CN104328095A - Phospholipase A2 with most appropriate pH being in acid range and application thereof - Google Patents

Abstract

The invention discloses a phospholipase A2 whose optimal pH is in the acid range and application thereof, belonging to the field of enzyme engineering. The invention optimizes the phospholipase A2 sequence derived from S. violaceoruber, clones the optimized sequence fragment into the expression vector pPIC9K to obtain a recombinant vector, and then linearizes the recombinant vector and transforms it into Pichia pastoris for expression. The optimal pH of the phospholipase A2 obtained by the invention is reduced to 6.0, which is suitable for degumming of vegetable oil, and can be applied in aspects such as enzyme preparation production, oil degumming and flour product processing.

Description

A kind of optimal pH is in the Phospholipase A2 of acid range and application thereof

Technical field

The present invention relates to a kind of optimal pH in the Phospholipase A2 of acid range and application thereof, belong to enzyme engineering field.

Background technology

Phospholipase A2 (PLA2; The ester bond that EC3.1.1.4) can be hydrolyzed on glyceryl phosphatide second generates free fatty acids and lysophospholipid, and plays a significant role in metabolism, host defense and signal transmission.In pancreatic juice and elapid venom, Late Cambrian phospholipase activity, subsequently, extracts Phospholipase A2 from different types of snake, meltittin venom and mammalian pancreas, obtains and study widely in structure and mechanism etc.2002, Japanese scholars is separated to a strain bacterium red-purple streptomycete Streptomyces violaceoruber A-2688 from soil can secrete Phospholipase A2.This is in prokaryotic organism, detect that Phospholipase A2 is active first time.

Phospholipase A2 can be used for the Degumming Procedures of vegetables oil.Enzymatic degumming is the fatty acid chain generation lysophospholipid being cut away non-hydratable phospholipid by Phospholipid hydrolase hydrolytic action, and lysophospholipid has very strong wetting ability, can be removed easily by hydration, enzymatic degumming wide adaptability, reaction conditions is gentle, greatly can save the consumption of chemical substance, produce waste water hardly, in environmental protection, economy, quality etc., there is potential advantage, utilize enzymatic degumming to be the development trend of fats and oils processing.

Come unstuck is in sour environment, and derive from the optimal pH of the Phospholipase A2 of S.violaceoruber at alkaline range 7.3 ~ 8.3, enzyme work is 0.5U/mL, this gene is expressed in muta lead mycillin, optimal pH is 8.0, and enzyme is lived as 1.04U/mL, and the expression product in intestinal bacteria detects activity in alkaline environment, enzyme work is 7.45U/mL, and when this enzyme being used for vegetable oil degumming, enzyme activity loss is larger.Derive from the Phospholipase A2 of muta lead mycillin, optimal pH is 8.0, and enzyme work is 0.2U/mL, more stable under higher than the condition of pH6.3, very low lower than this range stabilises, is unsuitable for the application of vegetable oil degumming.

Pichia pastoris phaff (Pichia.pastoris) is a kind of expression system of energy high expression foreign protein, have that inheritance stability, expression level are high, albumen can post translational processing, product can secrete, can many advantages such as high density fermentation, application is very extensive, and existing hundreds of foreign protein obtains expression within the system.Express the phospholipase A_2 gene in S.violaceoruber source with P.pastoris, make the optimal pH of this enzyme be reduced to 6.0, be suitable for plant oil degumming.

Summary of the invention

The invention provides a kind of optimal pH in the Phospholipase A2 of acid range and application thereof.

First object of the present invention is to provide a kind of preparation method of Phospholipase A2, that the gene fragment clone of the sequence of encoding amino acid sequence as shown in SEQ ID NO.1 is obtained recombinant vectors to expression vector, obtain genetic engineering bacterium by being transformed in Pichia yeast after recombinant vectors again, namely abduction delivering obtains Phospholipase A2.

The optimal pH of described Phospholipase A2 is at acid range.

Described Phospholipase A2 optimal pH be 6.

Encode the nucleotide sequence of described Phospholipase A2 as shown in SEQIDNO.2.

Described expression vector is any one in pPIC9K, pPIC9, pPIC Z α, pGAPZ α, pPIC3.5, pPIC3.5K, pYAM75P6.

Described Pichia yeast can be following any one: GS115, KM71, SMD1168.

Described expression vector is pPIC9K in one embodiment of the invention.

Described Pichia yeast is P.pichia GS115 in one embodiment of the invention.

Second object of the present invention is to provide a kind of Phospholipase A2 obtained according to aforesaid method

3rd object of the present invention is to provide a kind of genetic engineering bacterium of expressing described Phospholipase A2.

The construction process of described genetic engineering bacterium, the nucleotide fragments of aminoacid sequence shown in coding SEQ ID NO.1 is cloned in expression vector form recombinant vectors, again recombinant vectors is transformed in Host Strains, screening positive clone, namely obtains the genetic engineering bacterium expressing described Phospholipase A2.

The gene order of described nucleotide fragments is as shown in SEQ ID NO.2.

The construction process of described genetic engineering bacterium, is specially: the gene fragment shown in (1) chemical synthesis synthesis SEQ ID NO.2; (2) use primer 80PLA2F (sequence is as shown in SEQ ID NO.3) and the fragment that obtains of 81PLA2R (sequence is as shown in SEQ ID NO.4) amplification step 1, and be connected in E. coli cloning vector and obtain recombinant plasmid; (3) recombinant plasmid and pPIC9K are cut to be connected with Not I enzyme with EcoR I simultaneously obtain recombinant vectors pPIC9K-PLA2; (4) with Sal I, linear for pPIC9K-PLA2 rear electricity is proceeded to P.pichia GS115, screening positive clone.

4th object of the present invention is to provide a kind of method utilizing described engineering bacteria fermentation to produce described Phospholipase A2, be by genetic engineering bacterium in BMGY substratum, OD2 ~ 6 are cultured under temperature 30 DEG C, rotating speed 200rpm, collect thalline and be resuspended in BMMY substratum, cultivate under temperature 28 DEG C, rotating speed 200rpm, add methanol induction to express, fermentation supernatant is namely containing described Phospholipase A2.

In described method, the addition manner of methyl alcohol is the methyl alcohol that every 24h adds 1%.

The methyl alcohol of described 1%, refers in every 100mL fermented liquid and adds 1mL methyl alcohol.

The present invention also provides a kind of described Phospholipase A2 for the method for vegetable oil degumming, get vegetables oil 50g, be preheated to 80 DEG C, add 2.5mol/L citric acid solution 0.125mL, maintain after 45min in this temperature and temperature is reduced to 40 DEG C, add 5mol/LNaOH and mix and make pH value of solution be 5.0, add the citric acid-sodium citrate damping fluid of 4%pH5.0,253U enzyme liquid, 1.0mmol/LCa ²⁺, be placed in 40 DEG C of concussion reaction 6h, namely obtain the vegetables oil come unstuck through Phospholipase A2.

Phospholipase A2 provided by the invention, relative to the Phospholipase A2 in S.violaceoruber source, its optimal pH is reduced to 6 from 8, is suitable for plant oil degumming.The optimal pH of the enzyme of gained is reduced to 6.0, may be because to a certain degree glycosylation modified occurs expression product, the glycosylation modified hydrophobe character changing whole albumen, thus has influence on the charge environment of active centre microcosmic, and then changes optimal pH.The optimum temperuture of lipase A2 of the present invention is 50 DEG C, and stability is better under cryogenic, and the rising stability with temperature is on a declining curve, and show maximum activity and the most stable when pH6.0, under optimal pH and temperature condition, enzyme work reaches 35U/mL.Phospholipase A2 of the present invention can be applied in the aspects such as zymin production, fat degumming and flour products processing aspect.

Accompanying drawing explanation

Fig. 1: methanol induction of Pichia pastoris expression alien gene PLA ₂; A is thalli growth curve (OD ₆₀₀), B is cellular protein concentration curve, C is the outer supernatant phospholipase activity curves of born of the same parents.

Fig. 2: phospholipase A ₂aminoacid sequence; Underscore place is possible glycosylation site.

Fig. 3: the zymologic property curve of Phospholipase A2; A: optimum temperuture; B: temperature stability; C: optimal pH; D:pH stability.

Fig. 4: Streptomyces violaceoruber A-2688 Phospholipase A2 structure (PDB ID:1IT4), shown in spherical, amino acid is N-glycosylation site, and shown in shaft-like, amino acid is site, active centre.

Embodiment

Embodiment 1

In ncbi database S.violaceoruber Phospholipase A2 sequence (GenBank NO.AY359866.1) based on, codon-bias according to P.pastoris is optimized phospholipase A_2 gene, the aminoacid sequence of majorizing sequence is as shown in SEQ ID NO.1, nucleotide sequence, as shown in SEQ ID NO.2, is synthesized the nucleotide fragments of sequence shown in SEQ ID NO.2 by the Shanghai biological company limited of raw work.

The structure of the pichia yeast genetic engineering bacteria of embodiment 2 Phospholipase A2

According to the gene order (shown in SEQ ID NO.2) of Phospholipase A2, design primer (as shown in SEQ ID NO.3 and SEQ ID NO.4):

80PLA2F:5'-ATCA gAATTCgCTCCACCTCAGGCTGC-3'(dashed part is EcoR I restriction enzyme site)

81PLA2R:5'-CGTC gCGGCCGCtTAAAGAATTTTCACGGCCTGG-3'(dashed part is Not I restriction enzyme site)

With the nucleotide fragments of primer amplification containing sequence shown in SEQ ID NO.2, obtain object fragment, goal gene fragment after purifying is added A tail (TaKaRa Taq 0.5 μ L, 10 × PCR Buffer 5 μ L, dNTP Mixture 4 μ L, purified product 40.5 μ L reacts 15min under 72 DEG C of conditions) be connected to pMD-19T carrier and obtain recombinant plasmid pMD-19T-PLA2, plasmid pMD-19T-PLA2 and pPIC9K is cut to be connected with Not I enzyme with EcoR I simultaneously and obtains recombinant vectors pPIC9K-PLA2, with the linear rear electricity of SalI to proceed in P.pichiaGS115 (electric shifting method: get a pipe competent cell be transferred to enzyme cut concentrated after linearization plasmid in, Homogeneous phase mixing, transfer in the electric revolving cup of 2mm precooling, carry out electricity after placing 3-5min on ice to turn.Electricity conversion instrument parameters is: voltage 1500V, resistance 200 Ω, electric capacity 25 μ F.Electricity adds the Sorbitol Solution USP of 1mL 1mol/L immediately after transforming, be transferred in 1.5mLEP pipe after mixing, 30 DEG C of recovery 1h, except supernatant is to residue 100 μ L after centrifugal, be coated with MD flat board after resuspended thalline, after cultivating 3d in 30 DEG C of incubators, just can obtain a series of single colony transformation.), coat on the MD flat board containing G418 (0.75mg/mL) resistance, picking mono-clonal carries out positive verification, verifies that correct bacterial strain is the pichia yeast genetic engineering bacteria of the Phospholipase A2 successfully constructed.

The shake flask fermentation of embodiment 3 recombinant yeast pichia pastoris genetic engineering bacterium

By positive recombinant bacterium line and cultivation about 3 days on YPD flat board, picking mono-clonal with containing 25ml BMGY substratum 250mL triangular flask in, 30 DEG C, 200r/min cultivate 16 ~ 18h make OD600 reach 2 ~ 6, by centrifugal for bacterium liquid, thalline is resuspended in 100mL BMMY substratum, 28 DEG C, 200r/min cultivation, every 24h adds 1% methanol induction to express.Timing sampling measures its cell concentration, protein concentration and enzyme activity (Fig. 1).During fermentation 84h, enzyme activity reaches maximum.The SDS-PAGE display of fermented liquid supernatant, control strain (Pichia pastoris GS115) is without obvious band, and recombinant bacterium has a thick and heavy band at about 14.4kDa, be close with the Phospholipase A2 molecular weight 13.3kDa of Theoretical Calculation, this band through Mass Spectrometric Identification be recombinant bacterium express PLA2 product.

YDP culture medium prescription: 1% yeast powder, 2% Tryptones, 2% glucose.

BMGY culture medium prescription: 1% glycerine, 2% Tryptones, 1% yeast powder, 1.34%YNB, 4 × 10 ^-5% vitamin H, 10%pH6.0 phosphoric acid buffer.

BMMY culture medium prescription: 1% yeast powder, 2% Tryptones, 1.34%YNB, 4 × 10 ^-5% vitamin H, 10%pH6.0 phosphoric acid buffer, 1.5% methyl alcohol.

Determination of protein concentration method: 20 μ l fermented liquid supernatant are added in the hole of 96 orifice plates, then add 200 μ lG250 staining fluids, room temperature places 3-5 minute.The absorbancy of A595 wavelength is measured by microplate reader,

The protein concentration in sample is calculated according to typical curve.

Mass Spectrometric Identification method: cut the target stripe on glue with knife blade, as in EP pipe, cuts empty glue in contrast simultaneously; Add 200-400 μ L 100mmol/LNH ₄hCO ₃/ 30%ACN decolours, and cleaning, to transparent, removes supernatant, freeze-drying; Often pipe adds 90 μ L 100mmol/L NH ₄hCO ₃, 10 μ L 100mmol/L DTT, hatch 30 minutes, go back crude protein for 56 DEG C; Remove supernatant, often pipe adds 100 μ L 100%ACN, sucks after 5 minutes; Often pipe adds 70 μ L 100mmol/L NH ₄hCO ₃, 30 μ L 200mmol/L IAA, 20 minutes, dark place; Remove supernatant, often pipe adds 100 μ L 100mmol/L NH ₄hCO ₃, room temperature 15 minutes; Remove supernatant, add 100 μ L 100%ACN, suck after 5 minutes, freeze-drying; After freeze-drying, respectively add 5 μ L 2.5-10ng/ μ L Trypsin solution, be placed in 4 DEG C of 30-60 minute, make the abundant imbibition of blob of viscose; Add 20-30 μ about L 25mmol/L NH again ₄hCO ₃damping fluid, pH7.8-8.0,37 DEG C of reactions are spent the night, 20 hours; Sucking-off enzymolysis solution, is transferred in EP pipe, and former pipe adds 10 μ L 60%CAN, 0.1%TFA, ultrasonic 15 minutes, and sucking-off solution, is incorporated to previous solution, freeze-drying; Sample preparation completes, and can redissolve, and point sample carries out mass spectroscopy.

Use pichia yeast expression system to express Phospholipase A2, the method have that inheritance stability, expression level are high, albumen can post translational processing, product can secrete, can many advantages such as high density fermentation.

Embodiment 4 recombinant phospholipase A2 zymologic property measures

(1) Phospholipase A2 enzyme activity determination method

A. weighing polyvinyl alcohol (PVA) 40.0g, add water 800mL, heats, stirs, until all dissolve, be dissolved to 1000mL after cooling in boiling water bath.Filter with clean double gauze, it is for subsequent use to get filtrate;

B. phosphoric acid buffer and PVA take volume ratio as the ratio mixing of 19:1, make the final concentration of PVA be 0.2% (w/v)

C. taking soybean lecithin 3g is dissolved in the mixed solution of 60ml phosphoric acid buffer and PVA; Process 6min (point 2 times process, each 3min, interval 5min) with high-speed tissue mashing machine, oyster white PVA emulsion.

D. two 100mL triangular flasks are got, respectively in blank bottle (A) and sample bottle (B), respectively add substrate solution and phosphoric acid buffer mixed solution 9.00mL, 95% ethanol (3.3) 15.0mL is added again in A bottle, preheating 5min in 40 ± 0.2 DEG C of water-baths, then, in A bottle, add fermentoid liquid 1.00mL, add enzyme liquid 1ml to be measured in B bottle, mix timing immediately, accurate response 15min in 40 ± 0.2 DEG C of water-baths, in B cup, add 95% ethanol 15.0mL termination reaction immediately, take out;

E. in blank and sample solution, instructions phenolphthalein solution 2 is respectively added, with the titration of 0.05mol/L standard solution of sodium hydroxide, until blush 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 certain temperature and pH condition, and the enzyme amount required for titratable lipid acid that 1min hydrolysis substrate produces 1 μm of ol is 1 enzyme activity unit, represents with U/ml.

Phospholipase activity calculation formula:

$X_1=\frac{(V_1-V_2)\×c\×50\×n_1}{0.05}\×\frac{1}{15}$

In formula:

X ₁the enzyme activity of-sample, U/mL;

V ₁the volume of the standard solution of sodium hydroxide consumed during-titration sample, unit is (mL);

V ₂the volume of the standard solution of sodium hydroxide consumed when-titration is blank, unit is (mL);

C-Concentration of Sodium Hydroxide Solution Standard, unit is mole often liter (mol/L);

50-0.05mol/L sodium hydroxide solution 1.00mL is equivalent to lipid acid 50 μm of ol;

N ₁the extension rate of-sample;

0.05-Concentration of Sodium Hydroxide Solution Standard calculates coefficient;

15-reaction times 15min, in 1min.

(2) Phospholipase A2 zymologic property measuring method:

Temperature is on the impact of enzyme activity and stability thereof: (25 DEG C-65 DEG C) measure enzyme and live at different temperatures, with most high enzymatic activity for 100%, and the optimum temperuture of what enzyme activity was the highest be this enzyme.During testing temperature stability, enzyme liquid is incubated 1h under different temperature condition, after cooled on ice, measure enzyme under 40 DEG C of conditions live, uninsulated enzyme work is 100%.

PH is on the impact of enzyme activity and stability thereof: the different pH damping fluids of configuration 0.05M, substrate is configured with damping fluid, measure the enzyme of enzyme liquid under condition of different pH to live, the pH that most high enzymatic activity is corresponding is the optimal pH of this enzyme, when measuring pH stability, measure enzyme work under the optimum temperuture optimal pH condition measured above after enzyme liquid is placed 5h under condition of different pH, what the work of setting original enzyme liquid enzyme was maximum is 100%.

Measure the zymologic property of recombinant phospholipase A2, as shown in Figure 3.Fig. 3 A illustrates that the optimum temperuture of Phospholipase A2 is 50 DEG C, and this enzyme stability is better under cryogenic, the rising stability with temperature (Fig. 3 B) on a declining curve.When pH6.0, this enzyme shows maximum activity (Fig. 3 C), and Fig. 3 D shows that this enzyme is the most stable when pH6.0.The optimal pH of recombinant phospholipase A2 is 6.0, is comparatively applicable to applied research of coming unstuck under this pH condition.Be illustrated in figure 4 the structure (protein structure PDB database accession number: 1IT4) of Streptomyces violaceoruber A-2688 Phospholipase A2, Phospholipase A2 molecular structure is comparatively simple, be positioned at 3 glycosylation sites (shown in spherical amino acid) of protein surface if occur glycosylation modified, can to the hydrophobe character changing whole albumen, thus have influence on the charge environment of active centre (shown in shaft-like amino acid) microcosmic, and then change optimal pH.The enzyme optimal pH that the present invention obtains is 6, and may be that occur to a certain degree glycosylation modified causes, as shown in Figure 2, this Phospholipid hydrolase has 3 potential glycosylation sites.After measured, the enzyme of recombinant phospholipase A2 under optimal pH, optimum temperuture condition is lived as 35U/mL.

The vegetable oil degumming application of embodiment 5 recombinant phospholipase A2

Be the citric acid-sodium citrate damping fluid dialysis of 5.0 by the PLA2 fermented liquid supernatant 5LpH of genetic engineering bacterium, the phosphoric in removing fermented liquid, reduces the impact on degumming effect, does experiment of coming unstuck after super filter tube is concentrated.

Get level Four rapeseed oil 50g, be preheated to 80 DEG C, add 2.5mol/L citric acid solution 0.125mL, after this temperature maintains 45min, temperature is reduced to 40 DEG C, add 5mol/L NaOH to mix and make pH value of solution be 5.0, add the citric acid-sodium citrate damping fluid of 4%pH5.0,253U enzyme liquid, 1.0mmol/L Ca ²⁺, be placed in the centrifugal 20min of reaction 6h, 7000rpm in 40 DEG C of shaking tables, get upper oil phase and survey phosphorus content (mensuration of GB GBT5537-2008 grain and oil detection phospholipids content).Result of study shows that phospholipids content drops to 10mg/Kg from 130mg/Kg.

The application of embodiment 6 recombinant phospholipase A2 in bread baking

The impact of recombinant phospholipase A2 on bread specific volume is mainly have studied in bread baking experiment.

Bread preparation method: with reference to AACC method 10-10B, and made certain amendment.Flour 100%, salt 1%, white sugar 4%, butter 4%, yeast 1.5%, water 62.5% (in flour quality), the addition of recombinant phospholipase A2 is 250U/kg, does not add lipase in blank.After above-mentioned raw materials is stirred 10min in stirrer, leave standstill 10min, be divided into 100g/, rub circle with the hands, leave standstill 10min, shaping sabot, in 38 DEG C, proof 90min under the condition of relative humidity 85%, cure 25min (getting angry 170 DEG C, lower fiery 210 DEG C), for subsequent use after cooling.

Bread specific volume measures: adopt vegetable seed exclusive method to measure.Bread, after room temperature cooling 1h, measures volume and the quality of bread respectively.

Specific volume (mL/g)=volume (mL)/quality (g).

Specific volume increased value (%)=(sample specific volume-blank specific volume)/blank specific volume * 100%

Result of study shows, with the addition of that the specific volume of recombinant phospholipase A2 is maximum adds 22%.

Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. A preparation method for phospholipase A2 is to clone the gene fragment of the sequence shown in SEQ ID NO.1 into an expression vector to obtain a recombinant vector, and then transform the recombinant vector into the gene obtained in Pichia pastoris The engineering bacteria can obtain phospholipase A2 after induced expression; the optimum pH of the phospholipase A2 is in the acid range. 2. method according to claim 1, is characterized in that, the nucleotide sequence of coding described phospholipase A2 is as shown in SEQ ID NO.2. 3. The method according to claim 1, wherein the expression vector is any one of pPIC9K, pPIC9, pPIC Zα, pGAPZα, pPIC3.5, pPIC3.5K, pYAM75P6. 4. The method according to claim 1, characterized in that, the Pichia pastoris is any one of the following: GS115, KM71, SMD1168. 5. The method according to claim 3, wherein the expression vector is pPIC9K. 6. The method according to claim 4, characterized in that, the Pichia saccharomyces is P.pichia GS115. 7. A phospholipase A2 obtained by the method according to claim 1. 8. A genetically engineered bacterium expressing phospholipase A2 according to claim 7. 9. The genetically engineered bacterium according to claim 8, characterized in that, the genetically engineered bacterium is to clone the gene fragment of the amino acid sequence shown in encoding SEQ ID NO.1 into an expression vector to form a recombinant vector, and then the recombinant vector Transform into the host bacterium, and screen positive clones to obtain the genetically engineered bacterium capable of expressing the phospholipase A2 described in claim 7. 10. the application of the phospholipase A2 described in claim 7 in enzyme preparation production, grease degumming and flour product processing.