阅读说明：本技术 一类热稳定性提高的蛋白酶突变体及其编码基因和应用 (Protease mutant with improved thermal stability and coding gene and application thereof ) 是由 肖志壮 方安然 于 2020-04-27 设计创作，主要内容包括：本发明提供了一类热稳定性提高的蛋白酶突变体及其编码基因和应用,本发明使用易错PCR方法对<I>Bacillus licheniformis</I>来源的蛋白酶基因进行突变,再通过高通量筛选,获得蛋白酶突变体BLAPR1、BLAPR2、BLAPR3、BLAPR4,相比于未突变的蛋白酶,本发明得到的蛋白酶突变体的热稳定性得到显著提高,具有良好的市场应用前景和工业价值。(The invention provides a protease mutant with improved thermal stability, a coding gene and application thereof, and the invention is easy to useWrong PCR method pair Bacillus licheniformis The protease mutants such as BLAPR1, BLAPR2, BLAPR3 and BLAPR4 are obtained by mutating the protease genes and then screening the protease mutants with high flux.)

1. A protease mutant BLAPR1 with improved thermostability, which is characterized in that the amino acid sequence of the protease mutant is shown as SEQ ID NO: 3, the nucleotide sequence of the coding gene is shown as SEQ ID NO: 4, respectively.

2. A protease mutant BLAPR2 with improved thermostability, which is characterized in that the amino acid sequence of the protease mutant is shown as SEQ ID NO: 5, the nucleotide sequence of the coding gene is shown as SEQ ID NO: and 6.

3. A protease mutant BLAPR3 with improved thermostability, which is characterized in that the amino acid sequence of the protease mutant is shown as SEQ ID NO: 7, the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown in fig. 8.

4. A protease mutant BLAPR4 with improved thermostability, which is characterized in that the amino acid sequence of the protease mutant is shown as SEQ ID NO: 9, the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown at 10.

5. A recombinant expression vector comprising a gene encoding the protease mutant according to any one of claims 1 to 4.

6. Genetically engineered bacterium comprising a gene encoding a protease mutant according to any one of claims 1 to 4, characterised in that the genetically engineered bacterium is Bacillus subtilis or Bacillus licheniformis.

7. A method for producing the protease mutant according to any one of claims 1 to 4, characterized by comprising the steps of:

1) constructing recombinant gene engineering bacteria: connecting the coding gene of the protease mutant to a pUB110 vector, transforming the recombinant vector into bacillus subtilis, and screening positive clones by using a resistance marker;

2) shaking flask fermentation of recombinant gene engineering bacteria: inoculating the positive clone which is verified to be correct into a shake flask for fermentation, carrying out shake culture, and fermenting to generate a protease mutant;

3) amplifying and fermenting the recombinant strain: the genetically engineered strain expressing the protease mutants is inoculated into a fermenter, thereby producing the protease mutants BLAPR1, BLAPR2, BLAPR3 and BLAPR4 by fermentation.

8. The method for preparing the protease mutant according to claim 7, wherein the fermentation medium of the step (3) comprises the following components: 5-10% of soybean meal, 1-5% of corn flour, 1-1.0% of PPG-200000.1, 0.1-1.0% of protease, 0.1-1.0% of amylase and 0.2-0.5% of disodium hydrogen phosphate by mass ratio.

9. Use of the protease mutant according to any one of claims 1 to 4 for the preparation of feed additives and food additives.

10. Use of a protease mutant according to any of claims 1-4 for the preparation of detergents.

Technical Field

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a protease mutant with improved thermal stability as well as a coding gene and application thereof.

Background

Proteases are enzymes which catalyze the hydrolysis of peptide bonds in proteins, are widely present in animals, plants and microorganisms, have many different physiological functions, and are the first and most mature ones for the development of enzymology. The application of protease in various fields, such as food industry, brewing, detergent industry, feed industry, tanning industry, silk industry, pharmaceutical industry and the like, is more closely related to our life, and the environment puts higher demands on the yield and the characteristic improvement of protease.

At present, most of proteases on the market only have less than 20% of residual enzyme activity after being treated at 70 ℃, and the overall temperature resistance is poor, so that the wide use of the proteases is limited. For example, in the production of feed, the enzyme preparation is mixed with feed and then granulated at high temperature, during which the enzyme is easily inactivated. It is therefore of great importance to improve the thermostability of the protease.

The error-prone PCR technology is that when DNA polymerase is used for PCR reaction amplification of a target fragment, mutation frequency in the amplification process is increased by adjusting reaction conditions, so that mutation is randomly introduced into a target gene at a certain frequency, a mutant library is constructed, and a required forward mutant is screened. The error-prone PCR technique can be well applied to molecular modification of proteins.

Disclosure of Invention

The invention provides protease mutants with improved thermal stability, coding genes and application thereof, which are obtained by constructing a mutant library and performing directional screeningBacillus licheniformisAnd the protease gene from WX-02 is improved, and a mutant with improved heat stability is obtained by screening, so that the application effect of the mutant in the fields of feed, food or washing is improved.

In order to achieve the purpose of the invention, the invention is realized by adopting the following technical scheme:

the invention provides a protease mutant BLAPR1 with improved thermal stability, and the amino acid sequence of the protease mutant BLAPR1 is shown in SEQ ID NO: 3, the nucleotide sequence of the coding gene is shown as SEQ ID NO: 4, respectively.

The invention provides a protease mutant BLAPR2 with improved heat stability, and the amino acid sequence of the protease mutant BLAPR2 is shown in SEQ ID NO: 5, the nucleotide sequence of the coding gene is shown as SEQ ID NO: and 6.

The invention provides a protease mutant BLAPR3 with improved heat stability, and the amino acid sequence of the protease mutant BLAPR3 is shown in SEQ ID NO: 7, the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown in fig. 8.

The invention provides a protease mutant BLAPR4 with improved heat stability, and the amino acid sequence of the protease mutant BLAPR4 is shown in SEQ ID NO: 9, the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown at 10.

The invention also provides a recombinant expression vector containing the protease mutant coding gene.

The invention provides a genetic engineering bacterium containing a protease mutant coding gene, wherein the genetic engineering bacterium is bacillus subtilis and bacillus licheniformis.

The invention provides a preparation method of the protease mutant, which comprises the following steps:

1) constructing recombinant gene engineering bacteria: connecting the coding gene of the protease mutant to a pUB110 vector, transforming the recombinant vector into bacillus subtilis, and screening positive clones by using a resistance marker;

2) shaking flask fermentation of recombinant gene engineering bacteria: inoculating the positive clone which is verified to be correct into a shake flask for fermentation, carrying out shake culture, and fermenting to generate a protease mutant;

3) amplifying and fermenting the recombinant strain: the genetically engineered strain expressing the protease mutants is inoculated into a fermenter, thereby producing the protease mutants BLAPR1, BLAPR2, BLAPR3 and BLAPR4 by fermentation.

Further: the fermentation medium in the step (3) comprises the following components: 5-10% of soybean meal, 1-5% of corn flour, 1-1.0% of PPG-200000.1, 0.1-1.0% of protease, 0.1-1.0% of amylase and 0.2-0.5% of disodium hydrogen phosphate by mass ratio.

The invention provides application of the protease mutant in preparation of feed additives, food additives and detergents.

Compared with the prior art, the invention has the advantages and the technical effects that: the invention is provided withBacillus licheniformisBased on the protease gene derived from WX-02, single-site mutants BLAPR1 comprising A196C, double-site mutants BLAPR2 and BLAPR3 comprising I140C/A196C, K49E/A196C and triple-site mutant BLAPR4 comprising S191C/A196C/G308E are provided.

The heat stability of the modified mutant BLAPR1, BLAPR2, BLAPR3 and BLAPR4 at 75 ℃ for 3 minutes is respectively improved by 30.2%, 64.0%, 45.9% and 39.0% compared with the original protease. The thermal stability is obviously improved.

Therefore, the heat stability of the protease mutant obtained by the technical scheme of the invention is greatly improved compared with that of a wild type, so that the protease mutant has good application potential in the fields of feed, food or washing and the like. Has good market application prospect.

Drawings

FIG. 1 shows the fermentation data of the protease of the present invention in a 30L fermenter.

FIG. 2 shows the comparison of the heat resistance of the protease mutants of the present invention in a water bath at 75 ℃ for 3 min.

FIG. 3 shows the effect of the protease mutants of the present invention on the degradation of soybean meal resistant protein (1, 19 are protein markers; 2, 8, 12, 18 are soybean meal without enzyme addition; 3-7 are, respectively, 200U/g of BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR 4; 9-13 are, respectively, 400U/g of BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR 4; 13-17 are, respectively, 600U/g of BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR 4).

Detailed Description

In order to facilitate understanding of the invention, the invention will be described in more detail below with reference to the accompanying drawings and examples, but the scope of the invention is not limited to the following specific examples.

The molecular biological experiments, which are not specifically described in the following examples, can be performed by referring to the specific methods listed in molecular cloning, A laboratory Manual (third edition) J. SammBruker, or according to the kit and product instructions. Reagents and biomaterials used in specific examples are commercially available without specific recitation.

1 strains and vectors

Bacillus subtilis WB600, plasmid pUB110, Escherichia coli BL21, plasmid pET-21a (+) were purchased from Invitrogen.

2 reagents and culture media

Plasmid extraction kit, fragment purification recovery kit, restriction enzyme and the like are purchased from precious bioengineering (Dalian) Co., Ltd; the GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Biotechnology engineering (Shanghai) Co., Ltd; protein Marker: blue Plus II Protein Marker (14-120kDa) was purchased from Beijing Quanjin Biotechnology, Inc. LB culture medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.