阅读说明：本技术 一种抗自切和高比活力胰蛋白酶突变体 (Trypsin mutant with anti-autogenous cutting and high specific activity ) 是由 王洪彬 路福平 曾芳 冯永蕊 于 2020-05-15 设计创作，主要内容包括：本发明涉及一种抗自切和高比活力胰蛋白酶突变体,具体涉及通过分子改造和筛选获得胰蛋白酶的抗自切和高比活力突变体及其编码基因与应用,属于蛋白质和基因工程技术领域。本发明提供的胰蛋白酶突变体是在SEQ ID NO.2所示氨基酸序列的基础上发生至少一个以下位点的突变获得的：R107H、R107L、R115S、R115T、K133A、K133A、K147D、K157E、K208P、K210A。比原始酶相比,具有更好的抗自切性能及更高的比活力,相对于野生型胰蛋白酶,抗自切性能提高0.67-2.88倍,比酶活提高0.07-2.98倍,改善了胰蛋白酶储存和使用过程中容易自切降低活力的问题。(The invention relates to an anti-autogenous cutting and high specific activity trypsin mutant, in particular to an anti-autogenous cutting and high specific activity trypsin mutant obtained by molecular modification and screening, a coding gene and application thereof, belonging to the technical field of protein and gene engineering. The trypsin mutant provided by the invention is obtained by mutating at least one of the following sites on the basis of the amino acid sequence shown in SEQ ID NO. 2: R107H, R107L, R115S, R115T, K133A, K133A, K147D, K157E, K208P and K210A. Compared with the original enzyme, the trypsin has better anti-autogenous cutting performance and higher specific activity, compared with the wild trypsin, the anti-autogenous cutting performance is improved by 0.67 to 2.88 times, the specific enzyme activity is improved by 0.07 to 2.98 times, and the problem that the trypsin is easy to autogenously cut and reduce the activity in the storage and use processes is solved.)

1. An autocleavable trypsin mutant characterized by having a mutation at least one of the following sites based on the amino acid sequence shown in SEQ ID NO. 2: R107H, R107L, R115S, R115T, K133A, K133A, K147D, K157E, K208P and K210A.

2. The gene encoding the protease mutant according to claim 1.

3. The encoding gene of claim 2, which is represented by SEQ ID NO.3-22 of the sequence Listing.

4. A recombinant vector or recombinant strain comprising the gene of claim 2.

5. The recombinant vector of claim 4, wherein the expression vector is pHBM905A, pPIC9K, pPIC9K-His, pPIC3.5K, pPIC9, pPICZ α A, pAO815, pPIC9k-His, pHIL-S1, pPink hc, pGADT7, pGBKT7, pWB980 or pT 3.

6. The recombinant strain of claim 4, wherein the host cell is a Pichia pastoris host X33, KM71, KM71H, GS 115; saccharomyces cerevisiae strains YM4271, AH109, Y187 and Y190; coli BL 21, e.colix90; or bacillus subtilis SCK 6.

7. A method for producing trypsin by using the recombinant strain of claim 6, which comprises the following steps:

(1) recombinant pichia pastoris shake flask fermentation

Inoculating the strain to YPD liquid culture medium, and culturing at 28 deg.C and 200rpm for 24 hr; inoculating 2% of seed solution cultured in YPD liquid culture medium to BMGY culture medium, culturing at 28 deg.C and 200rpm to OD₆₀₀2-6, collecting thalli; transferring to BMMY medium, adding methanol to final concentration of 0.5%; adding methanol with the final concentration of 0.5% every 12h for induction culture for 96 h;

(2) activation of trypsinogen

Centrifuging to collect fermentation broth supernatant, adjusting pH to 7.5, adding enterokinase, and incubating to activate trypsinogen;

(3) and (5) purifying trypsin.

8. Use of the trypsin according to claim 1 in the fields of feed processing, food processing, leather processing, medicine, and scientific research.

9. The autocleavable trypsin mutant according to claim 1, which is a sequence having 80% or more homology with the amino acid sequence shown in SEQ ID No.2 and having the same function.

10. The autocleavable trypsin mutant according to claim 1, which is a trypsin sequence of porcine, bovine, ovine or rabbit origin comprising at least one of the following mutations: R107H, R107L, R115S, R115T, K133A, K133A, K147D, K157E, K208P and K210A.

The technical field is as follows:

the invention relates to an anti-autogenous cutting and high specific activity mutant of trypsin obtained by molecular modification and screening, and a coding gene and application thereof, belonging to the technical field of protein and gene engineering.

Background art:

trypsin (EC 3.4.21.4) is an alkaline serine proteolytic enzyme and is widely used in the fields of food processing, medicine, scientific research, and the like. The trypsin is used as digestive enzyme, can supplement the deficiency of endogenous enzyme of animals and promote the absorption of nutrient components by the animals; the trypsin can dilute blood clots and pus without damaging other tissues, accelerate wound healing, degrade intercellular matrix and adhesion protein, and has important medical value; the trypsin can clarify the wine and the beverage and can improve the elasticity and the softness of the leather; because of its strong amino acid site cleavage specificity, it is used as an important tool enzyme in the field of scientific research.

Traditionally, trypsin is mainly extracted from animal pancreas, raw materials are limited, separation and purification are difficult, and mixed miscellaneous enzymes such as chymotrypsin can seriously influence the specific hydrolysis effect of the enzymes. By adopting the genetic engineering technology to express and prepare the trypsin through microbial recombination, the yield and the purity of the trypsin can be improved, and the separation difficulty and the production cost are reduced. The animal-derived protein can be expressed by a prokaryotic expression system or a eukaryotic expression system. The host bacteria commonly used in the prokaryotic expression system are escherichia coli, which has the advantages of short period, low cost, diversified expression vector selection and the like, but also has the defects of high difficulty in denaturation and protein renaturation of inclusion bodies, difficulty in purification, difficulty in large-scale and continuous expression of target protein and the like. Animal-derived trypsin is more suitable for production and production using eukaryotic expression systems, and pichia pastoris is the most common and most sophisticated host for current eukaryotic expression systems.

The trypsin specifically hydrolyzes peptide bonds of arginine and lysine at the carboxyl terminal, and has strong amino acid site specificity. The trypsin can not only hydrolyze other protein molecules, but also attack lysine and arginine sites on the surface of the trypsin, so that the enzyme activity of the trypsin is rapidly reduced in the storage or use process, and the application efficiency and level of the trypsin are further influenced. The anti-autogenous cutting ability of the trypsin is improved through molecular modification, which is beneficial to improving the stability and hydrolysis efficiency of the trypsin.

In view of the problems of easy self-excision inactivation, difficult purification, high production cost and the like of the natural trypsin, a novel enzyme molecule with more excellent performance is obtained through microbial recombinant expression and molecular mutation modification research, and the method has great significance for promoting the application of the novel enzyme molecule in the fields of scientific research, medicines, foods and the like.

The invention content is as follows:

in order to solve the technical problem that trypsin is easy to self-cut and inactivate, the invention carries out site-directed mutagenesis modification on arginine and lysine sites in a porcine trypsin sequence (the nucleotide sequence of wild porcine trypsin is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2), researches prove that the obtained trypsin mutant obviously improves the self-cutting resistance, and simultaneously has higher specific activity, and the advantages enable the trypsin mutant to be better applied to the fields of medicine, scientific research and the like.

The invention aims to obtain a trypsin mutant with improved anti-autocleavage capability and activity. On the basis of a wild trypsin sequence SEQ ID NO.2, the arginine or lysine site is selected as a target mutation site, and site-directed mutation is carried out by an inverse PCR technology. Firstly, single-point mutation is carried out on a target mutation site, pichia pastoris is recombined for heterologous expression, 10 single-point mutants capable of effectively improving the anti-autogenous cutting capability and the specific activity are obtained by measuring the anti-autogenous cutting capability and the specific activity of a mutant enzyme and are respectively named as P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10, and the mutation sites are shown in Table 1. Then, the effective single-point mutations are adopted for combined mutation, and 10 multipoint combined mutants which can effectively improve the autocleavage resistance and specific activity of the trypsin are further obtained and are respectively named as P11, P12, P13, P14, P15, P16, P17, P18, P19 and P20, and the mutation combination is shown in Table 1.

TABLE 1 comparison of mutation sites

The constructed mutant engineering strain is fermented to synthesize trypsinogen. After the proenzyme is activated by enterokinase, trypsin is prepared by ultrafiltration, concentration condensation and purification. The enzyme activity is measured by measuring the change rate of absorbance under the wavelength of 253nm by using N-benzoyl-L-arginine ethyl ester (BAEE) as a substrate and an ultraviolet spectrophotometer. The enzyme activity residual rate after incubation for 120h at 37 ℃ and pH 8.0 is used as the evaluation index of the anti-autogenous cutting performance of trypsin.

The initial specific activities of the single-point mutants P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10 respectively reach 3.02 times, 1.67 times, 1.91 times, 2.49 times, 2.67 times, 2.06 times, 1.54 times, 2.79 times, 1.18 times and 1.14 times of the wild-type trypsin, and the residual rates of 120h enzyme activity respectively reach 1.67 times, 1.69 times, 2.03 times, 2.27 times, 2.70 times, 1.36 times, 2.14 times, 1.61 times, 1.09 times and 1.07 times of the wild-type trypsin, which shows that the single-point mutants have different degrees of improvement in specific activity and anti-self-excision capability compared with the wild-type enzyme molecules.

The specific activities of the multi-point combined mutants P11, P12, P13, P14, P15, P16, P17, P18, P19 and P20 respectively reach 1.73 times, 1.46 times, 2.42 times, 3.37 times, 2.37 times, 3.88 times, 3.71 times, 3.69 times, 3.56 times and 3.62 times of the specific activity of the wild-type recombinant trypsin, and the enzyme activity residual rates of the multi-point combined mutants after incubation for 120 hours are sequentially 2.62 times, 2.43 times, 2.24 times, 2.34 times, 3.2 times, 3.09 times, 3.32 times, 3.50 times, 3.69 times and 3.98 times of the enzyme activity residual rate of the wild-type recombinant trypsin, which shows that the self-cutting resistance of the combined mutants is further improved compared with that of single-point mutants.

The invention also provides a recombinant vector containing the trypsin mutant coding gene for improving the anti-autogenous cutting ability and specific activity;

further, the expression vector adopted by the recombinant vector can be pHBM905A, pPIC9K, pPIC9K-His, pPIC3.5K, pPIC9, pPICZ alpha A, pAO815, pPIC9k-His, pHIL-S1, pPink hc; pGADT7, pGBKT7, pWB980, pT3 and the like;

preferably, the expression vector is pPIC 9K;

the invention also provides a recombinant strain containing the trypsin mutant coding gene for improving the anti-autogenous cutting ability and specific activity;

further, the host cell adopted by the recombinant strain can be pichia pastoris host X33, KM71, KM71H and GS 115; saccharomyces cerevisiae strains YM4271, AH109, Y187, Y190; coli BL 21, e.coli X90; bacillus subtilis SCK6, etc.;

preferably, the host cell is pichia pastoris GS 115.

The invention also provides a preparation method of the trypsin mutant for improving the anti-autogenous cutting ability and the specific activity, which comprises the following steps:

(1) recombinant pichia pastoris shake flask fermentation

Inoculating the strain to YPD liquid culture medium, and culturing at 28 deg.C and 200rpm for 24 hr; inoculating 2% of seed solution cultured in YPD liquid culture medium to BMGY culture medium, culturing at 28 deg.C and 200rpm to OD₆₀₀2-6, and then collecting thalli; transferring to BMMY medium, adding methanol to final concentration of 0.5%; adding methanol with the final concentration of 0.5% every 12h for induction culture for 96 h;

(2) activation of trypsinogen

Centrifuging at 12000rpm for 10min to collect the supernatant, adjusting pH to 7.5 with 1M HCl or NaOH, adding enterokinase with final concentration of 2.5U/ml, and incubating at 25 deg.C for 30h to activate trypsinogen;

(3) purification of Trypsin

And after the zymogen is activated for 30 hours, centrifuging at 12000rpm for 15min, collecting supernatant, transferring to a 15ml 10kDa ultrafiltration centrifugal tube precooled at 4 ℃, centrifuging and concentrating at 2800rpm, adding a 50mM acetic acid solution into the trapped fluid, centrifuging and concentrating again, and repeating once to realize solution replacement, concentration and purification of the trapped fluid.

The trypsin can also be a sequence with the same function with more than 80 percent of homology of the amino acid sequence shown in SEQ ID NO. 2;

the trypsin can also be porcine, bovine, ovine, rabbit and other animal-derived trypsin sequences containing the same mutation site of P1-P20.

The invention also provides application of the anti-autogenous cutting trypsin mutant with high specific activity in the fields of feed processing, food processing, leather processing, medicine and scientific research.

Has the advantages that:

compared with the wild trypsin, the mutant obtained by the invention has better anti-autogenous cutting performance and higher specific activity, the anti-autogenous cutting performance is improved by 0.67-2.88 times, the specific activity is improved by 0.07-2.98 times, and the problem that the activity is easily reduced by autogenous cutting in the processes of storage and use of the trypsin is solved.

Description of the drawings:

FIG. 1: constructing a vector pPIC 9K-M-try;

FIG. 2: specific activities of wild trypsin and point mutants P1-P10 and residual enzyme activity rate after incubation for 120h

a: specific activity of wild trypsin and single point mutants; b: the enzyme activity residual rate after incubation for 120 h;

FIG. 3: specific activity of wild trypsin and combined mutants P11-P20 and residual enzyme activity rate of incubation for 120h

a: specific activity of wild trypsin and combination mutants; b: and the enzyme activity residual rate after incubation for 120 h.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

In the following examples, procedures and methods not described in detail are conventional methods commonly used in the art. The source, trade name and composition of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the time of first appearance unless otherwise specified.

The following definitions are used in the present invention:

1. nomenclature for amino acid and DNA nucleic acid sequences

The accepted IUPAC nomenclature for amino acid residues is used, in the form of the single letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification of Trypsin mutants

"amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the trypsin mutant. E.g., R107H, indicating a substitution of the amino acid at position 107 from wild-type R to H. The numbering of positions corresponds to SEQ ID NO: 2, amino acid sequence numbering of wild-type trypsin.

Experimental materials and reagents:

1) strains and vectors

Escherichia coli JM109 and Pichia pastoris GS115 are stored in the laboratory and can be purchased and obtained through commercial approaches; pMD19(T-simple) vector was purchased from Invitrogen; the pPIC9K vector was purchased from TaKaRa.

2) Enzyme and kit

The plasmid extraction kit and the genome extraction kit are purchased from Beijing Solaibao science and technology Limited; restriction enzymes were purchased from TaKaRa.

3) Culture medium

The Escherichia coli culture medium is LB, the yeast seed liquid culture medium is YPD, the yeast enrichment culture medium is BMGY, and the yeast induction culture medium is BMMY.

The trypsin enzyme activity determination method adopted by the invention comprises the following steps:

the enzyme activity is measured by measuring the change rate of absorbance under the wavelength of 253nm by using N-benzoyl L-arginine ethyl ester (BAEE) as a substrate through an ultraviolet spectrophotometer. The specific method comprises the following steps: a BAEE enzyme activity unit was determined by measuring the change in absorbance at 253nm, Δ 253nm, at a pH of 7.6 at 25 ℃ in a reaction system of 3.0mL (optical path 1cm) using 0.25mM of BAEE as a substrate and a trypsin sample solution addition of 75 μ L and an enzyme amount which increases the absorbance by 0.001 per minute. The trypsin enzyme activity and specific activity are respectively calculated by the following formulas:

the invention obtains the amino acid sequence (Entry number: P00761, SEQ ID NO.22) of wild porcine trypsinogen through a Unit database, sends the amino acid sequence to Jinzhi Biotechnology Limited company, and selects Pichia pastoris as an expression host to carry out codon optimization to obtain the trypsinogen Pichia pastoris optimized gene (SEQ ID NO. 1). The trypsinogen gene was amplified by PCR technique. Purifying and recovering the amplified product, connecting to a pMD19T cloning vector, transforming into competent cells of Escherichia coli JM109, and mutating 107 th, 115 th, 133 th, 147 th, 157 th, 208 th and 210 th amino acids by using an inverse PCR technology to obtain the trypsin mutant with improved self-excision resistance and specific activity.

The site-directed mutagenesis specifically comprises the following steps: taking constructed pMD19T-try (obtained by connecting wild-type porcine trypsin to pMD 19T) as a template, carrying out reverse PCR amplification by using a corresponding mutation primer, decomposing an original template by using restriction endonuclease DpnI, carrying out agarose electrophoresis verification on an amplified product, purifying and recovering the product, carrying out double enzyme digestion treatment on the constructed recombinant plasmid pMD19T-M-try (M represents a corresponding mutant) and an expression vector pPIC9K by using Ecol I and Not I respectively, recovering a target gene fragment and a vector fragment, and connecting the target gene fragment and the vector fragment by using T4 ligase to obtain an expression vector pPIC 9K-M-try; and (3) transferring the constructed expression vector into escherichia coli by a heat shock method, verifying a recombinant transformant through bacterial liquid, extracting a plasmid of the transformant which is verified to be correct, and sequencing to determine a corresponding mutant. Extracting mutant plasmids with correct sequencing, linearizing pPIC9K-M-try by Sac I, electrically transferring to the competence of Pichia pastoris GS115, selecting transformants, coating the transformants on an MD plate for high copy screening, and preserving the transformants with correct sequencing at-80 ℃.

The invention will be further explained below by means of specific examples.