CN105112320B - A kind of Burkholderia gladioli strain and its method for fermentation production of alkaline lipase - Google Patents
A kind of Burkholderia gladioli strain and its method for fermentation production of alkaline lipase Download PDFInfo
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Abstract
The present invention relates to a kind of high yield alkaline lipase Burkholder bacteria strain (Burkholderia gladioli BPS 1), are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.10533.The bacterium source is in corrupt onion, and producing enzyme is stable, raw material sources are extensive, low production cost.Present invention also offers a kind of method that bacterial strain prepares alkaline lipase, the step of comprising seed culture, fermented and cultured and isolating and purifying.Methods described optimizes fermentation medium, the lipase activity of preparation is strong, up to 145.67U/mL, fabulous in alkaline environment stability inferior, and there is good tolerance to short chain alcohol, it is with a wide range of applications in fields such as biomass energy, organic synthesis, medicine chiral resolution and toolenzymes.
Description
Technical field
The present invention relates to microorganism and enzyme, more particularly to a kind of Burkholder bacterial strain and its fermentation production of alkaline lipase
Method.
Background technology
Lipase (EC 3.1.1.3) is a kind of special ester linkage hydrolyzing enzyme, is the living things catalysis with multi-catalytic function
Agent, the hydrolysis of substrate, acidolysis, alcoholysis, ammonolysis, transesterification and lactate synthesis reaction can be catalyzed, be widely used in food industry,
Paper-making industry, detergent industry, field of medicaments and correlation industry and field in.
The mechanism of lipase-catalyzed reaction is similar with serine protease, by the hydroxyl pair on active site serine residue
Substrate carbonyl C-atom initiate nucleophilic attack, formed tetrahedron intermediate, alcohol radical come off from compound after formed acyl group-
Combined enzyme agent, it is the key intermediate of lipase difference catalytic reaction.In aqueous phase, lipase can be catalyzed the water of various greases
Solution.And in organic phase, the organic reaction of enzymatic has mild condition, non-environmental-pollution, speed fast, selective high excellent
Point, lipase can be catalyzed the reaction such as Lipase absobed, transesterification.Although there is nonaqueous phase enzymic catalytic reaction above advantage and catalysis to post-process
The advantages that facilitating, but the activity and stability of lipase are limited by traditional organic solvent.Finding one kind can be in organic solvent
Enzymatic activity is high and the good lipase of stability is with regard to necessary.
Lipase is very wide in microbial world distribution, has now been found that pseudomonas, aspergillus, mould, micrococcus luteus etc. are a variety of micro-
Biology can yielding lipase.According to incompletely statistics, bacterium has 28 category, 4 category of actinomyces, saccharomycete 10 category, other fungies 23
Individual category can yielding lipase.In the lipase in various sources, the reaction type that is catalyzed with comprising lipase of bacterial origin in organic phase is more,
Reactivity and stability highest, and Burkholderia (Burkholderia sp.) lipase caused by bacterial strain have it is good
Many advantages, such as good temperature stability, organic solvent tolerance, strict enantio-selectivity, educational circles and industry are caused
Pay high attention to, the company that commercially produces including Amano companies gives as a kind of special industry lipase preparation
It is very wide with exploitation, application prospect of the enzyme in fields such as biomass energy, organic synthesis, medicine chiral resolution and toolenzymes
It is wealthy.Therefore it provides a kind of Burkholder bacteria strain of high yield lipase and the side using the bacterium fermentation production of alkaline lipase
Method is significant.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of bacterial strain of energy high yield alkaline lipase and its prepare alkalescence
The method of lipase.
Technical solution of the present invention is as follows：
A kind of microbial strains of high yield alkaline lipase, from corrupt onion, the classification category Burkholderia of the bacterial strain
The Burkholderia gladioli of section (Burkholderiaceae) Burkholderia (Burkholderia)
(Burkholderia gladioli), Burkholderia gladioli BPS-1 are named as, in 2 months 2015 6
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number CGMCC
No.10533。
The morphological feature of the strain BP S-1 is as follows：Gram-negative bacteria, bacterium colony are whole in faint yellow, circle, edge
Together, smooth moistening, growth produces xanthein two days later, obligate aerobic, and cell is in bar-shaped, rich in motility, no gemma, also not
Spore is produced, growth temperature is 30-35 DEG C, and growth pH value is 5.0-8.0, and most suitable is that pH value is 6.5-7.5.
The strain BP S-1 is reflected using 16SrDNA molecular biology and Biolog automatic microbe identification systems
It is fixed, it is as a result as follows：
1. by the sequence alignment of the 16SrDNA to BPS-1, the sequence homology with Burkholderia gladioli
Up to 100%, specific DNA sequence dna refers to sequence table.
2.Biolog is accredited as Burkholderia gladioli, and SIM values (culture 24h) are 0.575.
A kind of method that alkaline lipase is prepared using strain BP S-1, includes following preparation process：
(1) seed culture：By the BPS-1 bacterial strains of preservation it is activated after, be inoculated in LB liquid medium, 30-35 DEG C training
12-18 hours are supported, obtain seed culture fluid；
(2) fermented and cultured：Seed liquor is inoculated into fermentation medium by 1-1.5% (V/V) amount, continuously cultivates 48-72
After hour, 8000r/mim centrifugation 10min, the fermented supernatant fluid rich in lipase is collected；The fermentation medium components are：Sugar
Sweet 0.5-1.0% (V/V), palm oil 0.25-0.5% (V/V)；Nitrogen source peptone 1-1.5% (W/V), KH2PO40.2-0.25%
(W/V)、MgSO4·7H2O 0.05-0.1% (W/V).After the fermentation medium triangle shake flask fermentation 72 hours, fermentation
Liquid lipase activity is up to 145.67U/mL.
Enzyme-activity unit (U) defines：The enzyme amount that decomposition pNPP per minute produces needed for 1 μm of ol p-nitrophenol is defined as one
Enzyme-activity unit.
(3) isolate and purify：Above-mentioned zymotic fluid is pure through ethanol precipitation, anion-exchange chromatography and the step of gel permeation chromatography three
Change and obtain electrophoretically pure alkaline lipase.Fatty specific activity of enzyme after purification reaches 443904.8U/mg.
The present invention has carried out zymologic property research to lipase.The optimal reaction pH of the results show lipase is
8.5, optimal reactive temperature is 50 DEG C, all has high stability in the range of pH8.0-11.0, to the tolerance of short chain alcohol
Well.
The advantages and positive effects of the present invention are：
(1) a kind of Burkholder bacterial strain BPS-1 of the high yield alkaline lipase from corrupt onion is provided.The bacterium
Strain producing enzyme is stable, raw material sources are extensive, low production cost.
(2) a kind of method using strain BP S-1 fermentation production of alkaline lipase enzymes is provided.Prepared using this method
Alkaline lipase enzyme activity is fabulous in alkaline environment stability inferior up to 145.67U/mL, and has to short chain alcohol good resistance to
By property, there is preferable industrial production potential in fields such as biomass energy, organic synthesis, medicine chiral resolution and toolenzymes
Be widely applied prospect.
(3) method of the present invention using strain BP S-1 fermentation production of alkaline lipase enzymes, utilizes sugar refinery production process
In principal by product cane molasses as fermentation raw material, can make full use of sugar refinery waste resource and can reduce tunning into
This, while be favorably improved in molasses also containing the trace element such as the biotin needed for microbial growth and nitrogen, phosphorus, potassium
The yield of lipase.
(4) method of the present invention using strain BP S-1 fermentation production of alkaline lipase enzymes, selected compounded carbonses
Palm oil also easily obtains as a kind of cheap edible oil, as long as and its nitrogen source then can be as the nitrogen substance in nitrogen source Tonghua
, this method raw material sources are cheap and extensive, to mass produce condition of providing convenience.
Brief description of the drawings
Fig. 1：The electrophoretogram of alkaline lipase prepared by the present invention
Fig. 2：Alkaline lipase optimal reaction pH schematic diagrames prepared by the present invention
Fig. 3：Alkaline lipase pH stability schematic diagrames prepared by the present invention
Fig. 4：Alkaline lipase optimal reactive temperature schematic diagram prepared by the present invention
Fig. 5：Alkaline lipase temperature stability schematic diagram prepared by the present invention
Fig. 6：Alkaline lipase prepared by the present invention is to organic solvent tolerance schematic diagram
Preservation information
Burkholderia gladioli (Burkholderia gladioli BPS-1), in preservation on the 6th in 2 months in 2015
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), preservation address is exposed to the sun for Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of area North Star West Road 1, deposit number are CGMCC No.10533.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1：Produce the screening and identification of alkaline lipase bacterial strain
1st, the preparation of directed screening flat board
Had the characteristics that according to burkholderia using special metabolite and the resistance to the action of a drug to antibiotic, design
The TB-T flat board directed screenings of improvement, screening burkholderia positive rate may be up to 100%.Detailed composition is as follows：1L is improved
TB-T directed screening flat boards in containing glucose 2g, altheine 0.5g, NaHCO30.5g、KH2PO40.25g、MgSO4·
7H2O 0.05g, platform phenol indigo plant 0.05g, tetracycline 0.01g, ampicillin 0.15g, agar powder 15g.
2nd, the preparation of flat board is identified
Contain peptone 5g, yeast extract 3g, NaCl 8g, rhodamine B solution in 1L rhodamine B identification flat board
(0.1%, W/V) 4mL, soya-bean oil emulsion 10mL.
The soya-bean oil emulsion, compound method are as follows：With emulsification after 3mL soya-bean oil and 9mL polyvinyl alcohol (2%, w/v) mixing
Machine emulsifies 10min, after placing 5 minutes, continues to emulsify 10min if any layering, until completely not stratified.
3rd, the screening of alkaline lipase bacterial strain is produced
It is suspended in after the onion of corruption is shredded in appropriate physiological saline, the 10 of gradient dilution to suspension-4-10-3
Times, it is coated on the TB-T directed screening flat boards of improvement, puts plate again after bacterium colony is grown in identification flat board.It will identify on flat board thoroughly
Bright circle obtains single bacterial strain after purification with colony radius than the bacterium colony more than 2, scribed line.By the inoculation that purifying obtains to liquid
In body fermentation medium, lipase activity is determined to the crude enzyme liquid of gained after 30 DEG C of fermentation 72h.
The initial formulation of liquid fermentation medium is：Glucose 0.5% (V/V), olive oil 0.25% (V/V)；Peptone
1% (W/V)；KH2PO40.2% (W/V), MgSO4·7H2O 0.05% (W/V).
Determination Methods for Lipase Activity is：
A liquid：16.5mmol/L pNPP (p-nitrophenyl palmitate) solution is prepared with isopropanol.
B liquid：0.05mol/L Tris-HCl containing 2% (w/v) TritonX-100 and 0.1% (w/v) Arabic gum delay
Fliud flushing (pH value 9.0).
A liquid and B liquid are mixed with 1: 9 (volume ratio) ratio during measure, the crude enzyme liquid for taking 50 μ L to dilute in right amount adds 450 μ L
In above-mentioned AB mixed liquors, 10min is reacted in 37 DEG C, adds 500 μ L10% (w/v) trichloroacetic acid terminating reaction, measure immediately
Preceding addition 1mL 0.5M Na2CO3Adjustment pH value reaches alkalescence.
Enzyme-activity unit (U) defines：The enzyme amount that decomposition pNPP per minute produces needed for 1 μm of ol p-nitrophenol is defined as one
Enzyme-activity unit.
Preliminary screening obtains the higher bacterial strain of 12 plants of yielding lipase vigor, selects one plant of high BPS-1 of genetic stability, just
Enzyme activity is 5.04U/mL after the fermentation of beginning fermentation medium.The bacterium preservation is to China General Microbiological culture presevation administrative center
(CGMCC), deposit number is CGMCC No.10533.
4th, the identification of alkaline lipase bacterial strain is produced
Strain BP S-1 identifies that SIM values are (24h cultures) 0.575 through Biolog automatic microbe assessing instruments, and qualification result shows
Burkholderia gladioli are shown as, by 16SrDNA sequence analyses, show bacterial strain and Burkholderia in database
Gladioli sequence homologies are up to 100%, and comprehensive Biolog and 16SrDNA analysis results identify that the bacterial strain is
Burkholderia gladioli, it is named as Burkholderia gladioli BPS-1.
Embodiment 2：Utilize BPS-1 bacterial strains and Optimal Medium fermenting and producing lipase
(1) seed culture：By BPS-1 bacterial strains it is activated after, be inoculated in LB liquid medium, 30-35 DEG C culture 12-18
Hour, obtain seed culture fluid；
(2) fermented and cultured：Seed liquor is inoculated into preliminary fermentation culture medium (such as embodiment 1 by 1-1.5% (V/V) amount
It is described), after continuously cultivating 72 hours, 8000r/mim, 10min is centrifuged, determine lipase activity.
Using carbon source (glucose, sucrose, molasses, dextrin, maltose, the wheat of single factor test Shift Method research fermentation medium
Bud medicinal extract, starch), nitrogen source (peptone, yeast extract, corn steep liquor, urea, NaNO3、NH4SO4) and derivant (olive oil, palm
Oil, corn oil, soybean oil, rice bran oil, castor oil, rapeseed oil) influence to producing enzyme, show that the component after optimization is molasses
1.0% (v/v), palm oil 0.25% (V/V), peptone 1.5% (W/V)；KH2PO40.2% (W/V), MgSO4·7H2O
0.05% (W/V).It is inoculated into 1% (V/V) inoculum concentration in above-mentioned fermentation medium, after continuously cultivating 72 hours, 8000r/
Mim, 10min is centrifuged, collect the fermented supernatant fluid rich in lipase, lipase activity is up to 145.67U/mL, with Preliminary fermentation
The fermentation broth enzyme vigor 5.04U/mL cultivated under culture medium prescription compares, and improves 28.9 times.
Embodiment 3：The purifying of alkaline lipase
1st, the zymotic fluid after optimization described in embodiment 2 is precipitated using final concentration of 60% (v/v) ethanol,
8000r/mim centrifugations 10min removes foreign protein, recycles final concentration of 95% (v/v) ethanol precipitation destination protein, 8000r/
Obtained precipitation is collected after mim centrifugations 10min, is dissolved, is obtained just using the 0.05mol/L piperazine buffer solutions of pH value 9.7
Walk the enzyme liquid of purifying.
2nd, the enzyme liquid of preliminary purification is entered through HiTrap Q ion-exchange chromatographies and the gel permeation chromatographies of superdex 75
One step obtains electrophoretically pure Lipase protein (as shown in Figure 1) after purification, and the molecular weight of the lipase is about 35kDa, after purification
Fatty specific activity of enzyme be 443904.8U/mg.
Embodiment 4：The zymologic property of alkaline lipase
1st, the detection of optimal reaction pH and pH stability
With the buffer solution substrate p-nitrophenyl palmitate of different pH value, lipase activity is determined, under the conditions of pH8.5
Lipase activity be control (100%), the lipase activity under different pH systems is as shown in Fig. 2 the optimum pH of the enzyme is
8.5.Lipase is diluted using pH value 2.2-12.0 buffer solution, after 25 DEG C of preservation 24h, determines residual enzyme activity, not add
The enzyme liquid of buffer solution preservation is control, and the pH stability of alkaline lipase is as shown in figure 3, the enzyme under alkaline environment, is particularly
PH8.0-11.0 shows extremely strong stability, and preserving 24h in the environment of pH11 still has more than 85% residual enzyme activity.
2nd, the detection of optimal reactive temperature and temperature stability
With pH8.5 buffer solution substrate p-nitrophenyl palmitate, lipase activity is determined under condition of different temperatures
Power, it is 50 DEG C as a result to show optimal reactive temperature, as shown in figure 4, the optimal reactive temperature of the enzyme is 50 DEG C.By what is diluted in right amount
Enzyme liquid determines remnant enzyme activity after preservation 1h at 25,30,35,40,45,50,55,60,65,70 DEG C, and with non-preservation
Enzyme liquid is control, and the temperature stability of alkaline lipase is as shown in figure 5, the good thermal stability of the enzyme, the preservation 1h at 70 DEG C
Still there is 50% residual enzyme activity.
3rd, the detection of organic solvent tolerance
After pH 8.5 Tis-HCl buffer solutions dilution enzyme liquid, be separately added into normal heptane, n-hexane, hexamethylene, methanol,
Ethanol, isopropanol, ethyl acetate, acetone, toluene, make final concentration of 30% (v/v) of organic solvent, at 25 DEG C, 300r/min
After concussion preserves 1h, determine the remnant enzyme activity of lipase, using not added with solvent enzyme liquid for control, as a result as shown in fig. 6, this
Enzyme is to the better tolerance of short chain alcohol, and enzyme activity is without significant change after being preserved 1 hour in the methanol, ethanol in 30% (v/v).
Claims (3)
1. a kind of microbial strains of high yield alkaline lipase, from corrupt onion, entitled gladiolus Burkholder of classifying
Bacterium (Burkholderiagladioli), bacterial strain number are BPS-1, and it is common to be deposited in China Committee for Culture Collection of Microorganisms
Microorganism center, deposit number CGMCCNo.10533.
A kind of 2. method that alkaline lipase is prepared using CGMCCNo.10533, it is characterised in that comprise the following steps：
(1) seed culture：After CGMCCNo.10533 is activated, LB liquid medium is inoculated in, 30-35 DEG C of culture 12-18 is small
When, obtain seed culture fluid；
(2) fermented and cultured：Seed liquor is inoculated into fermentation medium for 1-1.5% amount by volume, continuous culture 48-72 is small
Shi Hou, 8000r/mim centrifuge 10min, collect the fermented supernatant fluid rich in lipase；The fermentation medium components are：Volume
Source protein peptone that the palm oil for being 0.25-0.5% than the molasses for 0.5-1.0%, volume ratio, mass volume ratio are 1-1.5%,
Mass volume ratio is 0.2-0.25% KH2PO4, mass volume ratio be 0.05-0.1% MgSO4·7H2O；
(3) isolate and purify：Fermented supernatant fluid is obtained through the purifying of ethanol precipitation, anion-exchange chromatography and the step of gel permeation chromatography three
Obtain electrophoretically pure alkaline lipase.
3. the bacterial strain described in claim 1 prepares the application of alkaline lipase.
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| Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases;C. Elend,et al;《Applied and Environmental Microbiology》;20060531;第72卷(第5期);3637-3645 * |
| 一株高产脂肪酶产生菌16S rDNA的序列分析;孙新城等;《南方农业学报》;20121231;第43卷(第9期);1269-1272 * |
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