CN104328095B - Phospholipase A2 and its application of a kind of optimal pH in acid range - Google Patents
Phospholipase A2 and its application of a kind of optimal pH in acid range Download PDFInfo
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- CN104328095B CN104328095B CN201410504995.5A CN201410504995A CN104328095B CN 104328095 B CN104328095 B CN 104328095B CN 201410504995 A CN201410504995 A CN 201410504995A CN 104328095 B CN104328095 B CN 104328095B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01004—Phospholipase A2 (3.1.1.4)
Abstract
The invention discloses a kind of phospholipase A2 of optimal pH in acid range and its application, belong to enzyme engineering field.The present invention is will to be optimized from S.violaceoruber phospholipase A2 sequence, and the sequence fragment after optimization is cloned into Expression vector pPIC9K obtains recombinant vector, then is transformed into Pichia pastoris and is expressed after recombinant vector is linearized.The optimal pH for the phospholipase A2 that the present invention obtains is reduced to 6.0, suitable for the degumming of vegetable oil, can apply in terms of enzyme preparation production, fat degumming and Flour product processing etc..
Description
Technical field
The present invention relates to a kind of phospholipase A2 of optimal pH in acid range and its application, belong to enzyme engineering field.
Background technology
Phospholipase A2 (PLA2；EC3.1.1.4) can hydrolyze on glycerophosphatide second ester bond generation free fatty and
Lysophosphatide, and played a significant role in terms of metabolism, host defense and signal transmission.In pancreatic juice and the poison of cobra
Activity of phospholipase is found that in liquid first, then, is extracted from different types of snake, honey bee venom and mammalian pancreas
Phospholipase A2, extensive research is obtained in structure and mechanism etc..2002, Japanese scholars were separated to one plant from soil
Bacterium aubergine streptomycete Streptomyces violaceoruber A-2688 can secrete phospholipase A2.This is for the first time
Phospholipase A2 activity is detected in prokaryotes.
Phospholipase A2 can be used for the Degumming Procedures of vegetable oil.Enzymatic degumming be cut away by phosphatidase hydrolysis it is nonhydratable
The fatty acid chain generation lysophosphatide of phosphatide, and lysophosphatide has very strong hydrophily, can be by hydration side
Just remove, enzymatic degumming wide adaptability, reaction condition is gentle, can greatly save the consumption of chemical substance, be nearly free from
Waste water, there is potential advantage in environmental protection, economy, quality etc., be the development trend of fats and oils processing using enzymatic degumming.
Degumming be in sour environment, and from S.violaceoruber phospholipase A2 optimal pH in alkaline model
7.3~8.3 are enclosed, enzyme activity is 0.5U/mL, and the gene is expressed in muta lead mycillin, optimal pH 8.0, and enzyme activity is
1.04U/mL, the expression product in Escherichia coli detect activity in alkaline environment, and enzyme activity is 7.45U/mL, and the enzyme is used
Enzyme activity loss is larger when vegetable oil degumming.It is from the phospholipase A2 of muta lead mycillin, optimal pH 8.0, enzyme activity
0.2U/mL, it is relatively stable under conditions of higher than pH6.3, it is very low less than the scope stability, it is unsuitable for answering for vegetable oil degumming
With.
Pichia pastoris phaff (Pichia.pastoris) is a kind of expression system of energy high efficient expression foreign protein, is had
Have inheritance stability, expression be high, albumen can post translational processing, product can secrete, can high density fermentation many advantages, such as, application
Quite varied, existing hundreds of foreign proteins are expressed within the system.S.violaceoruber is expressed with P.pastoris
The phospholipase A_2 gene in source, the optimal pH of the enzyme is set to be reduced to 6.0, the degumming suitable for vegetable oil.
The content of the invention
The present invention provides phospholipase A2 and its application of a kind of optimal pH in acid range.
The present invention first purpose be to provide a kind of preparation method of phospholipase A2, be by encoding amino acid sequence such as
The gene fragment clone of sequence shown in SEQ ID NO.1 obtains recombinant vector to expression vector, then will be converted after recombinant vector
Genetic engineering bacterium is obtained into Pichia yeast, induced expression obtains phospholipase A2.
The optimal pH of the phospholipase A2 is in acid range.
The optimal pH of the phospholipase A2 is 6.
The nucleotide sequence of the phospholipase A2 is encoded as shown in SEQIDNO.2.
The expression vector is in pPIC9K, pPIC9, pPIC Z α, pGAPZ α, pPIC3.5, pPIC3.5K, pYAM75P6
Any one.
The Pichia yeast can be it is following any one：GS115、KM71、SMD1168.
The expression vector, it is in one embodiment of the invention pPIC9K.
The Pichia yeast, it is in one embodiment of the invention P.pichia GS115.
Second object of the present invention is to provide a kind of phospholipase A2 obtained according to the above method
Third object of the present invention is to provide a kind of genetic engineering bacterium for expressing the phospholipase A2.
The construction method of the genetic engineering bacterium, it is by the nucleotides piece of amino acid sequence shown in coding SEQ ID NO.1
Section, which is cloned into expression vector, forms recombinant vector, then recombinant vector is transformed into Host Strains, screening positive clone, that is, obtains
The genetic engineering bacterium of the phospholipase A2 can be expressed.
The gene order of the nucleotide fragments is as shown in SEQ ID NO.2.
The construction method of the genetic engineering bacterium, it is specially：(1) base shown in chemical synthesis synthesis SEQ ID NO.2
Because of fragment；(2) primer 80PLA2F (sequence is as shown in SEQ ID NO.3) and 81PLA2R (sequence such as SEQ ID NO.4 institutes are used
Show) the obtained fragment of amplification step 1, and be connected in E. coli cloning vector and obtain recombinant plasmid；(3) by recombinant plasmid and
PPIC9K connects to obtain recombinant vector pPIC9K-PLA2 with EcoR I with Not I digestions simultaneously；(4) Sal I are used by pPIC9K-
Electricity is transferred to P.pichia GS115, screening positive clone after PLA2 is linear.
Fourth object of the present invention is to provide a kind of produces the phospholipase A2 using the engineering bacteria fermentation
Method, it is by genetic engineering bacterium in BMGY culture mediums, is cultivated under 30 DEG C of temperature, rotating speed 200rpm to OD2~6, collect thalline
And be resuspended in BMMY culture mediums, cultivated under 28 DEG C of temperature, rotating speed 200rpm, addition methanol induction expression, fermentation supernatant is
Contain the phospholipase A2.
The addition manner of methanol is the methanol of the addition 1% per 24h in methods described.
Described 1% methanol, refer to add 1mL methanol in every 100mL zymotic fluids.
The present invention also provides a kind of method that phospholipase A2 is used for vegetable oil degumming, is to take vegetable oil 50g, is preheated to
80 DEG C, 2.5mol/L citric acid solution 0.125mL are added, temperature is reduced to 40 DEG C after the temperature maintains 45min, added
5mol/LNaOH is well mixed to make pH value of solution be 5.0, adds 4%pH5.0 citric acid-sodium citrate buffer solution, 253U enzyme liquids,
1.0mmol/LCa2+, 40 DEG C of concussion reaction 6h are placed in, that is, obtain the vegetable oil through phospholipase A2 degumming.
Phospholipase A2 provided by the invention, relative to the phospholipase A2 in S.violaceoruber sources, its optimal pH is from 8
6 are reduced to, the degumming suitable for vegetable oil.The optimal pH of the enzyme of gained is reduced to 6.0, it may be possible to because expression product occurs necessarily
Glycosylation modified, the glycosylation modified hydrophobe property for changing whole albumen of degree, it is microcosmic so as to have influence on activated centre
Charge environment, and then change optimal pH.The present invention lipase A2 optimum temperature be 50 DEG C, under cryogenic stability compared with
It is good, it is on a declining curve with the rise stability of temperature, maximum activity and the most stable is shown in pH6.0, optimal pH with
Under temperature conditionss, enzyme activity reaches 35U/mL.The phospholipase A2 of the present invention can be applied in enzyme preparation production, fat degumming and face
In terms of product processing etc..
Brief description of the drawings
Fig. 1：Methanol induction of Pichia pastoris expression alien gene PLA2；A is thalli growth curve (OD600), B be extracellular egg
White concentration curve, C are extracellular supernatant phospholipase activity curve.
Fig. 2：Phospholipase A2Amino acid sequence；It is possible glycosylation site at underscore.
Fig. 3：The zymologic property curve of phospholipase A2；A:Optimum temperature；B:Temperature stability；C:Optimal pH；D:PH is stable
Property.
Fig. 4：Streptomyces violaceoruber A-2688 phospholipase A2s structures (PDB ID：1IT4), spherical institute
It is N- glycosylation sites to show amino acid, and shaft-like shown amino acid is activated centre site.
Embodiment
Embodiment 1
It is with the phospholipase A2 sequence (GenBank NO.AY359866.1) of S.violaceoruber in ncbi database
Basis, phospholipase A_2 gene is optimized according to P.pastoris codon-bias, the amino acid sequence of optimization
As shown in SEQ ID NO.1, nucleotide sequence gives birth to work biology Co., Ltd as shown in SEQ ID NO.2, by Shanghai and synthesizes SEQ
The nucleotide fragments of sequence shown in ID NO.2.
The structure of the pichia yeast genetic engineering bacteria of the phospholipase A2 of embodiment 2
According to the gene order (shown in SEQ ID NO.2) of phospholipase A2, design primer (such as SEQ ID NO.3 and SEQ
Shown in ID NO.4)：
80PLA2F：5'-ATCAGAATTCGCTCCACCTCAGGCTGC-3'(dashed parts are EcoR I restriction enzyme sites)
81PLA2R：5'-CGTCGCGGCCGCTTAAAGAATTTTCACGGCCTGG-3'(dashed parts are Not I digestions
Site)
The nucleotide fragments of sequence, obtain purpose fragment, by mesh after purification shown in primer amplification ID containing SEQ NO.2
Genetic fragment add A tails (μ L of TaKaRa Taq 0.5, the μ L of 10 × PCR Buffer 5, μ L of dNTP Mixture 4, purified product
40.5 μ L react 15min under the conditions of 72 DEG C) it is connected to pMD-19T carriers and obtains recombinant plasmid pMD-19T-PLA2, by plasmid
PMD-19T-PLA2 connects to obtain recombinant vector pPIC9K-PLA2 with EcoR I with Not I digestions simultaneously with pPIC9K, uses SalI
Electricity is transferred in P.pichiaGS115 (electric shifting method after linear：A pipe competent cell is taken to be transferred to linear after digestion concentrates
Change in plasmid, uniformly mixing, is transferred in the electric revolving cup of 2mm precoolings, and progress electricity turns after placing 3-5min on ice.Electric conversion instrument
Arrange parameter is：Voltage 1500V, the Ω of resistance 200, the μ F of electric capacity 25.The sorbierite for adding 1mL 1mol/L after electricity conversion immediately is molten
Liquid, it is transferred to after mixing in 1.5mLEP pipes, 30 DEG C of recovery 1h, except supernatant to 100 μ L of residue after centrifugation, is coated with after thalline is resuspended
MD flat boards, a series of single bacterium colony transformants are just can obtain after cultivating 3d in 30 DEG C of incubators.), it is coated on containing G418 (0.75mg/
ML) on the MD flat boards of resistance, picking monoclonal carries out positive verification, and it is the phospholipase A2 successfully constructed to verify correct bacterial strain
Pichia yeast genetic engineering bacteria.
The shake flask fermentation of the recombinant yeast pichia pastoris genetic engineering bacterium of embodiment 3
By the line of positive restructuring bacterium with being cultivated 3 days or so on YPD flat boards, picking monoclonal and the culture mediums of BMGY containing 25ml
250mL triangular flasks in, 30 DEG C, 200r/min cultivate 16~18h OD600 is centrifuged bacterium solution, thalline is resuspended in up to 2~6
In 100mL BMMY culture mediums, 28 DEG C, 200r/min cultures, the expression of 1% methanol induction is added per 24h.Timing sampling determines it
Cell concentration, protein concentration and enzyme activity (Fig. 1).Enzyme activity reaches maximum during fermentation 84h.The SDS-PAGE of fermented liquid supernatant shows
Showing, control strain (Pichia pastoris GS115) is without obvious band, and recombinant bacterium has a thick and heavy band in 14.4kDa or so, with
The phospholipase A2 molecular weight 13.3kDa of theoretical calculation is close, and the band produces by the PLA2 that Mass Spectrometric Identification is recombinant bacterium expression
Thing.
YDP culture medium prescriptions：1% dusty yeast, 2% tryptone, 2% glucose.
BMGY culture medium prescriptions：1% glycerine, 2% tryptone, 1% dusty yeast, 1.34%YNB, 4 × 10-5% biologies
Element, 10%pH6.0 phosphate buffers.
BMMY culture medium prescriptions：1% dusty yeast, 2% tryptone, 1.34%YNB, 4 × 10-5% biotins, 10%
PH6.0 phosphate buffers, 1.5% methanol.
Determination of protein concentration method：20 μ l fermented liquid supernatants are added in the hole of 96 orifice plates, add 200 μ lG250 dyeing
Liquid, room temperature place 3-5 minutes.The absorbance of A595 wavelength is determined with ELIASA,
The protein concentration in sample is calculated according to standard curve.
Mass Spectrometric Identification method：The target stripe on glue is cut with knife blade, as in EP pipes, while the glue for cutting sky is made
For control；Add 200-400 μ L 100mmol/LNH4HCO3/ 30%ACN decolourizes, and cleans to transparent, removal supernatant, lyophilized；Often
Pipe adds 90 μ L 100mmol/L NH4HCO3, 10 μ L 100mmol/L DTT, 56 DEG C hatch 30 minutes, go back crude protein；Go
Clearly, often pipe adds 100 μ L 100%ACN, is sucked after 5 minutes；Often pipe adds 70 μ L 100mmol/L NH4HCO3, 30 μ L
200mmol/L IAA, dark place 20 minutes；Supernatant is removed, often pipe adds 100 μ L 100mmol/L NH4HCO3, room temperature 15 minutes；Go
Supernatant, 100 μ L 100%ACN are added, are sucked after 5 minutes, freezed；It is each to add 5 μ L 2.5-10ng/ μ L Trypsin after lyophilized
Solution, 4 DEG C of 30-60 minutes are placed in, make the abundant imbibition of blob of viscose；Add 20-30 μ L or so 25mmol/L NH4HCO3Buffer solution,
PH7.8-8.0,37 DEG C of reactions are stayed overnight, 20 hours or so；Enzymolysis liquid is suctioned out, is transferred in EP pipes, former pipe adds 10 μ L 60%
CAN, 0.1%TFA, ultrasound 15 minutes, solution is suctioned out, is incorporated to previous solution, freezed；Sample preparation is completed, and can be redissolved, point
Sample, carry out mass spectral analysis.
Phospholipase A2 is expressed using pichia yeast expression system, this method has inheritance stability, expression height, albumen
Can post translational processing, product can secrete, can high density fermentation many advantages, such as.
The recombinant phospholipase A2 zymologic property of embodiment 4 determines
(1) phospholipase A2 enzyme activity determination method
A. polyvinyl alcohol (PVA) 40.0g is weighed, adds water 800mL, heats, stir in boiling water bath, until all dissolvings,
1000mL is dissolved to after cooling.Filtered with clean double gauze, take filtrate standby；
B. phosphate buffer and PVA are using volume ratio as 19:1 ratio mixing, make PVA final concentration of 0.2% (w/v)
C. soybean lecithin 3g is weighed to be dissolved in 60ml phosphate buffers and PVA mixed liquor；Use high-speed tissue mashing machine
Handle 6min (points of 2 times processing, each 3min, be spaced 5min), milky PVA emulsions.
D. two 100mL triangular flasks are taken, respectively in blank bottle (A) and sample bottle (B), respectively add substrate solution and phosphoric acid to delay
Fliud flushing mixed liquor 9.00mL, 95% ethanol (3.3) 15.0mL is added in A bottles, 5min is preheated in 40 ± 0.2 DEG C of water-baths,
Then, in A bottles, add in inactivation enzyme liquid 1.00mL, B bottle and add enzyme liquid 1ml to be measured, timing is mixed immediately, in 40 ± 0.2 DEG C of water-baths
Middle accurate response 15min, add 95% ethanol 15.0mL terminating reactions immediately in B cups, take out；
E. respectively add instructions phenolphthalein solution 2 to drip in blank and sample solution, dripped with 0.05mol/L standard solution of sodium hydroxide
It is fixed, up to blush and keep 30s not take off for its terminal, the volume of record consumption 0.05mol/L standard solution of sodium hydroxide.
Phospholipase activity is defined as：Under the conditions of certain temperature and pH, 1min hydrolysis substrates produce 1 μm of ol titratable fat
Enzyme amount required for fat acid is 1 enzyme activity unit, is represented with U/ml.
Phospholipase activity calculation formula：
In formula：
X1The enzyme activity of-sample, U/mL；
V1The volume of the standard solution of sodium hydroxide consumed during-titration sample, unit is (mL)；
V2The volume of the standard solution of sodium hydroxide consumed during-titration blank, unit is (mL)；
C-Concentration of Sodium Hydroxide Solution Standard, unit are mole every liter (mol/L)；
50-0.05mol/L sodium hydroxide solutions 1.00mL is equivalent to 50 μm of ol of aliphatic acid；
n1The extension rate of-sample；
0.05-Concentration of Sodium Hydroxide Solution Standard calculates coefficient；
15-reaction time 15min, in terms of 1min.
(2) phospholipase A2 zymologic property assay method：
Influence of the temperature to enzyme activity and its stability：(25 DEG C -65 DEG C) measure enzyme activity at different temperatures, with highest enzyme
Vigor is 100%, and enzyme activity highest is the optimum temperature of the enzyme.During testing temperature stability, by enzyme liquid in different temperature
Under the conditions of be incubated 1h, determine enzyme activity under the conditions of 40 DEG C after cooled on ice, uninsulated enzyme activity is 100%.
Influences of the pH to enzyme activity and its stability：0.05M different pH buffer solutions are configured, substrate is configured with buffer solution,
Enzyme activity of the enzyme liquid under condition of different pH is determined, pH corresponding to highest enzyme activity is the optimal pH of the enzyme, determines pH stability
When, enzyme activity is determined under the conditions of enzyme liquid is placed into the optimum temperature optimal pH measured after 5h above under condition of different pH, is set
Original enzyme liquid enzyme activity it is maximum for 100%.
The zymologic property of recombinant phospholipase A2 is determined, as shown in Figure 3.Fig. 3 A illustrate that the optimum temperature of phospholipase A2 is 50
DEG C, the enzyme stability is preferable under cryogenic, and the rise stability with temperature is (Fig. 3 B) on a declining curve., should in pH6.0
Enzyme shows maximum activity (Fig. 3 C), and Fig. 3 D show that the enzyme is the most stable in pH6.0.The optimal pH of recombinant phospholipase A2 is
More it is adapted to degumming application study under the conditions of 6.0, the pH.It is illustrated in figure 4 Streptomyces violaceoruber A-
Structure (the protein structure PDB database accession numbers of 2688 phospholipase A2s：1IT4), phospholipase A2 molecular structure is relatively simple, is located at
3 glycosylation sites (amino acid shown in spherical) of protein surface are if it happens glycosylation modified, can be to changing whole albumen
Hydrophobe property, so as to have influence on the microcosmic charge environment in activated centre (amino acid shown in shaft-like), and then change optimal pH.This
It is 6 to invent obtained enzyme optimal pH, it may be possible to occur it is a certain degree of it is glycosylation modified caused by, as shown in Fig. 2 the phosphatidase
With 3 potential glycosylation sites.After measured, enzyme activity of the recombinant phospholipase A2 under the conditions of optimal pH, optimum temperature is
35U/mL。
The vegetable oil degumming application of the recombinant phospholipase A2 of embodiment 5
The PLA2 fermented liquid supernatants of genetic engineering bacterium are dialysed with the citric acid-sodium citrate buffer solution that 5LpH is 5.0, removed
The P elements gone in zymotic fluid, the influence to degumming effect is reduced, degumming experiment is done after super filter tube concentration.
Level Four rapeseed oil 50g is taken, is preheated to 80 DEG C, adds 2.5mol/L citric acid solution 0.125mL, is maintained in the temperature
Temperature is reduced to 40 DEG C after 45min, adding 5mol/L NaOH and being well mixed makes pH value of solution be 5.0, adds 4%pH5.0 lemon
Acid-sodium citrate buffer solution, 253U enzyme liquids, 1.0mmol/L Ca2+, it is placed in reaction 6h, 7000rpm centrifugations in 40 DEG C of shaking tables
20min, upper oil phase is taken to survey phosphorus content (measure of national standard GBT5537-2008 grain and oil detection content of phospholipid).Result of study shows
Content of phospholipid drops to 10mg/Kg from 130mg/Kg.
Application of the recombinant phospholipase A2 of embodiment 6 in bread baking
Influence of the recombinant phospholipase A2 to bread specific volume is mainly have studied in bread baking experiment.
Bread preparation method：With reference to AACC method 10-10B, and certain modification is made.Flour 100%, salt 1%, white granulated sugar
4%, butter 4%, yeast 1.5%, water 62.5% (in terms of flour quality), the addition of recombinant phospholipase A2 is 250U/kg,
Lipase is not added in blank control.After above-mentioned raw materials are stirred into 10min in mixer, 10min is stood, is divided into 100g/
It is individual, rub circle with the hands, stand 10min, be molded sabot, proof 90min under conditions of 38 DEG C, relative humidity 85%, bakee 25min (on
It is fiery 170 DEG C, lower fiery 210 DEG C), it is standby after cooling.
Bread specific volume determines：Determined using vegetable seed exclusive method.Bread measures the volume of bread respectively after room temperature cools down 1h
And quality.
Specific volume (mL/g)=volume (mL)/quality (g).
Specific volume value added (%)=(sample specific volume-blank control specific volume)/blank control specific volume * 100%
Result of study shows that the specific volume maximum that with the addition of recombinant phospholipase A2 adds 22%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (8)
1. a kind of preparation method of phospholipase A2, it is by the gene piece of encoding amino acid sequence sequence as shown in SEQ ID NO.1
Section is cloned into expression vector and obtains recombinant vector, then recombinant vector is transformed into the genetic engineering bacterium obtained in Pichia yeast,
Phospholipase A2 is obtained through induced expression；The optimal pH of the phospholipase A2 is in acid range；The expression vector be pPIC9K,
Any one in pPIC9, pPIC Z α, pGAPZ α, pPIC3.5, pPIC3.5K, pYAM75P6；The Pichia yeast be with
Descend any one：GS115、KM71、SMD1168.
2. method according to claim 1, it is characterised in that encode the nucleotide sequence such as SEQ ID of the phospholipase A2
Shown in NO.2.
3. according to the method for claim 1, it is characterised in that the expression vector is pPIC9K.
4. according to the method for claim 1, it is characterised in that the Pichia yeast is P.pichia GS115.
A kind of 5. phospholipase A2 that method according to claim 1 obtains.
A kind of 6. genetic engineering bacterium for expressing phospholipase A2 described in claim 5.
7. genetic engineering bacterium according to claim 6, it is characterised in that the genetic engineering bacterium is will to encode SEQ ID
The gene fragment clone of amino acid sequence shown in NO.1 forms recombinant vector into expression vector, then recombinant vector is transformed into place
In main bacterium, screening positive clone.
8. application of the phospholipase A2 in terms of enzyme preparation production, fat degumming and Flour product processing described in claim 5.
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| RU2766448C2 (en) * | 2019-12-27 | 2022-03-15 | Федеральное государственное учреждение "Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук" (ФИЦ Биотехнологии РАН) | OBTAINING PHOSPHOLIPASE A2 GENE WITH CHANGED pH OPTIMUM BY REMOVING GLYCOSYLATION SITES |
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| CN108048339B (en) * | 2017-12-07 | 2021-12-28 | 无锡蔚蓝生物科技有限公司 | Bacterial strain for recombinant expression of phospholipase and application thereof |
| RU2746817C1 (en) * | 2020-06-10 | 2021-04-21 | Общество с ограниченной ответственностью "Инновационный центр "Бирюч-новые технологии" | Method for producing phospholipase а2 komagataella phaffii (pichia pastoris) yib δleu2_pla2s production strain |
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