGTCGTAGTGGTCTTTCTTTCGATAGTACTAAGACCCCATCTCAAATTGGTCGTATGAAGGCTGATTTCAAACTTGTCAAACAAAATAGTTCCAATGTTGGTTATTCCTATGTTGGTGTTTACGGTTGGACTAGAAGTCCACTTGTCGAATACTACATTGTCGATAATTGGCTTAGTCCATTCCCACCAGGTGATTGGGTTGGTAACAAGAAGCATGGTTCTTTCACTATTGATGGTGCTCAATACACTGTTTATGAAAACACTAGGACTGGTCCATCTATTGATGGTGATACCACCTTCAATCAATACTTTAGTATTCGTCAACAAGCTCGTGATTGTGGTACCATTGATATTTCTGCTCACTTTGATCAATGGGAAAAGCTTGGTATGACTATGGGTAAATTACATGAAGCCAAGGTTTTAGGTGAAGCCGGTAACGTTAACGGTGGTGCCAGTGGTACCGCTGATTTCCCATACGCAAAGGTTTACATTGGTGATTAG G CGGCCGC

[0053] SEQ ID NO:4 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD

[0054] Example 2: Random mutagenesis based on the Xyn-M1 mutant

[0055] The optimized Xyn-M1 sequence was used as a template to randomly introduce mutations using the GeneMorph II Random Mutation PCR Kit (Stratagene) to construct a mutant library.

[0056] Primers: The underlined parts are the EcoR I and Not I restriction sites.

[0057] Table 2 Random mutagenesis primers

[0058]

[0059] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 60 s and extension at 72°C for 1 min, for a total of 30 cycles, post-extension at 72°C for 5 min, and storage at 15°C.

[0060] The mutant library construction process, materials, reagents and operating conditions were the same as in Example 1. Xyn-M1 was used as a control during mutant culture and screening. The enzyme activity of xylanase and the enzyme activity at pH 3.0 were tested, and the mutant genes with higher enzyme activity than Xyn-M1 were sequenced.

[0061] Finally, the following mutants with improved acid resistance were screened:

[0062] Xyn-M2 has a mutation pattern of L31M / S133V / K193Y, and an amino acid sequence as shown in SEQ ID No: 6;

[0063] Xyn-M3 has a mutation pattern of L31M / S133D / K193Y, and an amino acid sequence as shown in SEQ ID No: 8;

[0064] Xyn-M4 has mutation pattern of L31M / Y58T / S105N, and the amino acid sequence is shown in SEQ ID No: 10;

[0065] and the following mutants with improved enzyme activity:

[0066] Xyn-M5 has a mutation pattern of L31M / T71Y / Q168P, and an amino acid sequence as shown in SEQ ID No: 12;

[0067] Xyn-M6 has a mutation pattern of L31M / E145A / Q168P, and an amino acid sequence as shown in SEQ ID No: 14;

[0068] The mutation pattern of Xyn-M7 is L31M / E145Q / T149V / M189L, and the amino acid sequence is shown in SEQ ID No: 16;

[0069] SEQ ID NO:6 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGVFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGYLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD

[0070] SEQ ID NO:8 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGDFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGYLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD

[0071] SEQ ID NO:10 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDTLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRNPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD

[0072] SEQ ID NO:12 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKYPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQPARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD

[0073] SEQ ID NO:14 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYANTRTGPSIDGDTTFNQYFSIRQPARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD

[0074] SEQ ID NO:16 QSFCSSASHSGQSVKVTGNKVGTIGGVGYEMWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYQNTRVGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGLTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD。

[0075] Example 3: Expression of Xylanase Mutant in Pichia pastoris GS115

[0076] The mutants Xyn-M1 to Xyn-M7 of xylanase were selected and connected to the pPIC9K plasmid with EcoR I and Not I double restriction sites and transformed into Escherichia coli DH5α to obtain the expression vectors of each mutant in Pichia pastoris. The expression vectors of each mutant were linearized by Sal I and then electrotransformed into Pichia pastoris GS115. The transformants of each mutant expression strain were screened on MD plates and transferred to YPD plates for activation. The activated transformants were picked and inoculated into BMGY medium. After shaking culture at 30°C for 18 hours, the cells were obtained by centrifugation. An appropriate amount of cells were transferred to BMMY medium to make the cell concentration reach OD600=1. The shaking culture was continued, and 1% methanol of the culture volume was added every 24 hours. After induced expression for 96 hours, the culture solution was centrifuged to obtain the supernatant. By SDS-PAGE, each mutant was selected with a higher protein expression amount, that is, a darker band. The transformants can be considered as transformants with higher enzyme activity, and strain preservation and enzyme activity determination were performed.

[0077] After measuring at pH 3.0, the relative enzyme activity percentage was obtained by comparing it with the optimal enzyme activity (measured at pH 5.5). Figure 1 As shown, the original gene had only 4% of the optimal enzyme activity under pH 3.0 conditions, while the mutant genes Xyn-M1 to Xyn-M4 were 20%, 27%, 34% and 41.5% respectively, which significantly improved the enzyme activity under acidic conditions, had better acid resistance, and was more suitable for the animal gastrointestinal environment.

[0078] After enzyme activity determination, the enzyme activities of mutant genes Xyn-M5 to Xyn-M7 were 25%, 31%, and 21% higher than those of the control gene, respectively. Figure 2 shown.

[0079] Example 4: Fermentation and preparation of Xyn-M4 and Xyn-M6 mutants in a 30 L fermenter

[0080] The Pichia yeast in the glycerol tube was streaked on the YPD plate, inverted and cultured at 30℃ for 3 days to grow a single colony, and the single colony with good growth was picked and continued to be streaked on the YPD plate. The single colony of Pichia yeast obtained by activation for three generations was inoculated into 50mL BMGY medium and cultured at 30℃ and 200rpm for 24h. Inoculated into 1000mL BMGY medium with an inoculum of 2%, cultured at 30℃ and 200rpm until OD600 was 5, and used as seed liquid to inoculate the fermenter.

[0081] Fermentation production process: BSM medium, pH 4.8, temperature 30°C, stirring rate 500 rpm, ventilation volume 1.5 (v / v), dissolved oxygen controlled above 20%.

[0082] The fermentation process is divided into three stages: (1) bacterial culture stage: inoculate seed liquid at a ratio of 8%, and culture at 30°C for 20-24 hours to exhaust the glycerol in the fermentation liquid; (2) starvation stage: when the carbon source glycerol is exhausted, no carbon source is added for the time being, and the starvation stage ends when the dissolved oxygen rises to 80%; (3) induction expression stage: adjust the pH to the required value with ammonia or phosphoric acid, add methanol for induction, and keep the dissolved oxygen above 20%, and the induction time is 160-200 hours; after the fermentation is completed, the fermentation liquid is processed through a plate and frame filter to obtain a crude enzyme solution for enzyme activity, enzymatic properties and application tests.

[0083] The results of enzyme activity assay were as follows Figure 3 : The enzyme activities of mutants Xyn-M4 and Xyn-M6 reached the highest level around 176h of fermentation, among which the enzyme activity of Xyn-M6 was higher than that of Xyn-M4.

[0084] Example 5: Application of Xyn0, Xyn-M4 and Xyn-M6 mutants in piglet breeding

[0085] A total of 120 healthy weaned piglets with similar age, litter size and parity were selected and randomly divided into 4 groups, 30 in each group. The control group was fed with normal piglet diet, and the experimental group 1 was supplemented with 200 mg / kg of Xyn0 xylanase, the experimental group 2 was supplemented with 200 mg / kg of Xyn-M4, and the experimental group 3 was supplemented with 200 mg / kg of Xyn-M6. The pigs were fed separately for a 30-day experimental period. At 8:00 a.m. on the first day of the experiment and the 30th day of the experiment, the pigs in each pen were weighed on an empty stomach, the feed consumption was recorded, and the daily weight gain, daily feed intake and feed-to-weight ratio were calculated.

[0086] Depend on Figure 4 It can be seen that xylanase and its mutants can significantly increase the feed intake and daily weight gain of weaned piglets and reduce the feed-to-meat ratio, while reducing the loose stool rate of piglets during breeding, thereby reducing breeding costs. The feed-to-meat ratio of xylanase mutants Xyn-M4 and Xyn-M6 is lower than that of Xyn0. In particular, the xylanase mutant Xyn-M4 is more suitable for acidic environment, has better acid resistance, and can improve breeding results.

[0087] Example 6: Effects of Xyn0, Xyn-M4, and Xyn-M6 on Growth Performance of Crucian Carp

[0088] 400 crucian carps that had been temporarily raised to a weight of 8-9g / tail were selected and randomly divided into 4 treatment groups according to body weight using a single-factor experimental design. No xylanase was added to the control group, xylanase Xyn0 50mg / kg was added to the diet of the experimental group 1, xylanase Xyn-M4 50mg / kg was added to the diet of the experimental group 2, and Xyn-M6 50mg / kg was added to the diet of the experimental group 3. There were 5 replicates in each group, and 20 fish in each replicate. The basic diet (fish meal 12%, soybean meal 25%, rapeseed meal 20%, cottonseed meal 7%, corn germ cake 20%, second flour bran 16%) was used for feeding, and the breeding experiment lasted for 45 days.

[0089] After 45 days of breeding experiment, crucian carp were fasted for 24 h, fished out and the surface moisture of the fish body was gently absorbed with filter paper and then weighed to calculate the weight gain rate, specific growth rate and feed coefficient.

[0090] The formula for calculating weight gain rate is: WGR (%) = 100 × [(Wt-W0) / W0];

[0091] The specific growth rate is calculated as follows: SGR (%·day -1 )=100×[(lnWt-lnW0) / t];

[0092] The formula for calculating the feed coefficient is: FCR=F / (Wt-W0);

[0093] Where W0 is the initial average body weight (g), Wt is the final average body weight (g), t is the number of experimental days (d), and F is the feed intake (g, feed amount minus residual feed amount).

[0094] Table 3 Effects of different xylanases on weight gain rate, specific growth rate and feed conversion rate of crucian carp

[0095]

[0096] The results of the effects of xylanase and its mutants on the growth performance of crucian carp are shown in Table 3. After 45 days of feeding of the experimental crucian carp, xylanase and its mutants can significantly increase the weight gain rate and specific growth rate of crucian carp, and can also reduce its feed coefficient, and the effect of the mutant is better than the original xylanase.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A xylanase mutant Xyn-M1 with improved acid resistance, characterized in that: The amino acid sequence of the mutant Xyn-M1 is shown in SEQ ID NO: 4, which is obtained by changing the leucine at position 31 of the xylanase shown in SEQ ID NO: 2 to methionine.

2. A xylanase mutant Xyn-M2 with improved acid resistance, characterized in that: The amino acid sequence of the mutant Xyn-M2 is shown in SEQ ID NO: 6, which is obtained by changing the serine at position 133 of the mutant Xyn-M1 of claim 1 to valine and the lysine at position 193 to tyrosine.

3. A xylanase mutant Xyn-M3 with improved acid resistance, characterized in that: The amino acid sequence of the mutant Xyn-M3 is shown in SEQ ID NO: 8, which is obtained by changing the serine at position 133 of the mutant Xyn-M1 of claim 1 to aspartic acid and the lysine at position 193 to tyrosine.

4. A xylanase mutant Xyn-M4 with improved acid resistance, characterized in that: The amino acid sequence of the mutant Xyn-M4 is shown in SEQ ID NO: 10, which is obtained by changing the tyrosine at position 58 of the mutant Xyn-M1 of claim 1 to threonine and the serine at position 105 to asparagine.

5. A xylanase mutant Xyn-M5 with improved enzyme activity, characterized in that: The amino acid sequence of the mutant Xyn-M5 is shown in SEQ ID NO: 12, which is obtained by changing the threonine at position 71 of the mutant Xyn-M1 of claim 1 to tyrosine and the glutamine at position 168 to proline.

6. A xylanase mutant Xyn-M6 with improved enzyme activity, characterized in that: The amino acid sequence of the mutant Xyn-M6 is shown in SEQ ID NO: 14, which is obtained by changing the glutamic acid at position 145 of the mutant Xyn-M1 of claim 1 to alanine and the glutamine at position 168 to proline.

7. A xylanase mutant Xyn-M7 with improved enzyme activity, characterized in that: The amino acid sequence of the mutant Xyn-M7 is shown in SEQ ID NO: 16, which is obtained by changing the glutamic acid at position 145 of the mutant Xyn-M1 of claim 1 to glutamine, the threonine at position 149 to valine, and the methionine at position 189 to leucine.

8. A gene encoding the xylanase mutant according to any one of claims 1 to 7.

9. Use of the xylanase mutant according to any one of claims 1 to 7 in the preparation of an animal feed additive.

10. The use according to claim 9, characterized in that: The animals include pigs, chickens, ducks, fish, shrimps and crabs.

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