阅读说明：本技术 新型碱性蛋白酶抑制肽的筛选 (Screening of novel alkaline protease inhibitory peptides ) 是由 路福平 王洪彬 宋平 李雪 王玉迎 刘逸寒 于 2019-10-09 设计创作，主要内容包括：本发明涉及新型碱性蛋白酶抑制肽的筛选,通过体外RNA展示筛选技术获得了芽孢杆菌来源的碱性蛋白酶的新型八肽抑制肽,该抑制肽能够增强液体制剂中碱性蛋白酶的稳定性,有望应用于液体酶制剂和加酶液体洗涤剂。(The invention relates to screening of novel alkaline protease inhibitory peptides, and the novel octapeptide inhibitory peptides of alkaline protease from bacillus are obtained by an in vitro RNA display screening technology, can enhance the stability of the alkaline protease in a liquid preparation, and are expected to be applied to the liquid enzyme preparation and an enzyme-added liquid detergent.)

1. Novel alkaline protease inhibiting peptides characterized by: the inhibitor is polypeptide consisting of 8 amino acids.

2. The polypeptide inhibitor of claim 1, wherein: in the octapeptide sequence obtained by screening through an RNA in-vitro display technology, from an amino terminal, the first amino acid site is tyrosine or histidine, the second amino acid site is cysteine or tryptophan, the types of amino acids contained in the third to fourth amino acid sites comprise histidine, leucine, cysteine, arginine, serine, proline, tyrosine, phenylalanine, asparagine, glycine, threonine, tryptophan and glutamic acid, and the carboxyl terminal site of the polypeptide sequence is tyrosine, leucine or histidine.

3. The polypeptide inhibitor of claim 1, wherein: the following ten octapeptides have significant inhibitory activity against Bacillus-derived alkaline proteases, including YCLSRDWH, HCLSRDWH, YCLSRDCH, YCLSRDWY, HCNNNNNNH, HWNNNNNH, YCLSRDWH, YCLSHDWH, YCLSRAWH and YCNNNNNH. The corresponding relation of amino acid abbreviations is as follows: arginine, R; aspartic acid, D; cysteine, C; histidine, H; asparagine, N; leucine, L; tryptophan, W; tyrosine, Y. The amino acid sequence is written from N-terminus to C-terminus.

4. The polypeptide inhibitor of alkaline protease according to claim 1, characterized in that: the alkaline protease is bacillus-derived alkaline protease.

Technical Field

The invention belongs to the field of enzyme engineering, and particularly relates to screening of novel inhibitory peptides of alkaline protease derived from bacillus.

Background

Alkaline protease hydrolyzes protein most efficiently under alkaline conditions. The method is widely applied to industries such as washing, food, medicine, tanning and the like, wherein the consumption of the washing industry accounts for the global alkaline protease demand. The microbial alkaline protease mainly from bacillus is an enzyme for adding a main liquid detergent, has high dirt-removing capacity under alkaline conditions, and has the best effect on protein dirt such as blood stains, sweat stains, oil stains and the like. Protease is inactivated by autogenous cleavage, so that the liquid enzyme-containing detergent needs to be added with an inhibitor during storage to avoid degradation of the washing performance by hydrolysis of the alkaline protease itself. The protease inhibitor widely used in the liquid detergent at present is a boric acid inhibitor, is not friendly to the environment and human health, and animal toxicity tests show that the boric acid compound is a second class of reproductive toxicity compound, so that a very large reproductive toxicity risk exists after long-term use. Therefore, there is an urgent need to develop novel alkaline protease inhibitors that are green, environmentally friendly and safe, and polypeptide inhibitors are currently a major research direction.

The work of the present invention attempted to obtain inhibitory peptides of alkaline protease by screening through the mRNA in vitro display technique. The mRNA in vitro display technology has the advantages of high library capacity, high sensitivity and the like on the screening efficiency. The core technology is characterized in that an RNA sequence of a peptide library and a translated corresponding amino acid sequence form mRNA-linker-polypeptide fusion molecules through a linker (linker), the mRNA-linker-polypeptide fusion molecules are used for affinity peptide screening, and the obtained affinity peptide sequence can realize ultrahigh-sensitivity identification through PCR amplification and high-throughput sequencing of covalently linked cDNA. During the screening process, mRNA is covalently bound to its encoded polypeptide or protein to form mRNA-protein fusions that can be used in large-capacity polypeptide libraries (10)¹³～10¹⁵) Screening for polypeptides and proteins having specific biological functions. However, successful establishment and application of mRNA in vitro display technology is extremely challenging. No report on screening the inhibitory peptide of the alkaline protease by using the mRNA in vitro display technology exists at home and abroad.

Disclosure of Invention

The present invention aims to screen novel polypeptide inhibitors for bacillus-derived alkaline proteases by an mRNA in vitro display screening technique.

The technical scheme for realizing the purpose of the invention is as follows:

primarily screening candidate inhibitory peptides of the alkaline protease by an mRNA in vitro display method, and determining and verifying the inhibitory peptides of the alkaline protease by measuring the enzyme activity inhibition rate, which comprises the following steps:

(1) a specially designed and synthesized DNA library is subjected to steps of in vitro transcription, linker photocrosslinking, in vitro translation, purification, reverse transcription and the like to obtain a genotype phenotype fusion molecule library, namely an mRNA-linker-polypeptide fusion molecule library;

(2) immobilizing biotin-modified bacillus-derived alkaline protease by streptavidin magnetic beads;

(3) and then, carrying out incubation screening on the mRNA-linker-polypeptide fusion molecule library and magnetic bead immobilized alkaline protease, amplifying DNA of the screened affinity peptide through PCR, and then carrying out next screening.

(4) And finally obtaining the high-affinity fusion molecules by multi-round screening, and realizing the sequence identification of the candidate inhibitory peptides through PCR amplification and DNA sequencing.

(5) And (3) measuring the inhibition rate of the inhibitory peptide on the enzyme activity of the spore-derived alkaline protease, and confirming the inhibition performance of the inhibitory peptide on the alkaline protease.

The inhibitory peptide of bacillus-derived alkaline protease obtained by mRNA in vitro display screening is characterized in that the amino acid at the N1 position from the N terminal is tyrosine or histidine; position N2, cysteine or tryptophan; the amino acid types contained in the N1-N7 positions comprise histidine, leucine, cysteine, arginine, serine, proline, tyrosine, phenylalanine, asparagine, glycine, threonine, tryptophan and glutamic acid, and the carboxyl terminal position C1 of the polypeptide sequence is tyrosine, leucine or histidine. The amino acid positions in the octapeptide sequence were designated: the amino acid positions are N1, N2, N3, N4, N5, N6, N7 and C1 in sequence from the N segment.

Wherein the following ten octapeptides have high inhibitory activity against alkaline protease, including YCLSRDWH, HCLSRDWH, YCLSRDCH, YCLSRDWY, HCNNNNNNH, HWNNNNNH, YCLSRDWH, YCLSHDWH, YCLSRAWH and YCNNNNNNNH. The corresponding relation of amino acid abbreviations is as follows: arginine, R; aspartic acid, D; cysteine, C; histidine, H; asparagine, N; leucine, L; tryptophan, W; tyrosine, Y. The amino acid sequence is written from N-terminus to C-terminus.

The invention has the beneficial effects that:

(1) a plurality of rounds of screening of mRNA in vitro display successfully obtain novel octapeptide inhibitors of a plurality of alkaline proteases. The polypeptide inhibitor has high biological safety and small environment pollution. The protease inhibitor widely used in the liquid detergent at present is a boric acid inhibitor, is not friendly to the environment and human health, and animal toxicity tests show that the boric acid substance has a very large reproductive toxicity risk.

(2) The inhibition rate of inhibitory peptides such as YCLSRDWY and the like on the activity of alkaline protease from spores is close to the performance of the existing inhibitor 4-formylphenylboronic acid (4-FPBA), and the inhibitor is expected to be applied to liquid enzyme preparations and enzyme-added liquid detergents in the future.

Drawings

FIG. 1 is a diagram showing the distribution of amino acid species at different sites in octapeptide sequences screened by RNA in vitro display technology

FIG. 2 shows the inhibition rate of peptide fragments on the enzyme activity of alkaline protease

Detailed Description

The technical content of the present invention is further illustrated by the following examples, but the present invention is not limited to these examples, and the following examples should not be construed as limiting the scope of the present invention.