CN105316274A - Recombined bacillus subtilis with improved asparaginase secretion capacity and application thereof - Google Patents

Abstract

The invention discloses recombined bacillus subtilis with improved asparaginase secretion capacity and application thereof, and belongs to the field of enzyme engineering. On the basis of an efficient secretion signal peptide strain, through constructing a p43 strong promoter strain, asparaginase efficient expression in a food grade safe strain-bacillus subtilis is achieved, and a strain with the improved asparaginase secretion capacity is obtained. Compared with an original strain, the efficiency of L-asparaginase II extracellular secretion of recombined bacillus subtilis is improved by 1.4 times.

Description

The recombined bacillus subtilis that a kind of asparaginase secretion capacity improves and application thereof

Technical field

The present invention relates to recombined bacillus subtilis and the application thereof of the raising of a kind of asparaginase secretion capacity, belong to enzyme engineering field.

Background technology

L-ASP (EC3.5.1.1) is a kind of proteolytic enzyme having antitumour activity, can be hydrolyzed into aspartic acid and NH3 by single-minded catalysis altheine.The physiological action main manifestations of L-ASP is the restraining effect to some tumour, especially to acute leukemia and malignant lymphoma effective.L-ASP has become the very effective medicine for the treatment of leukemia, does not have restraining effect to medullary cell.

L-ASP has two types, L-ASP I and L-ASP II, Escherichiacoli, Erwiniachrysanthemi, B.subtilis etc. all comprise this two kinds of L-ASPs, research confirms that only L-ASP II has antitumous effect, be developed to the active drug for the treatment of acute lymphoblastic leukemia from Escherichiacoli and ErwiniachrysanthemiL-asparaginase II, the major part of research is at present the L-ASP II with antitumous effect.

L-ASP can reduce the generation of acrylamide in food.Acrylamide is mainly generated by Maillard reaction in high-temperature heating process by the reducing sugar in food raw material and l-asparagine, and in food, add asparaginase can be hydrolyzed l-asparagine, reduces the generation of acrylamide from source.Some microorganisms, Mammals and plant are proved containing L-ASP.Because L-ASP content is low in animal serum, and extraction process is complicated, microorganism has easy cultivation, low cost and other advantages, become the emphasis of scholar's research, the product L-ASP microorganism of current research mainly comprises Escherichiacoli, Erwiniacarotovora, Erwiniachrysanthemi etc., but wild strain L-ASP yields poorly, utilize genetic engineering technique in recent years by L-ASP gene clone in intestinal bacteria, obtain the high expression of L-ASP, utilizing works bacterium produces L-ASP has become an important sources.

How in food safety bacterial strain, to express L-ASP and improve L-ASP secretion capacity, be current problem demanding prompt solution.

Summary of the invention

In order to overcome the problems referred to above, based on efficient secretion signal peptide bacterial strain, by building p43 strong promoter bacterial strain, realizing the high expression of asparaginase in grade-safe bacterial strain-subtilis, obtaining the bacterial strain that asparaginase secretion capacity improves.

First object of the present invention is to provide the recombined bacillus subtilis that a kind of asparaginase secretion capacity improves, and described recombined bacillus subtilis for carrier, expresses the L-ASP gene containing WapA signal peptide with pP43NMK plasmid; The aminoacid sequence of the described L-ASP gene containing WapA signal peptide is SEQIDNO.1.

In one embodiment of the invention, the nucleotide sequence of the described L-ASP gene containing WapA signal peptide is SEQIDNO.2.

In one embodiment of the invention, the described L-ASP gene containing WapA signal peptide is the KpnI being connected to pP43NMK plasmid, PstI restriction enzyme site, is then transformed in subtilis WB600 and expresses.

In one embodiment of the invention, described asparaginase is L-ASP II.

The present invention also provides a kind of method utilizing described recombined bacillus subtilis fermentative production asparaginase.

Described method be by recombined bacillus subtilis seed liquor according to 4% inoculum size transfer in fermention medium, cultivate 24h for 37 DEG C, then fermented liquid is centrifugal, and get supernatant and be the outer crude enzyme liquid of born of the same parents, cytoclasis supernatant liquor is crude enzyme liquid in born of the same parents.

The present invention's asparaginase that also claimed described recombined bacillus subtilis and its production obtain is preparing the application in medicine.

Beneficial effect of the present invention:

The present invention is by building p43 strong promoter bacterial strain, realize the high expression of L-ASP II in grade-safe bacterial strain-subtilis, improve the secretion capacity of L-ASP II, compared with starting strain, the efficiency of recombinant bacterium exocytosis L-ASP II of the present invention improves 1.4 times.

Accompanying drawing explanation

Fig. 1: different strains fermentation asparaginase enzyme is lived;

Fig. 2: WB43H fed-batch fermentation.

Embodiment

Substratum:

LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.0;

Fermention medium: soy peptone 10g/L, corn steep liquor 5g/L, urea 1g/L, sucrose 35g/L, dipotassium hydrogen phosphate 2.3g/L, potassium primary phosphate 1.7g/L, magnesium sulfate 0.75g/L, sodium-chlor 5g/L; Regulate pH6.8-7.0.

The mensuration of asparaginase enzyme activity:

Spectrophotometry asparaginase enzyme is adopted to live.1 unit asparaginase enzyme is lived and is defined as: enzyme is lived and defined: under 37 DEG C of reaction conditionss, can the catalysis altheine enzyme amount discharged required for 1 μm of olNH3 be a Ge Meihuo unit (U/ml) in per minute.Enzyme activity determination condition: under 37 DEG C of conditions, 1ml10mMK2HPO4-KH2PO4 (PH7.5), 0.1ml189mM l-asparagine, 0.1ml fermented supernatant fluid, is incubated 30 minutes, 0.5ml1.5MTCA termination reaction.Utilize ShimadzuUV-1240 to measure light absorption value at 436nm place, by ammonium sulfate drawing standard curve, calculate enzyme according to typical curve and live.

Embodiment 1: the structure of recombinant bacterium

Upstream and downstream primer P1, P2 (as shown in table 1) of design L-ASP, the pMA0911-wapA-SP-ansZ built with early stage, for template, to be increased the L-ASP gene containing WapA signal peptide by the mode of PCR.PCR condition is: 98 DEG C of 3min, 98 DEG C of 30S, 55 DEG C of 90S, 72 DEG C of 90S, 34 circulations.PCR amplification system: template 1 μ L, upstream and downstream primer each 1 μ L, dNTPMix4 μ L, 5 × primeSTARBuffer10 μ L, the distilled water 32.5 μ L of sterilizing, primeSTARDNA polysaccharase 0.5 μ L.Adopt glue to reclaim test kit and carry out purifying and recovery to PCR primer, the concentration of product is reclaimed in electrophoresis inspection.KpnI, PstI enzyme is cut glue and is reclaimed product (goal gene L-ASP) and pP43NMK plasmid (expression vector), post reclaim test kit enzyme is cut after glue reclaim product carry out purifying and recovery, glue recovery test kit is cut rear plasmid to enzyme and is carried out purifying recovery, and the concentration of product is reclaimed in electrophoresis inspection.Goal gene L-ASP is connected with carrier pP43NMK, linked system: goal gene 4 μ L, carrier pP43NMK1 μ L, solutionI5 μ L, 16 DEG C of connections of spending the night.The recombinant plasmid pP43H connected is transformed into competence E.coilJM109, with ampicillin/LB plates, the positive bacterium colony of picking.37 DEG C of incubator overnight extract plasmid, called after pP43H after cultivating, and after digestion verification is correct, transformant is checked order by the raw work in Shanghai.Accordingly, order-checking recombinant plasmid pP43H is proceeded to subtilis WB600, again to obtain WB43H.

In addition, contriver also adopts similar method with the expression in WB600 of the L-ASP gene of the strong promoters such as PyxiE, PlapS, Pglv (being plasmid pPyxiEH, pPlapSH and pPglvH respectively) mediation containing WapA signal peptide, constructs recombinant bacterium WByxiEH, WBPlapSH and WBPglvH etc.

Table 1 primer

Embodiment 2: recombinant bacterium fermentative production L-ASP

Recombinant bacterium WB43H, WByxiEH, WBPlapSH and WBPglvH etc. that embodiment 1 is built, (take pMA0911 as carrier with starting strain pMA0911-wapA-SP-ansZ/B.subtilisWB600, HpaII is promotor) be inoculated in 10mL respectively and support in base containing the LB of kantlex, 37 DEG C of shaking culture are spent the night, next day by 4% inoculum size transfer in fermentation of bacillus subtilis substratum, cultivate 24h for 37 DEG C, get fermented liquid in 4 DEG C, the centrifugal 10min of 10000r/min, supernatant is the outer crude enzyme liquids of born of the same parents, for the mensuration of enzyme activity.

Fermention medium: soy peptone 10g/L, corn steep liquor 5g/L, urea 1g/L, sucrose 35g/L, dipotassium hydrogen phosphate 2.3g/L, potassium primary phosphate 1.7g/L, magnesium sulfate 0.75g/L, sodium-chlor 5g/L; Regulate pH6.8-7.0.

L-asparagine enzyme activity is measured, result shows: compare with starting strain, the extracellular enzyme vigor of the L-ASP of mutant strain WB43H improves 40%, reach 39.52U/ml, and the extracellular enzyme vigor of the L-ASP of mutant strain WByxiEH, WBPlapSH and WBPglvH only improves 7%, 5% and 12%.

On 3L fermentor tank, be fermentation strain with WB43H, by fed-batch fermentation, fermentation 40h enzymatic activities is up to 109.15U/ml (Fig. 2).

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 (9)

1. a recombined bacillus subtilis for asparaginase secretion capacity raising, is characterized in that, described recombined bacillus subtilis for carrier, expresses the L-ASP gene containing WapA signal peptide with pP43NMK plasmid; The aminoacid sequence of the described L-ASP gene containing WapA signal peptide is SEQIDNO.1.

2. recombined bacillus subtilis according to claim 1, is characterized in that, the nucleotide sequence of the described L-ASP gene containing WapA signal peptide is SEQIDNO.2.

3. recombined bacillus subtilis according to claim 1, it is characterized in that, the described L-ASP gene containing WapA signal peptide is the KpnI being connected to pP43NMK plasmid, PstI restriction enzyme site, is then transformed in subtilis WB600 and expresses.

4. recombined bacillus subtilis according to claim 1, is characterized in that, described asparaginase is L-ASP II.

5. one kind utilizes the method for the recombined bacillus subtilis fermentative production asparaginase described in claim 1.

6. method according to claim 5, it is characterized in that, described method be by recombined bacillus subtilis seed liquor according to 4% inoculum size transfer in fermention medium, cultivate 24h for 37 DEG C, then fermented liquid is centrifugal, get supernatant and be the outer crude enzyme liquid of born of the same parents, cytoclasis supernatant liquor is crude enzyme liquid in born of the same parents.

7. method according to claim 6, is characterized in that, described fermention medium contains: soy peptone 10g/L, corn steep liquor 5g/L, urea 1g/L, sucrose 35g/L, dipotassium hydrogen phosphate 2.3g/L, potassium primary phosphate 1.7g/L, magnesium sulfate 0.75g/L, sodium-chlor 5g/L.

8. the asparaginase that method described in claim 5-7 obtains is preparing the application in medicine.

9. recombined bacillus subtilis according to claim 1 is preparing the application in medicine.