CN102864163A - Method for efficiently expressing bacillus subtilis L-asparaginase - Google Patents
Method for efficiently expressing bacillus subtilis L-asparaginase Download PDFInfo
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- CN102864163A CN102864163A CN2012103605543A CN201210360554A CN102864163A CN 102864163 A CN102864163 A CN 102864163A CN 2012103605543 A CN2012103605543 A CN 2012103605543A CN 201210360554 A CN201210360554 A CN 201210360554A CN 102864163 A CN102864163 A CN 102864163A
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Abstract
A method for efficiently expressing bacillus subtilis L-asparaginase belongs to the field of enzyme gene engineering and enzyme engineering. Bacillus subtilis (6-7) total DNA (deoxyribonucleic acid) is used as a template to obtain coding gene ansZ of L-asparaginase by PCR (polymerase chain reaction) amplification, the coding gene is connected to an expression vector pMA5, and L-asparaginase genetically engineered microorganism pMA5-ansZ and bacillus subtilis 168 is built by using bacillus subtilis 168 as an expression host. Therefore, the L-asparaginase is expressed efficiently. The engineered microorganism is 22.4U/mL in enzyme activity, which is 574 times higher than that of an original strain.
Description
Technical field
A kind of method of high efficient expression subtilis L-ASP the invention belongs to enzyme genetically engineered and enzyme engineering field.Relate in particular to a kind of L-ASP genetic engineering bacterium and construction process thereof.
Technical background
L-ASP (EC 3.5.1.1) is a kind of proteolytic enzyme that antitumour activity is arranged, and can be hydrolyzed into aspartic acid and NH by single-minded catalysis altheine
3The physiological action main manifestations of L-ASP is the restraining effect to some tumour, and is especially effective to acute leukemia and malignant lymphoma.L-ASP has become the very effective medicine for the treatment of leukemia, and marrow is not had restraining effect.
L-ASP can reduce the generation of acrylamide in the food.Acrylamide mainly is to be generated by Maillard reaction in high-temperature heating process by the reducing sugar in the food raw material and l-asparagine, adds asparaginase and can be hydrolyzed l-asparagine in food, reduces the generation of acrylamide from the source.
Observe guinea pig serum from nineteen fifty-three Kidd the effect that destroys the Gardner cancer cells is arranged, Broom in 1961 confirms that the anti-tumor factor in the guinea pig serum is L-ASP, Mashburn in 1964 etc. from Escherichia coli Partial purification L-ASP, and confirm that this kind of enzyme also has the effect of anti-tumor factor, Amrein in 2004 etc. propose to replace additive method to reduce acrylamide content in the food with L-ASP, Hendriksen in 2009 etc. studies have shown that adding L-ASP when pyroprocessing food can not produce harm, has numerous scholars can reduce chrisps by the concrete L-ASP that experimental results show that simultaneously, potato flour, the content of acrylamide in the foods such as dough.These researchs have proved that not only L-ASP has anticancer effect, also are simultaneously clinical study and a large amount of condition of having created of producing.
Some microorganisms, Mammals and plant are proved and contain L-ASP.Because L-ASP content is low in the 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 research mainly comprises Escherichia coli at present, Erwinia carotovora, Erwinia chrysanthemi etc., but the wild strain L-ASP yields poorly, utilize in recent years genetic engineering technique with the L-ASP gene clone in intestinal bacteria, obtain the high efficient expression of L-ASP, utilizing engineering bacteria to produce L-ASP has become an important sources.
L-ASP has two types, L-ASP I and L-ASP II, Escherichia coli, Erwinia chrysanthemi, B.subtilis etc. all comprise this two kinds of L-ASPs, studies confirm that only L-ASP II has antitumous effect, be developed to the active drug for the treatment of acute lymphoblastic leukemia from the L-ASP II of Escherichia coli and Erwinia chrysanthemi, the major part of research is the L-ASP II with antitumous effect at present.
The present invention has realized the high efficient expression of B.subtilis 6-7L-asparaginase II (being called for short ansZ) by expression vector pMA5.Subtilis is trained as the industrial producing strain of safety and stability. and the condition of supporting is simple, and fermentation period is short, becomes the potential bacterial strain of Kinds of Microbes Producing L-asparaginase.
Summary of the invention
Bacterial classification used herein is B.subtilis 6-7, and this bacterial strain is used for the synthetic 2,3-butanediol of glucose fermentation in earlier stage, has been preserved in Chinese Typical Representative culture collection center, and deposit number is: CTCCM 2009200.
Main research of the present invention: the present invention utilizes molecular engineering to clone L-ASP gene from B.subtilis 6-7 (being called for short ansZ), make up recombinant expression vector pMA5-ansZ, and with its conversion B.subtilis168, successfully made up genetic engineering bacterium pMA5-ansZ/B.Subtilis168.The genetic engineering bacterium that makes up is carried out enzyme activity determination, find the enzyme 22.4U/mL of being alive of recombinant bacterium, the work of starting strain enzyme only is 0.039U/mL, and the recombinant bacterium enzyme bacterium that sets out alive has been improved 574 times.
The technical scheme that the present invention takes:
A kind of L-ASP genetic engineering bacterium is that starting strain B.subtilis 6-7L-asparaginase gene is connected on the expression vector pMA5, and transforms the engineering strain that B.subtilis168 obtains.
The recombinant bacterial strain construction process
(1) L-ASP design of primers
According to ansZ gene order in the full genomic nucleic acid sequence of NCBI subtilis, PCR primer P1 and the P2 of design L-ASP gene.
P1：5’-GAC?
GGA?TCC?ATG?AAA?AAA?CAA?CGA?ATG?CT-3’(BamH?I)
P2：5’-GGC?
ACG?CGT?TTAATA?CTC?ATT?GAAATAAG-3’(Mlu?I)
(2) structure of recombinant expression vector pMA5-ansZ
Reclaim the test kit specification sheets with reference to vast Imtech glue and reclaim the PCR product, glue recovery product spends the night with pMD18-T vector by a certain percentage and is connected, Transformed E .coli JM109 competent cell, use amicillin resistance plate screening recombinant bacterium, recombinant plasmid is cut through BamHI/Mlu I enzyme and is discharged the gene band that size is 2.7kb and 1.1kb, show the construction of recombinant plasmid success, recombinant plasmid called after pMD 18-T-ansZ.
Extraction is stored in plasmid pMD18-T-ansZ and the pMA5 among the E.coli JM109, plasmid pMD18-T-ansZ and pMA5 are through BamHI/Mlu I double digestion, glue reclaims purifying, 16 ℃ of connections of spending the night, with connector thermal shock Transformed E .coli JM109 competent cell, with kalamycin resistance plate screening positive transformant, extract the transformant plasmid, recombinant plasmid discharges size and is the gene fragment of 7.2kb and 1.1kb behind BamH I/Mlu I double digestion, the success of proof construction of recombinant plasmid, recombinant plasmid called after pMA5-ansZ.
(3) recombinant plasmid pMA5-ansZ transformation mode bacterial strain B.subtilis168
The recombinant plasmid pMA5-ansZ that empirical tests is successfully constructed is converted among the type strain B.subtilis 168 with chemical transformation.Method for transformation adopts improved Spizizen method.
(4) screening of recombinant bacterial strain B.subtilis168 positive transformant
Picking is at the bacterium colony that has kantlex microbiotic pressure flat and grow, and shake flask fermentation extracts plasmid and carries out enzyme and cut checking.
(5) restructuring B.subtilis168 enzyme activity determination
Enzyme activity determination method: adopt ammonia gas-sensing electrode to measure recombinant bacterium L-ASP enzyme and live.
Advantage of the present invention and positively effect are:
(1) the present invention has explored the expression of L-ASP gene in B.subtilis168 in subtilis source, made up the engineering strain of high yield L-ASP, be applicable to industrialized production and application, have certain theory value and using value.
(2) but the present invention explores the operand of L-ASP construction of genetic engineering, constructed engineering bacteria adopts the fermentation of bacillus subtilis substratum, the enzyme work that fermentation produces reaches 22.4U/mL, and is higher 574 times than starting strain.
Description of drawings
Fig. 1 Fig. 1 recombinant plasmid pMA5-ansZ schematic diagram.
The enzyme of Fig. 2 plasmid pMA5-ansZ is cut checking.
1：pMA5-ansZ/BamH?I；2：pMA5-ansZ/BamH?I+Mlu?I；3：DNA?Marker：DL?2000；4：DNA?Marker：λ-Hind?III
Embodiment
In conjunction with the embodiments, the present invention is further described.
Embodiment 1:L-asparaginase design of primers
According to ansZ gene order in the full genomic nucleic acid sequence of NCBI subtilis, PCR primer P1 and the P2 of design L-ASP gene.
P1：5’-GAC
GGA?TCC?ATG?AAA?AAA?CAA?CGA?ATGCT-3’(BamH?I)
P2：5’-GGC?
ACG?CGT?TTA?ATA?CTC?ATT?GAA?ATAAG-3’(Mlu?I)
Embodiment 2:L-asparaginase gene cloning
Take the total DNA of B.subtilis 6-7 as template, the primer that utilization provides is above done pcr amplification, and amplification condition is: 94 ℃ of denaturations, 5min, a circulation; 94 ℃ of sex change, 1min, 56 ℃ of annealing, 1min, 72 ℃ of extensions, 90s, 30 circulations; 72 ℃, 10min, a circulation; 15 ℃, 10min, a circulation.Pcr amplification system: template 2 μ L, each 0.5 μ L of upstream and downstream primer, dNTP Mix 4 μ L, 10 * Ex Taq Buffer, 5 μ L, the distilled water 37 μ L of sterilization, Ex Taq archaeal dna polymerase 1 μ L.Adopt gel to reclaim test kit the PCR product is carried out purifying and recovery, the concentration of product is reclaimed in the electrophoresis check.Reclaim product and leave in the centrifuge tube of 1.5mL ,-20 ℃ of Refrigerator stores are for subsequent use.Reclaiming product is connected with pMD18-T Vector, connect product Transformed E .coil JM109, the converted product coating contains the LB flat board of penbritin, through 37 ℃ of overnight incubation, picking colony extracts plasmid after 37 ℃ of shaking table incubated overnight in 10mL liquid LB substratum, called after pMD18-T-ansZ, after enzyme is cut the checking successful connection, add glycerine to final concentration 15%～20% (w/v) ,-70 ℃ of refrigerator preservations.
Embodiment 3: the structure of recombinant plasmid pMA5-ansZ
Extraction is stored in plasmid pMD18-T-ansZ and the pMA5 among the E.coli JM109, and carry out double digestion with BamH I and Mlu I respectively, utilizing gel to reclaim connects after test kit reclaims, linked system: the goal gene enzyme is cut product 7 μ L, the pMA5 enzyme is cut product 1 μ L, T4DNA ligase enzyme buffer 1 μ L, T4 dna ligase 1 μ L, 16 ℃ of connections of spending the night.The recombinant plasmid pMA5-ansZ that connects is transformed into competence E.coil JM109, dull and stereotyped with penbritin LB, the positive bacterium colony of picking.Extract plasmid after 37 ℃ of shaking table incubated overnight, after called after pMA5-ansZ, enzyme cut checking correctly, add glycerine to final concentration 15%～20% (w/v) ,-70 ℃ of refrigerator preservations are for subsequent use.
Embodiment 4: recombinant plasmid pMA5-ansZ transforms B.subtilis168
Picking B.subtilis168 is inoculated in a 5mL LB liquid nutrient medium test tube, and 37 ℃ of shaking table overnight incubation are got the bacterium liquid of 100 μ L incubated overnight, is seeded to among the 5mL SPI Medium, and 37 ℃ of shaking tables are cultivated, and begin to survey OD behind the 5h
600, when culture grows into logarithm during latter stage, fast fetching 200 μ L are inoculated among the 2mL SPII Medium, and 37 ℃ of 100r/min shaking tables are cultivated 1.5h.Add 20 μ L, 100 * EGTA solution, cultivate 10min in 37 ℃ of 100r/min shaking tables, be distributed into the every pipe of 500 μ L with the 1.5mL centrifuge tube, Xiang Guanzhong adds an amount of plasmid pMA5-ansZ, and mixing is cultivated 30min in 37 ℃ of 100r/min shaking tables gently.Centrifuge tube is transferred to the 250r/min shaking table, 37 ℃ of foster 1.5h, the centrifugal collection thalline of 4000r/min, abandon the part supernatant liquor, stay the resuspended thalline of 100 μ L, coat the kalamycin resistance flat board, 37 ℃ of incubated overnight, the positive bacterium colony of picking extracts the plasmid enzyme restriction checking, obtains recombinant bacterium pMA5-ansZ/B.subtilis168.
Embodiment 5: recombinant bacterium pMA5-ansZ/B.subtilis168L-l-asparagine enzyme activity determination
(1) the recombinant bacterium pMA5-ansZ/B.subtilis168 that embodiment 4 is made up, being inoculated in respectively the LB that 10mL contains kantlex with starting strain B.subtilis 6-7 supports in the base, 37 ℃ of shaking culture are spent the night, transfer in fermentation of bacillus subtilis substratum in by 4% inoculum size next day, cultivates 24h, get fermented liquid in 4 ℃ for 37 ℃, the centrifugal 10min of 10000r/min, supernatant is the outer crude enzyme liquids of born of the same parents, and the cytoclasis supernatant liquor is crude enzyme liquid in the born of the same parents, is used for the mensuration of enzyme activity.
Fermentation of bacillus subtilis substratum: soy peptone 10g/L, corn steep liquor 15g/L, urea 3g/L, glucose 40g/L, dipotassium hydrogen phosphate 2.3g/L, potassium primary phosphate 1.7g/L, sal epsom 0.75g/L, sodium-chlor 5g/L.Regulate pH 6.8-7.0.
(2) altheine enzyme catalysis altheine generates NH
3, can detect NH in the solution at the alkaline condition ammonia gas-sensing electrode
3Content according to this principle, is measured the activity of L-ASP.
Enzyme is lived and is defined: under 40 ℃ of reaction conditionss, can be converted into 1 μ mol NH by the catalysis altheine in the per minute
3Needed enzyme amount is enzyme unit alive.
Adopt 25mL to measure system, get two colorimetric cylinders, a conduct contrast, one is sample hose, all adds the Tris-HCl (pH7.5) of 9mL 50mM, the altheine substrate of 10mL 50mM, in 40 ℃ of preheatings, control tube adds the trichoroacetic acid(TCA) of 5mL 15%, and control tube and sample hose all add 0.5mL supernatant liquor or cytoclasis supernatant liquor, 40 ℃ of reaction 15min, sample hose adds the trichoroacetic acid(TCA) termination reaction of 5mL 15%.Change over to reaction soln in the 50mL beaker and add high density NaOH1mL, under the induction stirring state, detect the L-ASP enzyme with ammonia gas-sensing electrode and live.
The result shows the L-ASP 22.4U/mL of being alive that recombinant bacterium pMA5-ansZ/B.subtilis168 expresses, and has improved 574 times than starting strain B.subtilis 6-7L-l-asparagine enzyme activity.
Claims (3)
1. recombinant expression vector pMA5-ansZ is characterized in that:
Obtain to derive from the ansZ gene of B.subtilis 6-7 by round pcr, pMD18-T links to each other with cloning vector, obtains a large amount of clones of this gene, through double digestion, is connected with expression vector pMA5 behind the fragment purification, obtains recombinant plasmid pMA5-ansZ.
2. recombinant plasmid pMA5-ansZ transforms B.subtilis 168
Adopt improved Spizizen method that recombinant plasmid pMA5-ansZ is transformed B.subtilis168, the positive bacterium colony of picking, enzyme is cut checking, obtains pMA5-ansZ/B.subtilis 168.
3.L-the l-asparagine enzyme activity detects
PMA5-ansZ/B.subtilis 168 is inoculated in incubated overnight in the fermentation of bacillus subtilis substratum, in 10000r/min, 4 ℃ of centrifugal 10min, supernatant liquor is the outer crude enzyme liquids of born of the same parents, the cytoclasis supernatant liquor is crude enzyme liquid in the born of the same parents, adopting ammonia gas-sensing electrode to detect the L-ASP enzyme lives, the recombinant bacterium enzyme is lived and is 22.4U/mL after testing, and the bacterium that sets out is approximately improved 574 times.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103243063A (en) * | 2013-05-24 | 2013-08-14 | 江南大学 | High-efficiency expression of bacillus subtilis L-asparaginase (ansZ) in escherichia coli |
| CN105349515A (en) * | 2015-11-25 | 2016-02-24 | 江南大学 | Asparaginase mutant with increased secretion ability and application of asparaginase mutant |
| WO2017031839A1 (en) * | 2015-08-25 | 2017-03-02 | 江南大学 | L-asparaginase mutant with improved enzyme activity and construction method thereof |
| CN107828768A (en) * | 2017-12-13 | 2018-03-23 | 江南大学 | A kind of L asparagines enzyme mutant and its construction method |
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| WO2003083043A2 (en) * | 2002-04-01 | 2003-10-09 | Novozymes Biotech, Inc. | Methods for producing secreted polypeptides having l-asparaginase activity |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243063A (en) * | 2013-05-24 | 2013-08-14 | 江南大学 | High-efficiency expression of bacillus subtilis L-asparaginase (ansZ) in escherichia coli |
| WO2017031839A1 (en) * | 2015-08-25 | 2017-03-02 | 江南大学 | L-asparaginase mutant with improved enzyme activity and construction method thereof |
| CN105349515A (en) * | 2015-11-25 | 2016-02-24 | 江南大学 | Asparaginase mutant with increased secretion ability and application of asparaginase mutant |
| CN105349515B (en) * | 2015-11-25 | 2019-04-23 | 江南大学 | The asparagine enzyme mutant and its application that a kind of secretion capacity improves |
| CN107828768A (en) * | 2017-12-13 | 2018-03-23 | 江南大学 | A kind of L asparagines enzyme mutant and its construction method |
| CN107828768B (en) * | 2017-12-13 | 2020-10-09 | 江南大学 | L-asparaginase mutant and construction method thereof |
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