CN105018407A

CN105018407A - Bacillus subtilis of secretory expression proline aminopeptidase and application thereof

Info

Publication number: CN105018407A
Application number: CN201510497845.0A
Authority: CN
Inventors: 田亚平; 汪克红
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-08-13
Filing date: 2015-08-13
Publication date: 2015-11-04

Abstract

The invention discloses bacillus subtilis of secretory expression proline aminopeptidase and application thereof and belongs to the technical field of biological enzyme engineering. PMA5 serves as an expression vector of genes of coding proline aminopeptidase, the bacillus subtilis WB600 is imported, recombinant bacteria are obtained, the secretory expression is achieved, and the extracellular enzyme activity reaches 36.0 U/mL. The bacillus subtilis and the application thereof lay a good foundation for the industrial production of the proline aminopeptidase and application in the food industry.

Description

A kind of subtilis of secreting, expressing proline aminopeptidase and application thereof

Technical field

The present invention relates to a kind of subtilis and application thereof of secreting, expressing proline aminopeptidase, belong to bio-enzyme engineering technical field.

Background technology

Aminopeptidase (aminopeptidases, be called for short Aps, EC3.4.11) be a class from the exopeptidase of albumen or polypeptide N section hydrolysis amino acid one by one, at proteolysate debitterize, the N end order-checking of proteins and peptides, protein raw materials deep processing and biologically active peptides such as to prepare at the aspect important effect.Proline aminopeptidase (prolyl aminopeptidase, be called for short PAP, EC3.4.11.5) be the aminopeptidase with strict substrate specificity, the proline residue of hydrolyzed peptide or n-end of albumen specifically, in the removal of protein food hydrolysis processed products bitter taste, there is good application prospect the aspects such as collagen hydrolysate.

Mainly concentrate on gene clone, expression, purifying and characteristic present to the research of aminopeptidase at present, intestinal bacteria because culture condition is simple, growth and breeding is fast and become the most widely used expression system, but because it is pathogenic, more easily form inclusion body and lower secernment efficiency and limit its application in the food industry; Pichia spp can be modified heterologous protein as the simple single celled eukaryotic expression system of one and be secreted in substratum, therefore also have and to carry out expressing in pichia spp, but due to pichia spp need methanol induction and culture cycle long and its application is in the food industry restricted.

The expression of current proline aminopeptidase, mostly from wild mushroom, mainly concentrates on its character research and expression level is low, and most of proline aminopeptidase is all intracellular enzyme.So far, yet there are no the report that proline aminopeptidase is expressed in subtilis, the present invention is conceived to this and utilizes subtilis expression system to carry out high expression to proline aminopeptidase for low, that exocytosis ability the is weak shortcoming of current proline aminopeptidase expression efficiency, and makes it more be secreted into outside born of the same parents by suitable secretion strategy.

Summary of the invention

The present invention provide firstly a kind of recombined bacillus subtilis of secreting, expressing proline aminopeptidase, and described recombined bacillus subtilis is expression vector by the gene of coding proline aminopeptidase with pMA5, imports subtilis WB600 and the recombinant bacterium that obtains.

The nucleotide sequence of the gene of described coding proline aminopeptidase is as shown in SEQ ID NO.1.

Present invention also offers a kind of method building the recombined bacillus subtilis of described secreting, expressing proline aminopeptidase, that goal gene is connected with expression vector pMA5 by BamH I, Mlu I restriction enzyme site, be converted in subtilis WB600, screening obtains positive transformant.

Present invention also offers a kind of method applying described recombined bacillus subtilis production proline aminopeptidase, after recombined bacillus subtilis is activated, inoculum size by 1% be inoculated in secretion substratum in 37 DEG C, 220rpm cultivates 24h, and proline aminopeptidase is secreted into outside born of the same parents.Described secretion substratum with the addition of 5% sorbyl alcohol and 2mM CaCl ₂tB substratum.

Present invention achieves the secreting, expressing of proline aminopeptidase, fermentation 24h enzymatic activities reaches 36.0U/mL.

Accompanying drawing explanation

Fig. 1 proline aminopeptidase gene amplification result (M:2000DNA Marker, 1,2: goal gene PCR)

The single, double digestion verification of Fig. 2 recombinant plasmid (M1:10000DNA Marker, 1: recombinant plasmid Mlu I single endonuclease digestion, 2: recombinant plasmid double digestion, 3:PCR product, M2:2000DNA Marker)

The single, double enzyme of Fig. 3 subtilis recombinant plasmid is cut and is tested (M1:10000DNA Marker, 1,2 recombinant plasmid BamHI single endonuclease digestions, 3,4: recombinant plasmid double digestion, 5:PCR product)

Fig. 4 recombined bacillus subtilis expresses SDS-PAGE result, and ((a): WB600 (pMA5) contrasts, (b): WB600 (pMA5-pap) recombinant bacterium, M: low molecular weight protein (LMWP) standard, the full cell of 1:WB600 (pMA5), 2:WB600 (pMA5) fermented supernatant fluid, supernatant after 3:WB600 (pMA5) is broken; The full cell of 4:WB600 (pMA5-pap), 5:WB600 (pMA5-pap) fermented supernatant fluid, supernatant after 6:WB600 (pMA5-pap) is broken)

Fig. 5 secreting, expressing SDS-PAGE result (M: low molecular weight protein (LMWP) standard, 1:TB fermented supernatant fluid, 2:STB fermented supernatant fluid)

Fig. 6 second time Hitrap Q HP ion exchange chromatography

Fig. 7 recombinates proline aminopeptidase optimal pH

Fig. 8 recombinates proline aminopeptidase pH stability

Fig. 9 recombinates proline aminopeptidase optimum temperuture

Figure 10 recombinates proline aminopeptidase temperature stability

Figure 11 recombinates proline aminopeptidase kinetic parameter

Figure 12 recombinates proline aminopeptidase salt tolerance

Embodiment

Material and detection method

LB substratum: 1% Tryptones, 0.5% yeast extract, 1% sodium-chlor, pH7.0.

TB substratum: 1.2% Tryptones, 2.4% yeast powder, 72mM K ₂hPO ₄, 17mM KH ₂pO ₄, 0.4% glycerine.

Secretion substratum (STB): 1.2% Tryptones, 2.4% yeast powder, 5% sorbyl alcohol, 72mM K ₂hPO ₄, 17mMKH ₂pO ₄, 0.4% glycerine, 2mM CaCl ₂.

PMA5, PrimeSTARMax DNA polymerase, restriction enzyme BamH I and Mlu I, T4 ligase enzyme are all purchased from precious biotechnology (Dalian) company limited.

Proline aminopeptidase enzyme activity determination method: (substrate stock solution Tris-HCl 7.5 prepares for substrate with L-PROLINE-p-Nitroaniline, concentration is 4.25mM), reaction mixture comprises the enzyme liquid after 1mL dilution, 2mL Tris-HCl 7.5 damping fluid and 1mL substrate stock solution, 50 DEG C of water-bath 10min, measure light absorption value at 405nm place.One Ge Meihuo unit (U) is defined as 50 DEG C of per minutes and decomposes L-PROLINE-p-Nitroaniline and produce enzyme amount needed for 1 μM of p-Nitroaniline.

The construction process of embodiment 1 proline aminopeptidase subtilis

Primer is designed: upstream primer P1:5'CG according to proline aminopeptidase cDNA sequence (SEQ ID NO.1) gGATCCaTGGCTGCCAAAC 3'(BamH I); Downstream primer P2:5'CGCG aCGCGTcTAATCAATAGAGTC 3'(Mlu I).Increase the goal gene obtained with BamH I, Mlu I restriction enzyme site, goal gene and plasmid pMA5 BamH I and Mlu I are carried out double digestion, after glue reclaims purifying, 16 DEG C of connections are spent the night, and also coating is dull and stereotyped containing the LB of ammonia benzyl for transformation of E. coli JM109, bacterium colony PCR checking is carried out after growing transformant, extract the checking of plasmid double digestion after being cultivated by positive transformant, verify that correct recombinant plasmid pMA5-pap serves Hai Shenggong order-checking.

Be converted in subtilis WB600 by the recombinant plasmid pMA5-pap electricity built, kalamycin resistance flat board screens positive recombinant, and concrete steps are as follows:

(1) fresh plate chooses single colony inoculation in 3mL LB substratum, incubated overnight;

(2) get in 2.6mL overnight culture access GM substratum, 37 DEG C, 200rpm is cultured to OD ₆₀₀=0.85 ~ 0.95 (about 3 ~ 4h);

(3) by bacterium liquid ice-water bath 10min, in 4 DEG C, the centrifugal 5min of 5000g collects thalline;

(4) turn substratum ETM with the electricity of 50mL precooling and again blow outstanding thalline, 5000g, 5min, 4 DEG C centrifugal removes supernatant, rinsing like this 4 times;

(5) thalline after washing is blown be suspended from 1mL electricity and turn in substratum ETM, often pipe packing 60 μ L;

(6) 60 μ L competent cells add 1 μ L (50ng/ μ L) DNA, hatch 2min on ice, add in the electric revolving cup (1mm) of precooling, and electric shock once (12.5kv/cm, 25 μ F, 200 Ω, 4.5ms ~ 5.0ms);

(7) the complete taking-up cup that shocks by electricity also adds 1mL RM, 37 DEG C, 200rpm immediately, is coated with on kalamycin resistance LB flat board, 37 DEG C of incubated overnight after recovery 3h.

(8) picking transformant PCR fast verification, what the checking of extraction plasmid double digestion was correct is recombined bacillus subtilis.

Embodiment 2 recombined bacillus subtilis secretion expresses the method for proline aminopeptidase

Recombined bacillus subtilis list bacterium colony on picking LB flat board (containing 50 μ g/mL kantlex) in 50mL LB liquid nutrient medium (containing 50 μ g/mL kantlex) 37 DEG C, 220r cultivates 14h, to be inoculated in 50mL STB substratum (containing 50 μ g/mL kantlex) 37 DEG C by the inoculum size of 1% again, 220r cultivates 24h.After fermentation ends, 8000r, 4 DEG C of centrifugal 10min, gained fermented supernatant fluid is proline aminopeptidase crude enzyme liquid, records enzyme and lives as 36.0U/mL.

The characteristic present of proline aminopeptidase secreted by embodiment 3 recombined bacillus subtilis

In embodiment 2, gained crude enzyme liquid obtains and substantially reaches electrophoretically pure proline aminopeptidase after twice Hitrap Q HP ion exchange chromatography, and it is characterized, result shows: optimal reactive temperature is 50 DEG C, at 50 DEG C and show good temperature stability below; Optimal reaction pH is 7.5, has good pH stability between pH6-11; Strict substrate specificity is had to proline-para-nitroanilide; Michaelis-Menton constant Km and maximum speed of reaction Vmax is respectively 0.171mmol L ^-1with 55.99 μm of ol min ^-1.Basically identical with proline aminopeptidase characteristic in the born of the same parents of expression of recombinant e. coli, illustrate and change is not caused to the structure and characteristics of enzyme carry out secreting, expressing in subtilis after.Also the salt tolerance of restructuring proline aminopeptidase is characterized in addition, find that the kind of its salt tolerance and salt has nothing to do, the vigor under high salt concn not affecting this enzyme on the contrary enzyme activity increases, when NaCl concentration reaches 4.36M, residual enzyme is lived and is also had 109.95%, illustrates that this enzyme can tolerate very high salt concn.

Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims

1. a recombined bacillus subtilis for secreting, expressing proline aminopeptidase, is characterized in that, is be expression vector by the gene of coding proline aminopeptidase with pMA5, imports subtilis WB600 and the recombinant bacterium that obtains; The nucleotide sequence of the gene of described coding proline aminopeptidase is as shown in SEQ ID NO.1.

2. build a method for recombined bacillus subtilis described in claim 1, it is characterized in that, be connected with expression vector pMA5 by BamH I, Mlu I restriction enzyme site by goal gene, be converted in subtilis WB600, screening obtains positive transformant.

3. application rights requires that described in 1, recombined bacillus subtilis produces a method for proline aminopeptidase, is characterized in that, is, after being activated by recombined bacillus subtilis, to be inoculated in and additionally to the addition of 5% sorbyl alcohol and 2mM CaCl ₂tB substratum in carry out fermentation culture.

4. method according to claim 3, is characterized in that, is that after being activated by recombined bacillus subtilis, the inoculum size by 1% is inoculated in and additionally with the addition of 5% sorbyl alcohol and 2mM CaCl ₂tB substratum in, in 37 DEG C, 220rpm cultivates 24h; Collect from fermented supernatant fluid and obtain proline aminopeptidase.