CN113861266A

CN113861266A - Biosynthesis method of bacillus alkaline protease inhibitory peptide

Info

Publication number: CN113861266A
Application number: CN202111163155.3A
Authority: CN
Inventors: 路福平; 王洪彬; 李奕鸣; 邹晓桐
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-31

Abstract

The invention relates to a biosynthesis method of bacillus alkaline protease inhibitory peptide AAPF (alanine-proline-phenylalanine), which is characterized in that an AAPF multiple artificial sequence is artificially designed, and AAPF short peptide is prepared by microbial fermentation synthesis and chymotrypsin specific hydrolysis. Compared with the traditional chemical synthesis method, the AAPF short peptide biosynthesis method has the advantages of cheap and simple raw materials, easy realization of steps, environmental protection and the like, and is favorable for improving the application level of AAPF inhibitory peptides in the fields of bacillus alkaline protease liquid enzyme preparations and liquid enzyme-added detergents.

Description

Biosynthesis method of bacillus alkaline protease inhibitory peptide

Technical Field

The invention belongs to the field of enzyme engineering, and particularly relates to a biosynthesis method of bacillus alkaline protease inhibitory peptide.

Background

The alkaline protease is a hydrolase capable of degrading proteins under alkaline conditions, and is widely applied to industries such as food processing, detergents, leather, textiles and the like. The alkaline protease from bacillus and produced by fermentation has strong alkali resistance, heat resistance and hydrolysis activity, and is a main product in the protease preparation market. The bacillus alkaline protease has very strong dirt-removing capability, particularly for protein stains such as sweat stains, blood stains and the like, and therefore, the bacillus alkaline protease is also widely applied to the detergent industry, such as being added into liquid enzyme-added detergent to enhance the washing effect. The protease not only hydrolyzes other protein molecules, but also hydrolyzes self molecules to cause enzyme activity loss. Protease preparations are largely divided into solid preparations and liquid preparations. Since the autogenous inactivation of Bacillus alcalase in liquid preparations is an important factor affecting the stability of Bacillus alcalase, it is necessary to add a reversible inhibitor to the liquid preparations to improve the stability and prolong the storage time of Bacillus alcalase in liquid enzyme preparations and liquid detergents. Earlier researches find that carboxyl terminal aldehyde modified products of the short peptide AAPF (alanine-proline-phenylalanine) have strong inhibition effect on bacillus alkaline protease, and are considered to replace borax inhibitors and 4-formylphenylboronic acid (4-FPBA) inhibitors, so that the stability of the protease in liquid enzyme preparations and liquid enzyme-added detergents is improved. The commonly used method for synthesizing the short peptide is a chemical synthesis method, and has high requirements on raw materials, complicated steps and harsh conditions. Therefore, the invention explores and develops the biosynthesis method of the AAPF inhibitory peptide, has the advantage of environmental protection, and establishes a foundation for improving the production and application levels of the AAPF inhibitory peptide.

Disclosure of Invention

The invention aims to synthesize the short peptide AAPF by a biological method, and a peptide aldehyde inhibitor R-AAPF-H of the bacillus alkaline protease can be obtained by chemical modification on the basis (R represents that an amino terminal is modified by acylation, and H represents that a carboxyl terminal is reduced into aldehyde group).

The biosynthesis method of the inhibitory peptide AAPF comprises the following steps:

(1) an artificial amino acid sequence containing AAPF repetitive sequence is designed and named as AAPF multiple sequence, and is characterized in that a plurality of repeated AAPF sequences exist, when the number of repeated AAPF is more, and in order to reduce the whole hydrophobicity of the sequence, a hydrophilic amino acid sequence is inserted between partial AAPF repetitive sequences, thereby being beneficial to the normal expression of the artificial sequence in microorganisms.

(2) Entrusted gene synthesis company to synthesize the designed AAPF multiple sequence DNA sequence, and integrate it into the microbial expression plasmid, preferably select pET22b (+) or pPIC9K as expression plasmid;

(3) introducing the expression plasmid into engineering strain, fermenting and expressing AAPF multiple sequence polypeptide molecule, preferably selecting colibacillus or pichia pastoris as engineering strain.

(4) Adding chymotrypsin into the prepared AAPF polypeptide with multiple sequences for incubation and hydrolysis, obtaining AAPF short peptide by ultrafiltration, and confirming the synthesis of AAPF product by liquid chromatography mass spectrometry;

(5) entrusted to the polypeptide synthesis company, the N-terminal of the AAPF repeat polypeptide was chemically modified to add an acylated hydrophilic group and the C-terminal carboxyl group was modified to an aldehyde group.

The invention has the beneficial effects that:

compared with a chemical method, the biosynthesis method of the AAPF inhibitory peptide does not need high-purity amino acid and a catalyst as synthesis raw materials, has simple and easily realized steps and green and environment-friendly production process, and is expected to greatly reduce the production cost in the future, so that the application of the AAPF inhibitory peptide in a bacillus alkaline protease in a liquid enzyme preparation or a liquid enzyme-added detergent is promoted.

Drawings

FIG. 1, liquid chromatography mass spectrometry analysis of AAPF repeat sequence polypeptide chymotrypsin hydrolysate. The upper graph is a chromatogram, and the lower graph is a mass spectrum of AAPF.

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.

Example 1: design of AAPF multiple sequences

Two AAPF multiple sequences are respectively designed, each of which contains 10 AAPF sequences and 20 AAPF sequences and are named as 10-AAPF and 20-AAPF. The 10-AAPF is formed by directly connecting 10 AAPF sequences. 20-AAPF is characterized by that in addition to 20 AAPF sequences, hydrophilic sequences are inserted within a certain interval. 10-AAPF protein sequence:

AAPFAAPFAAPFAAPFAAPFAAPFAAPFAAPFAAPFAAPF20-AAPF protein sequence:

TDKDFAAPFAAPFAAPFAAPFAAPFSRKDTDFAAPFAAPFAAPFAAPFAAPFTRKDKFAAPFAAPFAAPFAAPFAAPFTDRDKFAAPFAAPFAAPFAAPFAAPF

example 2: biosynthesis and chemical modification of AAPF inhibitory peptides

1. The DNA sequences of 10-AAPF and 20-AAPF were synthesized by entrusted Gene Synthesis and inserted into pET22b (+) plasmid.

2. The constructed plasmid was transformed and introduced into E.coli BL21(DE3)

(1) Competent cells (100. mu.L) were removed from-80 ℃ and thawed in ice bath.

(2) Add 10. mu.L of ligation product or 2-5. mu.L of plasmid to be transformed to competent cells, mix gently, and stand in ice bath for 30 min.

(3) The mixture was heat-shocked in a water bath at 42 ℃ for 30S, and placed in an ice bath for 2 min.

(4) Adding 900 μ L LB culture medium preheated to 37 deg.C, shaking and culturing at 37 deg.C for 1 hr with shaking at 220 r/min.

(5) Centrifuging at 4000r/min for 5min to collect thallus, discarding part of supernatant, re-suspending thallus, taking appropriate amount of LB plate coated with Amp (ampicillin) resistance, and culturing the plate in an incubator at 37 ℃ overnight for 12-16 h.

3. Fermenting and inducing the strain after the plasmid is introduced

(1) Transferring the bacterial liquid into LB liquid culture medium containing Amp (ampicillin) resistance (the final concentration of Amp is 100 mug/mL), and culturing at 37 ℃ and 220r/min until the bacterial liquid OD600 is 0.6-0.8.

(2) Adding IPTG (final concentration of 0.5mmol/L), and inducing and culturing at 16 deg.C and 120r/min for 16-20 h.

4. Treatment of zymocyte liquid

Centrifuging the fermented bacteria liquid at 8000r/min for 30min, and collecting precipitate. And carrying out ultrasonic crushing treatment on the precipitate. The ultrasonication parameters are as follows: the crushing time is 3 s; the intermittent time is 4 s; the power is 300 w; the total time is 20 min; the temperature is 4 ℃; horn Φ 10.

5. Denaturation and renaturation of Inclusion bodies

The inclusion bodies were denatured by addition of 8M urea for about 12 h. Then removing urea by dialysis to obtain the polypeptide molecule with AAPF multiple sequences.

6. And incubating and hydrolyzing to obtain the AAPF short peptide.

Adding a proper amount of chymotrypsin into the AAPF repetitive sequence protein, hydrolyzing for 3h at 37 ℃, and ultrafiltering to obtain the AAPF short peptide.

7. And (3) carrying out mass spectrum detection on the hydrolyzed polypeptide.

Detecting hydrolysate by adopting an Agilent LC-ESI-Q-TOF-MS/MS liquid chromatography time-of-flight mass spectrometer, wherein the model of a chromatographic column is xbRIDGE peptide BEH C18(2.1mm x 150mm, 3.5 mu m), the mobile phase gradient elution conditions are shown in Table 1, and the parameters of a mass spectrometer include energy: 4500v, slope: 3.6, offset:4.8, Gas Temp (° c): 325, Gas flow (L/min): 13, flow rate: 0.25 ml/min.

The liquid chromatography mass spectrometry detection proves that the AAPF short peptide is successfully obtained, and the result is shown in the attached figure 1.

TABLE 1 gradient elution conditions for mobile phase of liquid chromatography mass spectrometry

8. The AAPF short peptide prepared by the peptide synthesis company was subjected to acylation modification at the N-terminus and aldehyde modification at the C-terminus.

Sequence listing

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Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe

1 5 10 15

Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe

20 25 30

Ala Ala Pro Phe Ala Ala Pro Phe

35 40

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Thr Asp Lys Asp Phe Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro

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Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe

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Ala Ala Pro Phe Thr Arg Lys Asp Lys Phe Ala Ala Pro Phe Ala Ala

50 55 60

Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe Thr Asp

65 70 75 80

Arg Asp Lys Phe Ala Ala Pro Phe Ala Ala Pro Phe Ala Ala Pro Phe

85 90 95

Ala Ala Pro Phe Ala Ala Pro Phe

100

Claims

1. A biosynthesis method of bacillus basic protease inhibitory peptide AAPF (alanine-proline-phenylalanine) is characterized by comprising the following steps:

(1) artificially designing an AAPF multiple artificial sequence, wherein the sequence contains 10-30 AAPF short sequences, and inserting a short sequence consisting of hydrophilic amino acids into partial intervals among repeated sequences of the AAPF multiple artificial sequence so as to reduce the hydrophobicity of the whole artificial sequence;

(2) constructing an expression plasmid and an engineering strain of the AAPF multiple artificial sequence, and fermenting and synthesizing a polypeptide molecule of the AAPF multiple artificial sequence;

(3) hydrolyzing chymotrypsin and performing ultrafiltration purification to prepare AAPF short peptide;

(4) and carrying out amino terminal acylation modification and carboxyl aldehyde group modification on the AAPF short peptide to obtain the bacillus basic protease inhibitory peptide.

2. The expression plasmid of claim 1, preferably the pET22b (+) or pPIC9k plasmid is selected.

3. The engineered strain of claim 1, preferably escherichia coli or pichia pastoris is selected as the engineered strain.