阅读说明：本技术 碱性蛋白酶的突变体及其应用 (Mutant of alkaline protease and application thereof ) 是由 张大伟 付刚 于 2020-01-20 设计创作，主要内容包括：本发明公开的碱性蛋白酶突变体,其相对于原始氨基酸序列,其在第113位氨基酸由Ile变为Ala,或更进一步地第140位氨基酸由Ile变为Glu。通过将突变体编码基因分别构建了表达所述突变体的重组菌枯草芽孢杆菌,其发酵上清液在pH7.0条件下的酶活分别为5673 U/mL和6733 U/mL,比表达野生型碱性蛋白酶subC的枯草芽孢杆菌酶活分别提高了130％和154％。(The alkaline protease mutant disclosed herein has an amino acid Ile-to-Ala transition at position 113 or further an amino acid Ile-to-Glu transition at position 140, relative to the original amino acid sequence. Recombinant bacillus subtilis for expressing the mutant is respectively constructed by encoding genes of the mutant, the enzyme activities of fermentation supernatant under the condition of pH7.0 are 5673U/mL and 6733U/mL respectively, and the enzyme activities are respectively improved by 130 percent and 154 percent compared with the enzyme activities of the bacillus subtilis for expressing wild alkaline protease subC.)

1. An alkaline protease mutant is characterized in that the amino acid sequence of the alkaline protease mutant is shown as SEQ ID NO. 2 or SEQ ID NO. 3.

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

3. A recombinant expression vector carrying a gene encoding the alkaline protease mutant of claim 2.

4. A recombinant host cell transformed/transfected with the recombinant expression vector of claim 2.

5. The recombinant host cell of claim 4, wherein the host cell is Bacillus subtilis.

6. Use of the alkaline protease mutant according to claim 1 as an additive for detergents.

7. A detergent composition comprising the alkaline protease mutant according to claim 1.

8. A detergent composition as claimed in claim 7, which is a liquid composition.

9. The detergent composition of claim 7 or 8, wherein the alkaline protease mutant is added to the detergent composition in a fermented crude enzyme solution, or in a purified form.

Technical Field

The invention belongs to the field of biotechnology and protein engineering, and particularly relates to an alkaline protease mutant protein.

Background

The alkaline protease (alkaline protease) is an enzyme capable of hydrolyzing peptide bonds of proteins under alkaline conditions (the pH is within the range of 9-11), the main component of the alkaline protease is endoprotease, the catalytic site is serine, and the alkaline protease is widely applied to industries such as detergents, foods, medical treatment, brewing, silk and leather making. The alkaline protease adopted at present is a proteolytic enzyme which is cultured by a bacterial protoplast mutagenesis method and is derived from bacillus subtilis 2709 and is prepared by deep fermentation, extraction and refining, belongs to serine alkaline protease, can hydrolyze peptide chains of protein molecules to generate polypeptide or amino acid, and has stronger capability of decomposing protein. The production process adopts the advanced technologies of microfiltration and ultrafiltration membrane separation, spray drying or vacuum freeze drying and the like. The alkaline protease is a popular washing additive in the current market, can greatly improve the washing and dirt removing capacity, particularly has unique washing effect on protein dirt such as blood stains, sweat stains, milk stains, oil stains and the like, is an enzyme occupying the largest proportion in industrial enzymes, and accounts for about 60 percent of the total annual sales volume all over the world. In production, the quality of the granular alkaline protease for the powder detergent is required to be dust-free, safe and sanitary; the compatibility with a detergent is good; good stability in detergents, etc. Therefore, there is a need to develop alkaline proteases with superior performance and better applicability.

Site-directed mutagenesis is the introduction of desired changes (usually changes that characterize favorable orientations) including base additions, deletions, point mutations, and the like, by Polymerase Chain Reaction (PCR) or the like, into a DNA fragment (which may be a genome or a plasmid) encoding a protein of interest. The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work. The in vitro site-directed mutagenesis technology is an important experimental means in the research of various fields of biology and medicine at present, is a convenient scheme for modifying and optimizing genes, and is a powerful tool for researching the complex relationship between the structure and the function of protein. The corresponding amino acid sequence and protein structure can be changed by site-directed alteration, deletion or insertion of specific base of a known gene, and the research of the expression product of mutant gene is helpful for human to understand the relationship between protein structure and function and investigate the structure/structural domain of protein. In recent years, the application of site-directed mutagenesis technology of enzyme mainly focuses on the aspects of improving the catalytic activity of enzyme, improving the substrate specificity, improving the thermal stability, enantioselectivity and the like. The site-directed mutagenesis technology of the enzyme opens up a novel approach for the structure and the function of the enzyme. For example, studies have been made to improve the cleaning performance of alkaline proteases by mutating the alkaline proteases to improve their specific activities, stability, etc. (Japanese patent laid-open No. 2010-273673 and Japanese patent laid-open No. 5202690). For example, CN109312323A discloses that mutants of alkaline proteases are obtained by substituting threonine for the amino acid residue at position 294 in the amino acid sequence of a parent alkaline protease, thereby improving the specific activity. CN105176951A based on the alkaline protease aprE from Bacillus clausii, three mutants were obtained by mutation, with 42%, 180% and 130% higher activity than the parent.

Therefore, when a specific protein is mutated, the selection of the mutation site and the determination of the mutation direction are often difficult to predict or unpredictable in advance, and particularly in the case of alkaline proteases, the prior art has limited knowledge of the relationship between the structure and the function of the enzyme, and therefore, a protein mutant having a desired functional property cannot be obtained by theoretical analysis in advance, and it is necessary to search in advance in order to obtain a mutation in a desired direction of performance.

Disclosure of Invention

The present invention has been made in an intensive study on alkaline protease, and finally, by finding out a suitable mutation site and a specific substituted amino acid, a mutant produced by Bacillus licheniformis (B.)Bacillus licheniformis) Starting from the mature peptide of the alkaline protease gene subC, the alkaline protease mutant with obviously improved enzyme activity is finally obtained.

On the basis of the alkaline protease with a parent amino acid sequence of SEQ ID NO. 1, the 113 th amino acid of the alkaline protease is changed from Ile to Ala, and the amino acid sequence of the alkaline protease mutant is SEQ ID NO. 2.

Further, the present invention provides an alkaline protease mutant, which is an alkaline protease having the parent amino acid sequence of SEQ ID NO. 1 in which amino acid position 113 is changed from Ile to Ala and amino acid position 140 is changed from Ile to Glu (i.e., an alkaline protease having the amino acid sequence of SEQ ID NO. 2 in which amino acid position 140 is further changed from Ile to Glu), and which has the amino acid sequence of SEQ ID NO. 3.

The invention also provides an alkaline protease mutant coding gene, and the amino acid sequence of the coded polypeptide is shown as SEQ ID NO. 2 or SEQ ID NO. 3.

Further, the present invention provides a recombinant expression vector carrying the gene encoding the above alkaline protease mutant.

Still further, the present invention provides a recombinant host cell transformed/transfected with the recombinant expression vector described above. Preferably, the recombinant host cell is Bacillus subtilis.

In still another aspect, the invention also provides the use of the alkaline protease mutant as an additive for detergents. Further, the present invention provides a detergent composition comprising the above alkaline protease mutant. The lotion composition may be a powder detergent composition, but is preferably a liquid detergent composition. Wherein the alkaline protease mutant is added into the detergent composition in a form of fermented crude enzyme solution or purified.

The invention is based on the alkaline protease subC from bacillus licheniformis, obtains two alkaline protease mutants subC 1 and subC M2 by a site-directed mutagenesis technology, and ferments the mutants in bacillus subtilis, wherein the enzyme activities of the fermentation supernatant are 5673U/mL and 6733U/mL respectively, which are 130% and 154% respectively higher than the enzyme activity of bacillus subtilis (4356U/mL) expressing wild alkaline protease subC, and the invention has wider application.

Drawings

FIG. 1: a plasmid map of the alkaline protease expression vector;

FIG. 2: bar chart of relative enzyme activity of alkaline protease mutants.

Detailed Description

The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope of the invention.

The experimental techniques and experimental procedures used in this example are conventional techniques, unless otherwise specified, such as the conditions described in molecular cloning guidelines written by J. Sambruka (Sambrook), et al, or the procedures recommended by the manufacturer. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.

The terms and associated assay methods referred to in the present invention are explained below:

(1) the protease activity determination method comprises the following steps: the method for determining the protease preparation is adopted (GB/T25327-2009).

(2) Definition of enzyme activity unit: 1g of solid enzyme powder (or 1mL of liquid enzyme) hydrolyzes casein for 1min under the conditions of certain temperature and pH value to generate 1 mu g of tyrosine, namely 1 enzyme activity unit expressed by U/g (U/mL).

(3) Alkaline protease the activity of the protease was determined using the forskolin method using a solution comprising: folin use solution (one commercial Folin solution was mixed with two portions of water, shaken up), sodium carbonate solution (42.4g/L), trichloroacetic acid (65.4g/L), gradient pH buffer, casein solution (10.0 g/L). The reaction process is as follows: adding 1mL enzyme solution into the test tube, performing warm bath at 40 deg.C for 2min, adding 1mL casein solution, shaking, performing warm bath at 40 deg.C for 10min, adding 2mL trichloroacetic acid solution, and shaking (adding trichloroacetic acid and casein solution into blank control). Taking out and standing for 10min, and filtering with slow qualitative filter paper. Taking 1mL of filtrate, adding 5mL of sodium carbonate solution, adding 1mL of forskolin reagent solution, developing at 40 ℃ for 20min, and measuring absorbance at 680nm wavelength by using a 10mm cuvette.

(4) The identification of the alkaline protease mutant refers to the amino acid mutated in the alkaline protease mutant by the "amino acid substituted at the original amino acid position". Such as Ile113Ala, the amino acid at position 113 is replaced by Ile of the parent alkaline protease to Ala, and the numbering of the positions corresponds to that of SEQ ID NO 1 of the appendix sequence Listing. Such as Ile113Ala/Ile140Glu, indicating that both amino acids at position 113 and 140 have been mutated.