Xylanase mutant with improved enzyme activity, encoding gene and application thereof

Abstract

The application discloses a xylanase mutant with improved enzyme activity, a coding gene thereof and application, and belongs to the technical field of genetic engineering.The xylanase mutants XYNS01, XYNS02, XYNS03, XYNS04, XYNS05 and XYNS06 with improved enzyme activity are screened by combining random mutation and site-directed saturation mutation of amino acids on the basis of a xylanase amino acid sequence XYNS0.Experiments prove that the enzyme activity of the xylanase mutants obtained by the application is significantly improved compared with the unmutated xylanase, and the xylanase mutants are beneficial to development and application in the field of feed.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a xylanase mutant with enhanced enzyme activity, its encoding gene, and its applications. Background Technology

[0002] Xylan is a polysaccharide found in plant cell walls, composed of xylose monomers polymerized through 1,4-β-D-glycosidic bonds. It is the second most abundant form of hemicellulose, accounting for a large proportion of its composition in nature, second only to cellulose. Microbial-produced xylanases can effectively degrade xylan. Xylanases, through random endo-hydrolysis of the 1,4-β-D-glycosidic bonds in the xylan backbone, produce xylose or xylooligosaccharides, and are key enzymes in the xylan degradation process.

[0003] In the livestock industry, xylan in feed cannot be effectively degraded in the animal's digestive system and also affects the absorption and utilization of other nutrients, significantly reducing feed conversion efficiency. Adding xylanase to feed to convert the anti-nutritional factor xylan is a very economical and effective way to improve feed conversion rate.

[0004] Most xylanases on the market have an optimal temperature between 40 and 70°C and an optimal pH between 5.0 and 7.0. They are easily inactivated by high temperatures or in the digestive tract. One way to solve this problem is to use coating agents and carriers to improve enzyme stability. However, this not only increases the production cost of enzyme preparations but also seriously affects their bioavailability. Another economical and effective method is to improve the stability and activity of enzymes by modifying their genes. This is of great significance for reducing the current production cost of feed xylanases and improving their utilization efficiency.

[0005] Error-prone PCR technology involves adjusting reaction conditions during PCR amplification of a target fragment to increase the mutation frequency, thereby introducing random mutations into the target gene and constructing a mutant library. Site saturation mutations can precisely select nucleotides for a chosen sequence. By regulating the mutation library with universal bases, saturation mutations allow each amino acid to be precisely substituted in a controlled manner, constructing a mutant library and thus screening for desired positive mutants. Summary of the Invention

[0006] Based on the above technical requirements, this invention provides a xylanase mutant with enhanced enzyme activity, its encoding gene, and its applications. The xylanase mutant provided by this invention is obtained through random mutation and site-directed saturation mutation. Verification has shown that the enzyme activity of the recombinant xylanase is improved, which is beneficial to improving its application effect in the feed industry.

[0007] To achieve the above-mentioned objectives, the present invention employs the following technical solution: This invention provides a xylanase mutant XYNS01 with enhanced enzyme activity. The amino acid sequence of the mutant XYNS01 is shown in SEQ ID NO: 3. It is obtained by changing the serine at position 148 of xylanase XYNS0, whose amino acid sequence is shown in SEQ ID NO: 1, to alanine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO: 4.

[0008] This invention provides a xylanase mutant XYNS02 with enhanced enzyme activity. The amino acid sequence of the mutant XYNS02 is shown in SEQ ID NO: 5. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to arginine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO: 6.

[0009] This invention provides a xylanase mutant XYNS03 with enhanced enzyme activity. The amino acid sequence of the mutant XYNS03 is shown in SEQ ID NO: 7. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to threonine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO: 8.

[0010] This invention provides a xylanase mutant XYNS04 with enhanced enzyme activity. The amino acid sequence of the mutant XYNS04 is shown in SEQ ID NO: 9. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to leucine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO: 10.

[0011] This invention provides a xylanase mutant XYNS05 with enhanced enzyme activity. The amino acid sequence of the mutant XYNS05 is shown in SEQ ID NO: 11. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to methionine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO: 12.

[0012] This invention provides a xylanase mutant XYNS06 with enhanced enzyme activity. The amino acid sequence of the mutant XYNS06 is shown in SEQ ID NO: 13. It is obtained by changing serine at position 18 to glycine and glycine at position 183 to cysteine ​​in the mutant XYNS01 of claim 1. The nucleotide sequence of its encoding gene is shown in SEQ ID NO: 14.

[0013] The present invention also provides a recombinant expression vector for the xylanase mutant encoding gene.

[0014] The present invention also provides a genetically engineered bacterium encoding the xylanase mutant gene.

[0015] Furthermore, the genetically engineered bacteria is Pichia pastoris GS115.

[0016] The present invention also provides the application of the xylanase mutant in the preparation of formulations that enhance enzyme activity.

[0017] Compared with the prior art, the advantages and technical effects of the present invention are: 1. This invention constructs a mutant library by combining random mutation and site-directed saturation mutation, thereby screening for desired positive mutants. Based on xylanase XYNS0, this invention provides a single-site mutant XYNS01 containing S148A; two-site mutants XYNS02, XYNS03, XYNS04, and XYNS05 containing S18R / S148A, S18T / S148A, S18L / S148A, and S18M / S148A respectively; and a three-site mutant XYNS06 containing S18G / S148A / G183C.

[0018] 2. The modified mutants XYNS01, XYNS02, XYNS03, XYNS04, XYNS05, and XYNS06 of this invention exhibit enzyme activities that are 39.86%, 94.64%, 103.56%, 93.53%, 125.60%, and 113.58% higher than the original xylanase XYNS0, respectively. The xylanase mutants obtained through the technical solution of this invention show significantly improved enzyme activity compared to the wild type, making them highly promising for applications in food, feed additives, and chemical industries. Attached Figure Description

[0019] Figure 1 This is a comparison of the average enzyme activity of xylanase mutants in this invention. Detailed Implementation

[0020] To facilitate understanding of the present invention, the invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0021] Molecular biology experimental methods not specifically described in the following examples can be performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions. Unless otherwise specified, the reagents and biological materials used in the specific examples are commercially available.

[0022] 1. Strains and vectors Pichia pastoris GS115, plasmid pPIC9K, Escherichia coli DH5α, Escherichia coli BL21, and plasmid pET 21a(+) were purchased from Invitrogen.

[0023] 2. Reagents and Culture Media Plasmid extraction kit, fragment purification and recovery kit, restriction endonucleases, etc. were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0024] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl; MD medium: 1.34% YNB, 0.4 mg / L biotin, 2% glucose; YPD medium: 1% yeast extract, 2% peptone, 2% glucose; 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; 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; BSM medium: 26.7 mL 85% phosphate, 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 mL LPM1.

[0025] When the above culture medium is solid, add 2% agar powder.

[0026] 3. Experimental Methods Culture conditions: Escherichia coli was cultured at 37°C, and yeast at 30°C. The shaking speed for liquid culture was 200 rpm.

[0027] Transformation method of Pichia pastoris GS115: Activated Pichia pastoris GS115 was inoculated into 20 mL of YPD liquid medium and cultured in a shake flask at 30°C until OD... 600After a 1.2–1.5°C incubation period, the bacterial cells were collected by low-temperature centrifugation. The cells were washed sequentially with 20 mL of ice-cold sterile water and 5 mL of ice-cold 1 mol / L sorbitol solution. Finally, the cells were resuspended in 1 mL of sorbitol solution to obtain a competent yeast suspension. 100 μL of competent yeast cells were mixed with 10 μL of linearized vector and transferred to a pre-chilled electroporation cuvette for electroporation at 1.5 kV for 6 msec. After electroporation, 1 mL of sorbitol solution was added, and the mixture was transferred to a 1.5 mL centrifuge tube and incubated at 30°C for 1 h. The cells were centrifuged at 5000 rpm for 5 min, collected, and plated onto MD selection plates for inverted culture until positive single colonies appeared.

[0028] Xylanase activity was detected according to the People's Republic of China National Standard GB / T 23874-2009. Xylanase activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 5.0 mg / mL xylan solution under the conditions of 37℃ and pH 5.5. This is one unit of xylanase activity, denoted by U.

[0029] Example 1: Mutation screening of a mutant library of xylanase XYNS0 This embodiment provides xylanase XYNS0, which is derived from Penicillium. Penicillium occitanis The amino acid sequence of the xylanase XYNS0 (SEQ ID NO: 1) was obtained through commercial channels. Based on the codon preference of Pichia pastoris, its DNA sequence was optimized (SEQ ID NO: 2). The optimized gene was synthesized, and a design was made at the 5' end. Eco RⅠ restriction enzyme cleavage site, 3' end design Not Ⅰ Restriction enzyme cleavage site.

[0030] Using the GeneMorph II random mutagenesis PCR kit, random mutations were performed using the xylan XYNS0 gene as a template. The primer sequences used are as follows: Table 1 Random Mutation Primers

[0031] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 60 s, 72℃ extension for 1 min (30 cycles), 72℃ extension for 5 min, and storage at 15℃.

[0032] The conditions for the overlap PCR reaction were: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 68℃ annealing for 1 min 30 s (two steps, 30 cycles), 72℃ extension for 5 min, and storage at 15℃.

[0033] The amplified random mutant PCR product was used... Eco RⅠ and NotI. After double enzyme digestion, purification and recovery, the gene was ligated into the pET-21a(+) vector and transformed into E. coli BL21-DE3. Positive clones were selected by ampicillin-resistant LB plates to obtain pET-XYNSx. The synthesized original gene was ligated into the pET-21a(+) vector and transformed into E. coli BL21-DE3 in the same way to obtain pET-XYNS0.

[0034] Selected single colonies were inoculated into 96-well deep-well plates. Two single colonies expressing XYNS0 were inoculated into each plate as controls. 300 μL of LB liquid medium (containing 100 μg / mL ampicillin) was added to each well, and the plates were incubated at 37°C with shaking at 200 rpm for 4 h. After incubation, 50 μL of bacterial culture was transferred to a new 96-well plate for preservation. 200 μL of LB-Amp medium containing IPTG was added to the remaining bacterial culture on the plate to bring the final IPTG concentration to 1 mM and the final ampicillin concentration to 100 μg / mL. The plates were then incubated at 37°C with shaking at 200 rpm for 10 h to induce xylanase expression.

[0035] The induced bacterial culture was repeatedly freeze-thawed to disrupt it. The supernatant from the disrupted cell slurry was collected by centrifugation, and the xylanase activity was measured. The gene of mutants with higher enzyme activity than the control was sequenced.

[0036] The mutant S148A (named XYNS01) with enhanced enzyme activity was obtained by screening with XYNS0 as the starting template. Its amino acid sequence is shown in SEQ ID NO: 3, and its encoded nucleotide sequence is shown in SEQ ID NO: 4.

[0037] Example 2: Obtaining a site-directed saturation mutant of xylanase Using the xylanase gene XYNS01 screened in Example 1 as a template, 18 sites were selected for saturation mutation after online software analysis to construct a mutant library. The saturation mutation primers are shown in Table 2.

[0038] Table 2 Saturation Mutant Primers

[0039] The construction process of the mutant library, as well as the materials, reagents, and operating conditions used, were the same as in Example 1. XYNS01 was used as a control during mutant culture and screening. After detecting the enzyme activity of xylanase, the mutant genes with enzyme activity higher than the control were sequenced.

[0040] The following mutants with increased enzyme activity were finally screened: The XYNS02 mutation mode is S18R / S148A, and the amino acid sequence is shown in SEQ ID No: 5; The XYNS03 mutation mode is S18T / S148A, and the amino acid sequence is shown in SEQ ID No: 7; The XYNS04 mutation mode is S18L / S148A, and the amino acid sequence is shown in SEQ ID No: 9; The XYNS05 mutation mode is S18M / S148A, and the amino acid sequence is shown in SEQ ID No: 11; The XYNS06 mutation mode is S18G / S148A / G183C, and the amino acid sequence is shown in SEQ ID No: 13.

[0041] Example 3: Validation of expression of xylanase mutant with enhanced enzyme activity in Pichia pastoris The xylanase mutant gene selected above was used Eco RⅠ and Not I. The double restriction enzyme sites were ligated to the pPIC9K plasmid and transformed into *E. coli* DH5α. Expression vectors pPIC-XYNm for each mutant were screened in *Pichia pastoris* and verified by sequencing. The expression vectors of each mutant were then... Sal I. After linearization by enzyme digestion, the transformants were electroporated into Pichia pastoris GS115. Transformants of each mutant expression strain were screened using MD plates and then transferred to YPD plates for activation (48 transformants were selected from each mutant). The activated transformants were inoculated into 48-well plates (1 mL BMGY medium per well) and cultured at 30°C with shaking for 18 h. Then, 1% methanol was added for induction, and the culture was continued with shaking. Subsequently, 1% methanol (by volume of the culture) was added every 24 h. After 96 h of induction, the culture medium was centrifuged to obtain the supernatant. The average enzyme activity of the supernatant from the fermentation broth of each mutant was measured (those without enzyme activity were not included). The results are shown below. Figure 1 The shake-flask activities of the xylanases XYNS01, XYNS02, XYNS03, XYNS04, XYNS05, and XYNS06 obtained after mutation were increased by 39.86%, 94.64%, 103.56%, 93.53%, 125.60%, and 113.58%, respectively, compared with XYNS0.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A xylanase mutant XYNS01 with enhanced enzyme activity, characterized in that: The amino acid sequence of the mutant XYNS01 is shown in SEQ ID NO:

3. It is obtained by changing the serine at position 148 of xylanase XYNS0, whose amino acid sequence is shown in SEQ ID NO: 1, to alanine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:

4.

2. A xylanase mutant XYNS02 with enhanced enzyme activity, characterized in that: The amino acid sequence of the mutant XYNS02 is shown in SEQ ID NO:

5. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to arginine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:

6.

3. A xylanase mutant with enhanced enzyme activity, XYNS03, characterized in that: The amino acid sequence of the mutant XYNS03 is shown in SEQ ID NO:

7. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to threonine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:

8.

4. A xylanase mutant with enhanced enzyme activity, XYNS04, characterized in that: The amino acid sequence of the mutant XYNS04 is shown in SEQ ID NO:

9. It is obtained by changing the 18th serine of the mutant XYNS01 of claim 1 to leucine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:

10.

5. A xylanase mutant with enhanced enzyme activity, XYNS05, characterized in that: The amino acid sequence of the mutant XYNS05 is shown in SEQ ID NO:

11. It is obtained by changing the 18th serine residue of the mutant XYNS01 described in claim 1 to methionine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:

12.

6. A xylanase mutant with enhanced enzyme activity, XYNS06, characterized in that: The amino acid sequence of the mutant XYNS06 is shown in SEQ ID NO:

13. It is obtained by changing the 18th serine of the mutant XYNS01 according to claim 1 to glycine and the 183rd glycine to cysteine. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:

14.

7. A recombinant expression vector comprising the xylanase mutant encoding gene of any one of claims 1-6.

8. Genetically engineered bacteria comprising the xylanase mutant encoding gene of any one of claims 1-6.

9. The genetically engineered bacterium according to claim 8, characterized in that, The genetically engineered bacteria is Pichia pastoris GS115.

10. The use of the xylanase mutant according to any one of claims 1-6 in the preparation of formulations that enhance enzyme activity.

Images