CN103243117B - Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis - Google Patents
Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis Download PDFInfo
- Publication number
- CN103243117B CN103243117B CN201310196207.6A CN201310196207A CN103243117B CN 103243117 B CN103243117 B CN 103243117B CN 201310196207 A CN201310196207 A CN 201310196207A CN 103243117 B CN103243117 B CN 103243117B
- Authority
- CN
- China
- Prior art keywords
- lipase
- lipa
- bacillus subtilis
- serratia marcescens
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis, belonging to the fields of gene engineering and enzyme engineering. The method comprises the following steps of: firstly acquiring gene amplification of Serratia marcescens lipase (lipA), and then realizing overexpression of genes in type strain Bacillus subtilis 168 for the first time by utilizing pMA-5 plasmids. According to the method, Bacillus subtilis engineering strains which produce the Serratia marcescens lipase are constructed for the first time, after carrying out research on enzyme activities and fermenting properties of the strains and optimization on culture medium components and culture conditions, the activity of the lipase is 98.6 U/mL which is equal to 12 times of that, namely 8.57 U/mL, of a starting strain, and compared with the starting strain, the activity of the lipase is remarkably improved; and the method plays an active role in guiding the industrial production of the lipase by utilizing a microbiological fermentation method and can be applied to the field of food industry.
Description
Technical field
Utilize a method for restructuring Bacillus subtili clonal expression serratia marcescens lipase, especially a kind of expression method that can be used for the lipase of foodstuffs industry safely and efficiently, belongs to genetically engineered and enzyme engineering field.
Technical background
Lipase (Lipase, full name Triacylglycerol acylhydrolase) EC3.1.1.3 is the special ester linkage hydrolyzing enzyme of a class.Can catalytic hydrolysis triacylglycerol be lipid acid, Diglyceride, monoglyceride and glycerine, its natural substrate be generally the longer chain fatty acid acyl ester of water insoluble solution.Lipase is widespread in nature, and not only has and participates in metabolic important physiological action, and have very large industrial value, comprise some special organic synthesis, the hydrolysis of fat and oils, the improvement of spices in development of food industry, and chemical analysis etc.Lipase has good prospects for commercial application, generally obtained by microorganism such as bacterium, fungi, yeast, the actinomycetes etc. of product extracellular lipase, it is widely used in the synthesis, milk industry, agrochemical industries, papermaking, trophology, makeup, pharmaceutical industry etc. of the production of the processing treatment of organic chemical, washing agent, bio-surfactant are a lot, can also promote the degraded of lipid rubbish and Polyurethanes.
Lipase is present in animals and plants and microorganism widely.The production method of current lipase has three kinds:
(1) extraction method: automatically extract enzyme in plant organ or tissue;
(2) chemical synthesis: by the amino acid composition order of enzyme analysis, then chemically synthesize;
(3) fermentation method: the vitality utilizing microorganism, obtains the enzyme needed for people by Artificial Control.
Microbial lipase has the action pH wider than andvegetable fats enzyme, temperature range and better Substratspezifitaet, is widely used in tradition and infant industry catalytic field.Current lipase mainly obtains fast development in the industry such as food-processing, cosmetics of everyday use, and studies less in chiral drug resolution, biofuel direction.
Serratia marcescens can secrete extracellular lipase, and research shows (Zhang-De Long, Jian-He Xu, Li-Li Zhao.Overexpression ofSerratia marcescens lipase in Escherichia coli for efficient bioresolution ofracemic ketoprofen.Journal ofMolecular Catalysis B:Enzymatic47 (2007) 105-110) this lipase has higher stereoselectivity, if key intermediate racemization trans-4-methoxyphenyl glycidic acid methyl esters (MPGM) in narrow spectrum splitting diltiazem building-up process, obtain (-)-MPGM, the latter can synthesis of chiral cis (+) Odizem further.There is the lipase gene of research display serratia marcescens after expression in escherichia coli, have higher methanol tolerance simultaneously.Illustrate that the lipase of serratia marcescens can well be applied to chiral drug synthesis and field of biodiesel oil, simultaneously in order to expand its application in fields such as food-processings, we have carried out good in food safety to it and the simple Bacillus subtilis genes of industrial fermentation condition is high efficiency recombinant expressed, for solid technical foundation has been established in the final formation of lipase-catalyzed dose and industrialization.
The present invention is by plasmid pMA-5, achieve the overexpression of serratia marcescens lipase in subtilis 168, through single factor experiment and orthogonal test, the nutrient media components of recombinant bacterium and culture condition are optimized, make the work of its enzyme reach 98.6U/mL, be about 12 times of the work of original bacteria enzyme.Subtilis is as the industrial producing strain of safety and stability simultaneously, and culture condition is simple, and fermentation period is short, is the potential production bacterial strain of high-quality in microbial lipase fermentation industry.
Summary of the invention
The object of the present invention is to provide: a kind ofly obtained the method for microorganism with safe and efficient fermentative production serratia marcescens lipase ability by genetic engineering means.
Technical scheme of the present invention: be take genetically engineered as the recombined bacillus subtilis that means build a strain fermentative production serratia marcescens lipase, by PCR and digestion verification, screening positive recombinant, fermentation condition optimization is carried out to the subtilis of restructuring, is determined the vigor of its target enzyme by the detection of enzyme activity and leavening property.The present invention successfully constructs the subtilis engineering strain of a plant height yielding lipase and optimizes its fermentation condition, and its lipase activity comparatively starting strain has very big raising.
Recombinant bacterial strain construction process:
(1) clone of lipase (lipA) gene complete sequence
Restriction enzyme site design lipA gene primer on the Serratia marcescens lipA gene order announced according to GENBANK website and pMA-5 plasmid.With the serratia marcescens chromosomal DNA of preparation for template, go out lipA complete sequence by pcr amplification.
PCR reaction system: 10 × ExTaq Buffer2.5 μ L, dNTP2 μ L, template DNA 1 μ L, each 0.5 μ L of upstream and downstream primer, ExTaq enzyme 0.5 μ L, ddH
2o polishing is to cumulative volume 25 μ L.PCR reaction conditions: 94 DEG C of 4min, 94 DEG C of 90s, 59 DEG C of 90s, 72 DEG C of 120s, circulate 30 times, 72 DEG C of 10min, 15 DEG C of 10min.
(2) structure of recombinant expression vector pMA-5-lipA
Reclaim test kit specification sheets with reference to vast Imtech glue and reclaim PCR primer, 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/Nde I enzyme and is discharged the gene band that size is 2.7kb and 1.8kb, show construction of recombinant plasmid success, recombinant plasmid called after pMD18-T-lipA.
Extract plasmid pMD18-T-lipA and pMA-5 be stored in E.coli JM109, plasmid pMD18-T-lipA and pMA-5 is through BamHI/Nde I double digestion, and glue reclaims purifying, T
4dNA ligase spends the night connection two fragment, by connector thermal shock Transformed E .coli JM109 competent cell after spending the night, uses kalamycin resistance plate screening positive transformant.Extract transformant plasmid, recombinant plasmid discharges the gene fragment that size is 7.5kb and 1.8kb after BamH I/NdeI double digestion, proves construction of recombinant plasmid success, recombinant plasmid called after pMA-5-lipA.
(3) in recombinant plasmid pMA-5-lipA chemical transformation transformation mode bacterial strain Bacillus subtilis168
Recombinant plasmid pMA-5-lipA chemical transformation empirical tests successfully constructed is converted in type strain Bacillus subtilis168 expresses.Method for transformation is adopt the Spizizen method improved.
(4) screening of recombinant bacterial strain B.subtilis168/pMA-5-lipA positive transformant;
Picking has the bacterium colony that kantlex antibiotic pressure flat board grows, shake flask fermentation, extracts plasmid and carries out digestion verification.
(5) optimization of recombinant bacterial strain B.subtilis168/pMA-5-lipA culture medium condition:
A) determine that the best plants age according to seed growth curve.
B) interpolation Zulkovsky starch, maltose, cyclodextrin, glucose, sucrose, lactose is selected to be the impact that carbon source analyzes on biomass and lipase output.After being defined as a certain carbon source, to obtain the carbon source concentration of best biomass and lipase output from 10g/L-60g/L by changing this carbon source concentration simultaneously.
C) interpolation yeast extract, beef extract, dregs of beans, soy peptone, corn steep liquor is selected to be the impact that organic nitrogen source analyzes on biomass and lipase output.After being defined as a certain organic nitrogen source, to obtain the nitrogen concentration of best biomass and lipase output from 5g/L-30g/L by changing this nitrogen concentration simultaneously.
D) interpolation urea, ammonium chloride, ammonium sulfate, Secondary ammonium phosphate, ammonium nitrate is selected to be the inorganic nitrogen-sourced impact analyzed biomass and lipase output.When be defined as a certain inorganic nitrogen-sourced after, to obtain the nitrogen concentration of best biomass and lipase output from 0.5g/L-3g/L by changing this nitrogen concentration simultaneously.
E) dipotassium hydrogen phosphate/potassium primary phosphate adding magnesium sulfate, calcium chloride, Repone K, sodium-chlor, saltpetre, SODIUMNITRATE and 1: 1 ratio is selected to be the impact that inorganic salt analyze on biomass and lipase output.After being defined as a certain inorganic salt, to obtain the inorganic salt concentration of best biomass and lipase output from 1g/L-6g/L by changing this inorganic salt concentration simultaneously.
F) on above monofactorial basis, design orthogonal test carrys out the composition of Optimal Medium." four factor three levels " scheme that experiment adopts, the optimum carbon source chosen, optimum organic nitrogen source, optimum inorganic nitrogen-sourced, optimum inorganic salt are investigation factor, test.
(6) optimization of recombinant bacterial strain B.subtilis168/pMA-5-lipA culture condition:
A) on the basis of optimization culture based component, investigate the impact of initial pH on cellular biomass and AD transformation efficiency, initial for substratum pH is set respectively pH5.0-8.0 and test its impact on the enzyme scale of construction and lipase output.
B) on the basis of optimization culture based component and initial pH, investigate the impact of different vaccination amount on the enzyme scale of construction and lipase, inoculum size is set 1%-6% respectively.
(7) recombinant bacterium B.subtilis168/pMA-5-lipA enzyme activity determination
Enzyme activity determination method: adopt para-nitrophenol method.Concrete grammar is as follows: the aqueous isopropanol of the p-NP cetylate (p-NPP) of A liquid: 16.5mmol/L.B liquid: the 50mmol/L Tris-HCI damping fluid (pH8.0) containing 0.4%Triton X-100 and 0.1% Sudan Gum-arabic.During mensuration, corresponding A liquid and B liquid are mixed with 1: 9 (volume ratio) ratio, gets 100 μ L crude enzyme liquids and add in the above-mentioned mixed solution of 900 μ L, in 40 DEG C of reaction 10min.Add 1mL dehydrated alcohol termination reaction immediately, under 410nm, measure absorbance value.Enzyme (U) unit definition alive: per minute decomposes the p-NPP enzyme amount produced needed for 1 μm of ol p-NP (yellow) and is defined as 1 Ge Meihuo unit.
Beneficial effect of the present invention: to be increased existing this enzyme gene with the serratia marcescens of lipase ability of this experiment by genetic engineering means, by the withered grass expression system that this laboratory has, achieve the overexpression of lipase gene in type strain subtilis 168 in serratia marcescens source first.By this inoculation in being inoculated in 50mL LB substratum with 2% inoculum size, cultivate 36h, from carbon source, organic nitrogen source, angle that is inorganic nitrogen-sourced and inorganic salt, medium component is optimized, obtain optimum optimizing condition, determine optimal culture condition, then centrifuging and taking supernatant, thalline washes twice simultaneously, suspend with suitable PBS (PH7.5) damping fluid, ultrasonic disruption cell.Total enzyme is lived and is about 98.6U/mL, is 3 times before fermentation condition optimization, is about 12 times of bacterium 8.57U/mL of setting out.
The extracellular lipase that serratia marcescens produces can be applicable to the asymmetric synthesis of chipal compounds.As can efficiently selectivity splitting diltiazem (Diltiazem) produce in key intermediate-Xiao Xuan body trans-4-methoxyphenyl glycidic acid methyl esters (MPGM), obtain (2R, 3s)-4-methoxyphenyl glycidic acid methyl esters-(-)-MPGM, the latter can direct synthesis of chiral cis (+) Odizem as initial synthesis material.In addition, serratia marcescens lipase has higher tolerance to the organic solvent such as methyl alcohol, ethanol.Therefore biofuel and medical in have very large application prospect, simultaneously because its expression in withered grass system makes it have the application prospect in the fields such as potential food-processing.
Accompanying drawing explanation
The digestion verification of Fig. 1 recombinant bacterium B.subtilis168/pMA-5-lipA
M1: λ HindIII DNA Marker; 1:pMA-5-lipA single endonuclease digestion; 2:pMA-5-lipA double digestion; M2:DS2000Marker
The SDS-PAGE checking of Fig. 2 recombined bacillus subtilis B.subtilis168/pMA-5-lipA
M:Unstained protein MW marker of Takara, 1:B.subtilis168/pMA5-lipA cytoclasis supernatant, 2:B.subtilis168 cytoclasis supernatant, 3:B.subtilis168/pMA5-lipA fermented liquid supernatant, 4:B.subtilis168 fermented liquid supernatant
Specific implementation method
Embodiment 1: the structure of restructuring B.subtilis168 bacterial strain
1) clone of lipase (lipA) gene complete sequence: the restriction enzyme site design lipA gene primer on the Serratia marcescenslipA gene order announced according to GENBANK website and pMA-5 plasmid.Primer sequence following (wherein italic underscore part is restriction enzyme site, and primer synthesizes by matching Parkson company).
lipA F·5’-ATC
GAATTCATGGGCATCTTTAGCTATA-3’
lipA R：5’-TGACT
GCGCCGGCTTAGGCCAACACCACCTG-3’
The chromosomal DNA of extraction, with reference to Shanghai Sheng Gong company chromosomal DNA extraction test kit (bacterium) working instructions in a small amount, is placed in-20 DEG C of refrigerators stand-by by the extraction preparation of serratia marcescens chromosomal DNA.With the serratia marcescens chromosomal DNA of preparation for template, with lipAF, lipAR for primer, go out lipA complete sequence by pcr amplification.
PCR reaction system: 10 × ExTaq Buffer2.5 μ L, dNTP2 μ L, template DNA 1 μ L, each 0.5 μ L of upstream and downstream primer, ExTaq enzyme 0.5 μ L, ddH2O polishing is to cumulative volume 25 μ L.PCR reaction conditions: 94 DEG C of 4min, 94 DEG C of 90s, 59 DEG C of 90s, 72 DEG C of 120s, circulate 30 times, 72 DEG C of 10min, 15 DEG C of 10min.
2) structure of recombinant expression vector pMA-5-lipA: reclaim test kit specification sheets with reference to vast Imtech glue and reclaim PCR primer, the PCR primer of recovery is by carrying out 0.8% agarose gel electrophoresis detection.Glue recovery product spends the night with pMD18-T vector by a certain percentage and is connected, and by the PCR primer of detection in following ratio, be connected with cloning vector, 16 DEG C are spent the night.Linked system: Solution I5 μ L; PCR primer 4.8 μ L; PMD18-T0.2 μ L.Transformed E .coli JM109 competent cell, use amicillin resistance plate screening recombinant bacterium, recombinant plasmid is cut through BamH I/Nde I enzyme and is discharged the gene band that size is 2.7kb and 1.8kb, shows construction of recombinant plasmid success, recombinant plasmid called after pMD18-T-lipA.
Extract plasmid pMD18-T-lipA and pMA5 be stored in E.coli JM109, plasmid pMD18-T-lipA and pMA-5 is through BamH I/Nde I double digestion, glue reclaims purifying, T4DNA ligase enzyme spends the night connection two fragment, by connector thermal shock Transformed E .coli JM109 competent cell after spending the night, use kalamycin resistance plate screening positive transformant.Extract transformant plasmid, recombinant plasmid discharges the gene fragment that size is 7.5kb and 1.8kb after BamH I/NdeI double digestion, proves construction of recombinant plasmid success, recombinant plasmid called after pMA-5-lipA.
3) in recombinant plasmid pMA-5-lipA chemical transformation transformation mode bacterial strain B.subtilis168: recombinant plasmid pMA-5-lipA chemical transformation empirical tests successfully constructed is converted in type strain B.subtilis168 expresses.Method for transformation is adopt the Spizizen method improved.First night, picking Host Strains was inoculated in 10mL LB liquid nutrient medium, 37 DEG C of shaking table overnight incubation.Get the bacterium liquid of 100 μ L incubated overnight the next morning, be inoculated in 5mL SP I substratum, 37 DEG C of shaking tables are cultivated, start to survey OD600 after 5 hours, when culture grows into the logarithm art phase, fast fetching 200 μ L is inoculated in 2mL SP H substratum, 37 DEG C, 160rpm shaking table cultivation 1.5h.Add 20 μ L100 × EGTA solution, cultivate 10min in 37 DEG C of 160rpm shaking tables, be distributed into 500 μ L with 1.5mL centrifuge tube and often manage.Xiang Guanzhong adds the plasmid (5-10 μ L) verified in right amount, mixes gently, cultivates 2h in 37 DEG C of 160rpm shaking tables.With 8000rpm collected by centrifugation thalline, remove part supernatant liquor, stay the resuspended thalline of 200 μ L, coat kalamycin resistance flat board, 37 DEG C of incubated overnight.By the recombinant plasmid pMA-5-lipA verified, transform B.subtilis168 according to above-mentioned method for transformation, build recombined bacillus subtilis recombined bacillus subtilis B.subtilis168/pMA-5-lipA.
4) screening of recombinant bacterial strain B.Subtilis168/pMA-5-lipA positive transformant: picking has the bacterium colony that kantlex antibiotic pressure flat board grows, be forwarded to the LB liquid nutrient medium containing 50 μ g/mL penbritins, 37 DEG C, 160rpm shaking table cultivation 10-12h fermentation, extract transformant plasmid and carry out digestion verification, obtain the digestion verification figure (as Fig. 1) of recombinant bacterium.
5) recombined bacillus subtilis B.subtilis168/pMA-5-lipA produces enzyme analysis: by the bacillus subtilis strain B.subtilis168/pMA-5-lipA of the restructuring verified with in the 10mL LB liquid nutrient medium be seeded to containing 50 μ g/mL kantlex, 37 DEG C of shaking culture 24h, then be forwarded to fresh 50mL LB liquid nutrient medium by 1% inoculum size, cultivate 24h.Get the bacterium liquid 50mL cultivating 24h, 4 DEG C, the centrifugal 10min of 8000rpm collects thalline, with the 50mM PBS buffer solution secondary of 50mL pH7.4, is resuspended in this damping fluid of 4ml.In ice bath, 40% power ultrasonic is broken, work 2s interval 5s, working hour 10min.The centrifugal 30min of 10000rpm obtains supernatant liquor.The centrifugal rear supernatant of fermented liquid, in cytoclasis, cleer and peaceful cytoclasis precipitation carries out SDS-PAGE (as shown in Figure 2) and enzyme activity determination respectively.
Embodiment 2: the optimization of recombinant bacterial strain B.subtilis168/pMA-5-lipA fermentation condition
The optimization of recombinant bacterial strain B.subtilis168/pMA-5-lipA culture medium condition:
A) determine that the seed growing 13-15h plants age for best according to seed growth curve.
B) interpolation Zulkovsky starch, maltose, cyclodextrin, glucose, sucrose, lactose is selected to be the impact that carbon source analyzes on biomass and lipase output.Concentration 2%, Preliminary fermentation substratum is LB substratum.Owing to not having proper carbon source in LB substratum, alternative carbon source directly can be added as sole carbon source, other components remain unchanged.Inoculum size 2%, 37 DEG C, 160rpm cultivates 36h, measure its biomass and measure lipase activity when finding that sucrose is carbon source, lipase output is the highest, and biomass is relatively high.In the constant situation of other conditions, be respectively 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L cultivation by changing sucrose concentration simultaneously, find that biomass and lipase output raise in rising trend with sucrose concentration when sucrose concentration is 10-30g/L, start subsequently to decline, determine that sucrose concentration is the raising that 30g/L is conducive to biomass and lipase output.C) interpolation yeast extract, beef extract, dregs of beans, soy peptone, corn steep liquor is selected to be the impact that organic nitrogen source analyzes on biomass and lipase output.Concentration 2%, Preliminary fermentation substratum is LB substratum, replaces the nitrogenous source in original culture medium with organic nitrogen source to be selected, and other components are constant.Inoculum size 2%, measures its biomass and lipase activity finds that corn steep liquor is best by 37 DEG C, 160rpm cultivates 36h.Simultaneously be respectively 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L by changing corn steep liquor concentration to cultivate discovery concentration be that the corn steep liquor of 25g/L can obtain best biomass and lipase output in the constant situation of other conditions.
D) interpolation urea, ammonium chloride, ammonium sulfate, Secondary ammonium phosphate, ammonium nitrate is selected to be the inorganic nitrogen-sourced impact analyzed biomass and lipase output.Concentration 2%, Preliminary fermentation substratum is LB substratum, and with the inorganic nitrogen-sourced nitrogenous source replaced in original culture medium to be selected, other components are constant.Inoculum size 2%, measures its biomass and lipase activity finds that ammonium sulfate is best by 37 DEG C, 160rpm cultivates 36h.Simultaneously in the constant situation of other conditions, be respectively in 0.5g/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L that to cultivate discovery concentration be that the ammonium sulfate of 1.5g/L can obtain best biomass and lipase output by changing ammonium sulfate concentrations.
E) dipotassium hydrogen phosphate/potassium primary phosphate adding magnesium sulfate, calcium chloride, Repone K, sodium-chlor, saltpetre, SODIUMNITRATE and 1: 1 ratio is selected to be the impact that inorganic salt analyze on biomass and lipase output.Concentration 0.25%.Preliminary fermentation substratum is LB substratum.Owing to there is inorganic salt sodium-chlor in LB substratum, so the inorganic salt chosen directly replace original inorganic salt, other component all remains unchanged.Inoculum size 1%, culture temperature 37 DEG C, shaking speed 160rpm, cultivates, and measures its biomass and lipase output discovery calcium chloride the best.Simultaneously in the constant situation of other conditions, be respectively in 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L that to cultivate discovery concentration be that the calcium chloride of 4g/L can obtain best biomass and lipase output by changing calcium chloride concentration.
F) on above monofactorial basis, design orthogonal test carrys out the composition of Optimal Medium." four factor three levels " scheme that experiment adopts, the optimum carbon source chosen, optimum organic nitrogen source, optimum inorganic nitrogen-sourced, optimum inorganic salt are investigation factor, test.Finally determine sucrose 35g/L, corn steep liquor 27.5g/L, ammonium sulfate 1.25g/L, calcium chloride 4g/L is that medium component is best.Adopt the fermention medium of this optimum combination to cultivate thalline, survey its yielding lipase and live.
The optimization of recombinant bacterial strain B.subtilis168/pMA-5-lipA culture condition:
A) on the basis of optimization culture based component, investigate the impact of initial pH on biomass and lipase output, initial for substratum pH is set respectively pH5.0,6.0,7.0,8.0 and cultivate, when pH is 5.0, thalli growth is suppressed, and lipase output is lower simultaneously; When pH is 6.0-7.0 biomass and lipase output higher, for best when wherein pH is 7.0.
B) on the basis of optimization culture based component and initial pH, investigate the impact of different vaccination amount on the enzyme scale of construction and lipase, inoculum size is set 1%, 2%, 3%, 4%, 5%, 6% respectively, and obtaining optimum inoculation amount is 4%.
Embodiment 3: the enzyme activity determination of recombinant bacterial strain
Bacterial strain cultivates 36h in LB substratum, and the centrifugal 10min of 8000rpm, gets supernatant.The aqueous isopropanol of the p-NP cetylate (p-NPP) of A liquid: 16.5mmol/L.B liquid: the 50mmol/LTris-HCI damping fluid (pH8.0) of the Sudan Gum-arabic containing 0.4%Triton X-100 and 0.1%.During mensuration, corresponding A liquid and B liquid are mixed with 1: 9 (volume ratio) ratio, gets 100 μ L crude enzyme liquids and add in the above-mentioned mixed solution of 900 μ L, in 40 DEG C of reaction 10min.Add 1mL dehydrated alcohol termination reaction immediately, under 410nm, measure absorbance value.Enzyme (U) unit definition alive: per minute decomposes the p-NPP enzyme amount produced needed for 1 μm of ol p-NP (yellow) and is defined as 1 Ge Meihuo unit.
By coming into being, the inoculum size of bacterium by 2% (V/V) is transferred in Preliminary fermentation substratum, 37 DEG C, 160rpm cultivates 24h and measure its enzyme and live as 8.57U/mL.Carrying out enzyme activity determination to recombined bacillus subtilis B.subtilis168/pMA-5-lipA product enzyme is afterwards 32.24U/mL.Under the condition of optimum fermention medium, 37 DEG C, 160rpm shaking table is cultivated 33h recombinant bacterium lipase output and can be reached 98.6U/mL fermented liquid, is about 3 times before optimizing is about 12 times of bacterium of setting out.
Claims (1)
1. one kind utilizes the method for recombined bacillus subtilis yielding lipase, it is characterized in that: obtained the lipase gene lipA deriving from serratia marcescens by round pcr, be connected with cloning vector pMD-18T, obtain a large amount of clones of this gene, through double digestion, be connected with expression vector pMA5 after lipA fragment purification, the recombinant vectors pMA5-lipA built, be converted into type strain Bacillus subtilis 168 with chemical transformation, obtain recombined bacillus subtilis B.subtilis 168/pMA5-lipA; Recombined bacillus subtilis B.subtilis 168/pMA5-lipA fermentation medium components is: sucrose 35g/L, corn steep liquor 27.5g/L, ammonium sulfate 1.25g/L, calcium chloride 4g/L, initial pH7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310196207.6A CN103243117B (en) | 2013-05-24 | 2013-05-24 | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310196207.6A CN103243117B (en) | 2013-05-24 | 2013-05-24 | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103243117A CN103243117A (en) | 2013-08-14 |
| CN103243117B true CN103243117B (en) | 2015-07-08 |
Family
ID=48922996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310196207.6A Active CN103243117B (en) | 2013-05-24 | 2013-05-24 | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103243117B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107217025A (en) * | 2017-06-09 | 2017-09-29 | 盐城工学院 | A kind of bacillus subtilis JG 1 for producing endo-inulinase and its preparation method and application |
| CN108118016A (en) * | 2018-02-08 | 2018-06-05 | 上海化工研究院有限公司 | A kind of Serratia lipase and its application in (±)-MPGM is split |
| CN108517329A (en) * | 2018-03-27 | 2018-09-11 | 杭州柏普泰生物科技有限公司 | A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application |
| CN109852633A (en) * | 2019-03-20 | 2019-06-07 | 淮海工学院 | A method of based on Bacillusexpression system high efficient expression Bacterium prodigiosum Non-specific nuclease |
| CN114381445B (en) * | 2020-10-19 | 2023-12-12 | 中国科学院分子植物科学卓越创新中心 | Serratia lipase with plasmodium killing effect and encoding gene thereof |
| CN112852703A (en) * | 2021-03-17 | 2021-05-28 | 上海交通大学 | Culture medium for improving expression quantity of recombinant protein in bacillus subtilis, preparation method and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102051342A (en) * | 2010-11-05 | 2011-05-11 | 广西科学院 | Engineering bacterium producing lipase and method for preparing lipase catalyst and fatty acid methyl ester by using same |
| CN102703494A (en) * | 2012-06-20 | 2012-10-03 | 江南大学 | Method for generating ADD by utilizing recombined Bacillus subtilis whole-cell converted AD |
| CN102876704A (en) * | 2012-09-20 | 2013-01-16 | 江南大学 | Method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently |
| CN103074290A (en) * | 2012-12-18 | 2013-05-01 | 江南大学 | Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli |
-
2013
- 2013-05-24 CN CN201310196207.6A patent/CN103243117B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102051342A (en) * | 2010-11-05 | 2011-05-11 | 广西科学院 | Engineering bacterium producing lipase and method for preparing lipase catalyst and fatty acid methyl ester by using same |
| CN102703494A (en) * | 2012-06-20 | 2012-10-03 | 江南大学 | Method for generating ADD by utilizing recombined Bacillus subtilis whole-cell converted AD |
| CN102876704A (en) * | 2012-09-20 | 2013-01-16 | 江南大学 | Method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently |
| CN103074290A (en) * | 2012-12-18 | 2013-05-01 | 江南大学 | Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli |
Non-Patent Citations (1)
| Title |
|---|
| 枯草芽孢杆菌分泌载体构建及其对脂肪酶A 的分泌表达;夏雨;《安徽农业科学》;20111231;第39卷(第30期);18435-18437页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103243117A (en) | 2013-08-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103243117B (en) | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis | |
| Fabiano et al. | Thermodynamic study and optimization of hydrogen production by Enterobacter aerogenes | |
| CN103981203B (en) | 5 amino-laevulic acid superior strains and its preparation method and application | |
| CN105506010B (en) | A kind of method of multiplex-enzyme extraction microalgae grease | |
| CN105368882A (en) | Method for producing ethyl alcohol through crop stalks by use of recombinant zymomonas mobilis | |
| Diaz et al. | Simultaneous saccharification and fermentation of industrial sweetpotatoes for ethanol production and anthocyanins extraction | |
| CN109777793A (en) | A kind of GDSL lipase, genetic engineering bacterium and its application | |
| CN103352016B (en) | Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha | |
| CN106947772A (en) | A kind of carbonyl reduction enzyme mutant and its application in chiral alcohol preparation | |
| CN103642859B (en) | A kind of fermention medium and purposes of producing gamma-linolenic acid output for improving volume branch Mucor | |
| CN103131659B (en) | Organic solvent-resistant lipase, as well as encoding gene, producing strain and application thereof | |
| CN102634553B (en) | Hainanmycin fermentation method | |
| CN103045546A (en) | Method for producing feeding compound enzyme by taking lotus seed shells as raw material | |
| CN105695429A (en) | Fermentation medium and method for fermenting nicotinamide monoucleotide (NMN) transferase by same | |
| CN105199974A (en) | Recombinant saccharomyces cerevisiae industrial strain for secreted expression of beta-glucosidase and application of recombinant saccharomyces cerevisiae industrial strain | |
| CN109321617A (en) | A method of Natamycin is synthesized using industrial or agricultural by-product solid state fermentation | |
| CN104450825A (en) | Double-phase fermentation preparation condition optimizing method for rhamnolipid | |
| CN106011186B (en) | A kind of method of brevibacterium fermenting and producing succinic acid | |
| CN103497941A (en) | Method for preparing cellulase through trichoderma viride high-efficiency fermentation | |
| CN103621762A (en) | Method for preparing low-molecular-weight oyster peptide by employing bacillus natto fermentation process | |
| CN109161570B (en) | Method for improving fermentation production of N-acetylneuraminic acid and fermentation liquor | |
| CN104789489A (en) | Arginine deiminase high-yielding Bacillus cereus and application thereof | |
| CN102191293A (en) | Method for inversing microbe to prepare ethyl (S)-3-hydroxy-3-(2-thienyl)-propanoate | |
| CN105219748A (en) | A kind of fermentation process of Escherichia coli fermentation Restruction arylsulfatase | |
| JP5303543B2 (en) | Decomposition treatment agent and decomposition treatment method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |