阅读说明：本技术 一种洗涤用的碱性蛋白酶突变体及其在液体洗涤剂中的应用 (Alkaline protease mutant for washing and application thereof in liquid detergent ) 是由 石亚伟 周桂旭 魏婷婷 文阳宣 于 2019-11-26 设计创作，主要内容包括：本发明属于蛋白质工程改造技术领域,提供一种洗涤用的碱性蛋白酶突变体及其在液体洗涤剂中的应用。所述碱性蛋白酶突变体的亲本蛋白酶为枯草芽孢杆菌PB92的蛋白酶,所述碱性蛋白酶突变体至少包含以下氨基酸的置换：V262I,其中所述位置对应于氨基酸序列SEQ ID NO：1的多肽的氨基酸位置。使其能更好的适用于工业领域,特别是洗涤剂行业。使碱性蛋白酶在碱性pH条件下以及耐热性方面的酶活有所提高。为其更好的适应工业化生产奠定了基础。(The invention belongs to the technical field of protein engineering modification, and provides an alkaline protease mutant for washing and application thereof in a liquid detergent. The parent protease of the alkaline protease mutant is a protease of bacillus subtilis PB92, and the alkaline protease mutant at least comprises the following amino acid substitutions: V262I, wherein the position corresponds to the amino acid sequence SEQ ID NO:1, or a pharmaceutically acceptable salt thereof. So that it can be better applied to the industrial field, in particular to the detergent industry. The enzyme activity of the alkaline protease under the alkaline pH condition and the heat resistance is improved. And lays a foundation for better adapting to industrial production.)

1. A mutant alkaline protease for washing characterized by: the parent protease of the alkaline protease mutant is a protease of bacillus subtilis PB92, and the alkaline protease mutant at least comprises the following amino acid substitutions: V262I, wherein the position corresponds to the amino acid sequence SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.

2. A mutant alkaline protease for washing use according to claim 1, which comprises: the alkaline protease mutant further comprises a combination of the substitutions A188P + V262I.

3. A mutant alkaline protease for washing use according to any one of claims 1 to 2, which comprises: the parent protease is compared with the amino acid sequence SEQ ID NO:1 has at least 95% sequence identity.

4. A mutant alkaline protease for washing use according to any one of claims 1 to 2, which comprises: the parent protease has an amino acid sequence represented by SEQ ID NO 2.

5. A mutant alkaline protease for washing use according to claim 4, which comprises: the parent protease is compared with the amino acid sequence SEQ ID NO:2 having at least 97% sequence identity.

6. A liquid detergent composition characterized by: comprising the protease mutant of claim 1.

Technical Field

The invention belongs to the technical field of protein engineering modification, and particularly relates to an alkaline protease mutant for washing and application thereof in a liquid detergent.

Background

Alkaline Protease (AP) refers to a protease having a high activity in neutral to Alkaline environments, and can effectively hydrolyze peptide bonds, ester bonds, and amide bonds. Widely exists in plants, animals and microorganisms. The strains currently used for industrial production are mainly Bacillus licheniformis, Bacillus alkalophilus, Bacillus subtilis, etc. (Tekin N et al. Pol JMicrobiol, 2017, 66(1): 39-56).

Alkaline proteases find application in many fields including industrial fields such as detergents, pharmaceuticals, leather, soy processing, breweries, meat tenderization, waste management, photography, diagnostics, etc. Alkaline proteases alone account for 25% of the global enzyme market (Mikkelsen M L et al, Food and Chemical Toxicology, 2015: 07-21).

The catalytic activity and the thermal stability of the enzyme can be effectively improved and the substrate specificity can be improved by means of protein engineering (Johannes TW et al curr. Microbiol, 2006, 9: 261-. The protein engineering means opens up a new way for improving the functions of the enzyme and has great success in the fields of industry, agriculture and the like.

Disclosure of Invention

The invention aims to provide an alkaline protease mutant with improved heat stability and alkali resistance, which can be better suitable for the industrial field, in particular the detergent industry. The invention provides an alkaline protease mutant for washing, which improves the enzyme activity of alkaline protease under the alkaline pH condition and the heat resistance. And lays a foundation for better adapting to industrial production.

The invention is realized by the following technical scheme: an alkaline protease mutant for washing, the parent protease of which is the protease of bacillus subtilis PB92, comprising at least the following amino acid substitutions: V262I, wherein the position corresponds to the amino acid sequence SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.

The alkaline protease mutant further comprises a combination of the substitutions A188P + V262I.

The parent protease is compared with the amino acid sequence SEQ ID NO:1 has at least 95% sequence identity.

The parent protease has an amino acid sequence represented by SEQ ID NO 2.

The parent protease is compared with the amino acid sequence SEQ ID NO:2 having at least 97% sequence identity.

A liquid detergent composition comprising said protease mutant.

The liquid detergent compositions prepared according to the present invention use the MGDA and STPP standards.

The invention improves the enzyme activity of the alkaline protease under the alkaline pH condition and the heat resistance. The mutant enzyme has better activity retention than the parent protease under extreme conditions when used as a detergent, the protease can be used at higher temperature and stronger alkaline environment, and the test shows that: on the premise of not adding any protective agent and stabilizing agent, the alkaline protease and the mutant thereof are insulated for half an hour at 50 ℃, so that the residual activity of the mutants V262IA and 188P + V262I is obviously higher than that of the wild strain, even if the temperature is kept for a longer time, the residual activity of the mutants is always higher than that of the wild strain. Without adding any protective agent and stabilizer, the alkaline protease and the mutant thereof are incubated for 1 hour at different pH values, and it is obvious that the residual activity of the mutant A188PV262I is higher than that of the wild enzyme after the pH value is more than 9. On the premise of not adding any protective agent and stabilizing agent, the same addition amount of the alkaline protease and the mutant thereof is added into MGDA and STPP washing systems, so that the enzyme activity of both parent enzyme and mutant enzyme is obviously improved. The mutant enzymes have better stability than the parent enzyme in both wash systems. Is beneficial to expanding the application range of the alkaline protease and lays a foundation for better adapting to industrial production.

Drawings

FIG. 1: amino acid sequence alignment chart of subtilisin PB92 (SEQ ID NO: 1) and subtilisin mutant (SEQ ID NO: 2).

FIG. 2: SDS-PAGE electrophoresis of subtilisin PB92 (SEQ ID NO: 1) protein purification.

FIG. 3: SDS-PAGE electrophoresis of protein purification of subtilisin mutant (SEQ ID NO: 2).

Detailed Description

The experimental procedures of the present invention are further illustrated below with reference to examples, in which the procedures used are, unless otherwise specified, conventional procedures for molecular cloning, protein purification, and enzyme analysis.

The invention relates to a labeling and related enzyme activity determination method of alkaline protease mutants, which comprises the following steps:

labelling of alkaline protease mutants: "amino acid substituted at the original amino acid position" is used to indicate a mutated amino acid in the alkaline protease mutant. As shown in S259K/R, the amino acid at position 259 is replaced by lys or Arg from Ser of the original alkaline protease, and the numbering of the position corresponds to that in SEQ ID NO:1 of the attached sequence Listing.

The method for measuring the enzyme activity of the alkaline protease comprises the following steps: the method is carried out according to GB/T23527 appendix B Folin method, and the specific reaction process is as follows: a series of empty tubes were first removed, with one tube in each group labeled as the control group and the remaining three tubes labeled as the experimental group. Adding 0.5mL of 1% casein solution prepared by buffer solution into all test tubes, and keeping the test tubes at 40 ℃ for 2 min; adding 0.5mL of crude enzyme solution into the test tube except the blank to allow the enzyme solution and the substrate to react for 10 min; adding 1mL0.4mol/L of trichloroacetic acid to stop the reaction; adding 1mL of enzyme solution into a control group; standing for 10min, centrifuging, and placing 1mL of supernatant in new test tubes; 5mL of sodium carbonate and 1mL of Folin reagent are added; developing at 40 deg.C for 20 min. Absorbance was measured at 680 nm. The enzyme activity calculation formula is as follows: x = a × K × 4/10 × n, where K represents the absorption constant (laboratory measurement K = 97).