阅读说明：本技术 新型抗自切碱性蛋白酶的筛选 (Screening of novel autocleavage-resistant alkaline protease ) 是由 路福平 王洪彬 邱悦悦 李雪 王玉迎 刘逸寒 于 2019-11-29 设计创作，主要内容包括：本发明属于酶的基因工程技术领域,具体涉及一种新型的能够抗自切的碱性蛋白酶突变体的筛选。本发明通过对克劳氏芽孢杆菌来源的碱性蛋白酶进行改造和筛选获得了抗自切性能优异的突变体,突变体的基因在第493-495位突变为GAT,第799-801位为GAT,相应的氨基酸序列分别是在第161位突变为Thr,第183位为Asp。在40℃恒温孵育48h后,碱性蛋白酶突变体的酶活保留率比野生型碱性蛋白酶的酶活提高了40％以上。抗自切碱性蛋白酶分子的筛选,有助于改善碱性蛋白酶的储存和应用效果。(The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to screening of a novel alkaline protease mutant capable of resisting autogenous cutting. The invention obtains the mutant with excellent anti-autocleaving performance by modifying and screening the alkaline protease from the Bacillus clausii, the gene of the mutant is mutated into GAT at the 493-and 495-positions, GAT at the 799-and 801-positions, the corresponding amino acid sequences are respectively mutated into Thr at the 161-position and Asp at the 183-position. After incubation for 48h at the constant temperature of 40 ℃, the enzyme activity retention rate of the alkaline protease mutant is improved by more than 40 percent compared with the enzyme activity of wild alkaline protease. The screening of the anti-self-cutting alkaline protease molecule is beneficial to improving the storage and application effects of the alkaline protease.)

1. Screening of novel autocleavable alkaline protease, characterized in that: novel autocleavage resistant alkaline protease mutants are obtained by virtual amino acid mutation.

2. The novel autocleavage resistant alkaline protease mutant of claim 1 having one or both of the following mutations: the 733-rd and 735-th mutation of the gene sequence is ACC, the 799-th and 801-th mutation is GAT, the corresponding amino acid sequence mutation is Thr at the 161-th mutation and Asp at the 183-th mutation.

3. The novel autocleavage resistant mutant of alkaline protease as claimed in claim 1, wherein: the alkaline protease mutant has enzyme activity retention rates which are respectively improved by more than 40 percent compared with wild type after being incubated for 48 hours under the condition of optimal enzyme activity.

Technical Field

The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to screening of novel anti-autogenous cutting alkaline protease

Background

Alkaline protease (alkaline protease) is an enzyme that hydrolyzes peptide bonds of proteins at a pH slightly in the alkaline range, has an optimum pH of usually 9 to 11, belongs to the class of serine proteolytic enzymes in endopeptidases, and has a molecular weight of about 27 kDa. The alkaline protease is mainly applied to the enzyme-added detergent industry, is also widely applied to the industries of food, medical treatment, brewing, silk, leather making and the like, and is the enzyme occupying the largest proportion in industrial enzymes.

However, the self-cutting phenomenon of the alkaline protease in the substrate hydrolysis is also intensified due to the wide range of the hydrolysis sites, particularly, after the amino acid existing in the loose secondary structure of β -turn and loop is cut, the active center of the alkaline protease is exposed and is rapidly inactivated after being recognized and cut, the self-cutting easily occurs, a great amount of loss of enzyme activity is caused, and the production cost is increased for maintaining the activity of the alkaline protease for a long time.

The invention aims at searching and analyzing the surface specific amino acid of the alkaline protease to determine the easy self-cutting site, and then performs virtual amino acid mutation by using Discovery Studio software to determine the type of the optimally mutated amino acid. The present invention provides an alkaline protease mutant which is resistant to spontaneous cleavage without decreasing the enzyme activity. The mutant is obtained by starting from a mature peptide of an alkaline protease gene aprE of Bacillus clausii (Bacillus clausii) through technologies such as molecular simulation, site-directed mutagenesis and the like.

Disclosure of Invention

The invention aims to provide an alkaline protease mutant capable of resisting autogenous cutting. The invention is based on alkaline protease aprE from Bacillus clausii, determines an easy self-cutting site by searching and analyzing hydrophobic amino acid on the surface of the alkaline protease, and then performs virtual amino acid mutation by using Discovery Studio software to determine the type of the optimal mutated amino acid. Then, 2 alkaline protease mutants pQY-5 and pQY-6 are obtained by designing primers and introducing mutation sites by reverse PCR technology, recombinant bacteria of bacillus subtilis QY-5 and QY-6 for expressing the mutants are respectively constructed, the enzyme activities are respectively 27U/mL and 31U/mL after fermentation for 48 hours, and the enzyme activities of wild alkaline protease aprE expressed by the recombinant bacteria are respectively improved by 17 percent and 38 percent. The enzyme activity retention rate of the alkaline protease mutant is improved by 41 percent and 70 percent compared with the enzyme activity of the wild alkaline protease.

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

the easy self-cutting site is determined by the search and analysis of the hydrophobic amino acids on the surface of the alkaline protease, and then the type of the amino acid with the optimal mutation is determined by virtual amino acid mutation performed by Discovery Studio software. Taking a genome of bacillus clausii as a template, and performing amplification according to GenBank: the method comprises the steps of cloning FJ940727.1 to obtain an alkaline protease gene aprE, constructing the alkaline protease gene aprE on an expression vector pWB600, introducing a mutation site through an inverse PCR technology, carrying out site-specific mutation on the wild alkaline protease gene to obtain a mutant gene (SEQ ID NO: 3, 5), constructing a recombinant vector, and transforming the recombinant vector into Bacillus subtilis WB600 for fermentation enzyme production experiment verification to obtain the alkaline protease mutant capable of effectively resisting autogenous cutting.

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 a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification 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. Like Leul61Thr, indicates that the amino acid at position 161 is replaced by Leu to Thr of the wild-type alkaline protease. The numbering of positions corresponds to SEQ ID NO: 2, the amino acid sequence of the wild-type alkaline protease. Nucleotide changes are also denoted by "original nucleotide position substituted nucleotide" and the position numbering corresponds to that of SEQ ID NO: 1, nucleotide sequence number of wild-type alkaline protease.

In the present invention, aprE represents the gene sequence of wild type alkaline protease, i.e., the original sequence (shown in SEQ ID NO: 1), and aprEm represents the gene sequence of alkaline protease mutant (shown in SEQ ID NO: 3, 5); APRE represents wild type alkaline protease (the amino acid sequence is shown as SEQ ID NO: 2), APREM represents alkaline protease mutant (the amino acid sequence is shown as SEQ ID NO: 4, 6)

Alkaline protease	Base	Amino acids
			APREM-1	733 + 735 th position ACC	Thr at position 161
APREM-2	799 along position 801 as GAT	Asp at position 183

The expression host of the alkaline protease mutant is Bacillus subtilis WB60, and the expression vector is pWB 980.

The experimental steps of the invention are as follows:

1. downloading analyzed crystal structures 4cfz, 4cfy, 3bx1 and 1TK2 from a PDB structure database, and finding out an amino acid residue with the surface containing F, Y, W as a candidate mutation site;

2. the virtual amino acid mutations at positions 161 and 183 of 4cfz were performed by using the discovery studio software, and the best results were calculated as candidate mutant amino acids. The specific parameters are that in a queue status module in the status Energy of the discovery studio, the status: single details, details: candidate sites, All, CHARMm Polar H is selected in a force field, the influence of PH and temperature on mutation performance is ignored, and sites which are easy to self-cut by alkaline protease are obtained.

3. Construction of recombinant Bacillus subtilis strain containing wild type alkaline protease gene

Carrying out enzyme digestion on the wild type alkaline protease coding gene aprE obtained by amplification (figure 3), and connecting the wild type alkaline protease coding gene aprE with an expression vector pWB980 (figure 4) subjected to enzyme digestion to obtain a new recombinant vector; and (3) transforming the recombinant vector into the bacillus subtilis WB600, and obtaining a wild alkaline protease expression strain by screening kana resistance and determining enzyme activity of the obtained recombinant strain.

4. Construction of Bacillus subtilis recombinant Strain containing mutant alkaline protease Gene

Designing a mutation site in a primer, and introducing the mutation site by reverse PCR by using a recombinant plasmid aprE-pWB980 containing a wild-type alkaline protease coding gene as a template. Obtaining mutant alkaline protease aprEM gene, and performing self-connection on the linear vector obtained by amplification to obtain the recombinant vector of the alkaline protease mutant gene. And (3) transforming the recombinant vector into the bacillus subtilis WB600, and sequencing the obtained recombinant strain to obtain the alkaline protease mutant.

5. Fermentation preparation of wild type alkaline protease and mutant alkaline protease

6. Comparing the enzyme activity retention rates of different mutants through a stability investigation experiment to obtain the mutant with higher enzyme activity stability

7. Sequencing the obtained mutant strain plasmid with higher enzyme activity stability to obtain the high-stability alkaline protease mutant gene sequence.

The invention has the beneficial effects that: the wild alkaline protease from the Bacillus clausii has fast enzyme activity loss in the storage or use process due to autogenous cutting, and the mutant gene with excellent autogenous cutting resistance is obtained through gene mutation and screening, so that the application level of the alkaline protease is improved.

Drawings

FIG. 1 alkaline protease surface specific amino acid search

FIG. 2 alkaline protease surface A: 161(Y), B: 183(F)

FIG. 3 is a PCR amplification electrophoretogram of the wild-type alkaline protease gene of the present invention

Wherein: m is DNA Marker, 1 is aprE gene;

FIG. 4 is a diagram illustrating the restriction enzyme digestion of pWB980 of the present invention

Wherein: m is DNA Marker, 1 is pWB 980-aprE and is subjected to double enzyme digestion by BamHI and XbaI;

FIG. 5 enzyme activities of wild-type alkaline protease and alkaline protease mutant

FIG. 6 enzyme activity retention rates of wild-type alkaline protease and alkaline protease mutant

The specific implementation mode is as follows:

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.