CN102002486A - Phospholipase B from pseudomonas fluorescens and production method thereof - Google Patents

Abstract

The invention relates to a phospholipase B derived from Pseudomonas fluorescens and a production method thereof, belonging to the fields of enzyme genetic engineering and enzyme engineering. The phospholipase B is produced by Pseudomonas fluorescens. The full length of the gene is 1272bp, encoding 423 amino acids. The theoretical molecular weight of the enzyme protein is 45.8kDa, of which 1-69bp encodes the signal peptide of phospholipase B, and 70-1272bp encodes the mature phospholipase B peptide. The expression vector and recombinant host thereof can be obtained by using the existing molecular biology method, that is, the Escherichia coli recombinant plasmid containing the phospholipase B gene and the recombinant Escherichia coli can be obtained. The phospholipase B of the present invention has good activity and stability at low temperature, and has no lipase activity, can hydrolyze and catalyze two fatty acyl groups in complete phospholipids, and can be used in industrial processes such as oil refining and modification of phospholipid emulsifiers. It has wide application, simple preparation method, high product quality and low production cost.

Description

A kind of phospholipase B and production method thereof that derives from Pseudomonas fluorescens

Technical field

The present invention relates to a kind of phospholipase B and production method thereof that derives from Pseudomonas fluorescens, belong to enzyme genetically engineered and enzyme engineering field.

Background technology

Phosphatide is extensive in distributed in nature, all contains phosphatide in all cells, and phosphatide is biomembranous ultimate constituent.Phosphatide plays metabolism and structure formation effect in vital process, be important living matter.Phospholipid hydrolase is the enzyme of the ester bond of hydrolytic phosphatide, extensively is present in eukaryote and the prokaryotic organism, influence the katabolism of phosphatide, biomembranous formation and reorganization, and Phospholipid hydrolase also relates to the cascade of signal.According to its action site difference, Phospholipid hydrolase is divided into phospholipase A ₁, A ₂, B, C, D etc.Phospholipase A wherein ₁And A ₂Hydrolysis Sn-1 and Sn-2 position acyl group produce lysophospholipid and lipid acid respectively.Phospholipase B has the activity of lytic enzyme and lysophospholipase.The activity of lytic enzyme is hydrolysis Sn-1 and Sn-2 position acyl group simultaneously, and the activity of lysophospholipase then can the remaining acyl group of hydrolysis lysophospholipid.

Phospholipid hydrolase has multiple application in industry, comprise the modification of the phospholipid emulsifier that is applied to food and non-food product, increases the emulsification of oil/water mixture; Phospholipid hydrolase can be used for the enzymatic degumming in the vegetables oil course of processing; The subsequent disposal of starch hydrolysate (the particularly hydrolysate of wheat starch) is to improve the penetrating ability of filtering.In addition, Phospholipid hydrolase is effective especially for improving dough/pasta or baked product quality in curing application.The suitableeest enzyme of low temperature phospholipase B is lived temperature generally below 40 ℃; it is low that it has the suitableeest enzyme temperature alive; have higher characteristics such as catalytic efficiency at low temperatures, thereby be applied to simplify production technique, energy-saving consumption-reducing in the industrial production such as vegetables oil enzymatic degumming and decorated phospholipid emulsifying agent, improve the quality of products, protect environment, reduce production costs.

Phospholipase B obtains from different source such as animal, plant, microorganism.Animal comprises guinea pig [A.Gassama-Diagne etc., (J.Biol.Chem.), 264,9470-9475,1989]; Mouse [S.Pind et al., (Biochem.Cell.Biol.), 69,346-357,1991]; Rabbit [W.Boll et al., (J.Biol.Chem.), 268,12901-12911,1993].Plant has broad bean [Y.Lee et al., (FEBS Lett.), 343,213-218,2001].Microorganism comprise yeast saccharomyces cerevisiae [M.Ichimasa et al., (Agric.Biol.Chem.), 49 (4), 1083-1089,1985; F.Paultaufet al., (J.Biol.Chem.), 269,19725-19730,1994]; Schizosaccharomyces pombe [H.Oishi.et al., (Biosci.Biotech.Biochem.), 60 (7), 1087-1092,1996]; Penicllium chrysogenum [N.Kawasaki.et al., (J.Biol.Chem.), 77,1233-1244,1975; N.Masuda.etal., (Eur.J.Biochem.), 202,783-787,1991]; Ox Mohs bacillus [J.M.Tennent et al., (J.Bacteriol.), 183,6717-6720,2001]; Mycobacterium leprae murium [Shinji Maeda et al., (Biochimica et Biophysica Acta), 1303,31-38,1996].But animals and plants phospholipase B source is limited, and costs an arm and a leg.The application for Phospholipid hydrolase of discovering of microorganism phospholipase B provides new source, and its fermentation condition is simple, greatly reduces production cost, for later suitability for industrialized production is laid a good foundation.Up to the present, the relevant research that produces the Pseudomonas fluorescens of low temperature phospholipase B does not appear in the newspapers both at home and abroad as yet.

Summary of the invention

The objective of the invention is for a kind of phospholipase B and production method thereof that derives from Pseudomonas fluorescens is provided; this phospholipase B shows activity at low temperatures; and the activity of fat-free enzyme; two fatty acyl group groups in can the complete phosphatide of hydrolyzation catalysis, this method is simple, quality product is high, production cost is low.

The objective of the invention is to be achieved through the following technical solutions.

A kind of phospholipase B that derives from Pseudomonas fluorescens (Pseudomonas fluorescens) of the present invention, this phospholipase B is produced by Pseudomonas fluorescens, and phospholipase B has good activity and stable at low temperatures.Phospholipase B full length gene 1272bp, 423 amino acid of encoding, the zymoprotein theoretical molecular is 45.8kDa, 1～69bp coding phospholipase B signal peptide wherein, 70～1272bp coding phospholipase B mature peptide; Utilize existing molecular biology method can obtain its expression vector and recombinant host, promptly obtain to contain the intestinal bacteria recombinant plasmid and the recombination bacillus coli of phospholipase B gene.

A kind of production method that derives from the phospholipase B of Pseudomonas fluorescens of the present invention is cultivated the Pseudomonas fluorescens strain system that can produce phospholipase B in suitable fermention medium.By optimization to fermention medium and fermentation parameter, select cheap common nutritive ingredient for use, adopt conventional single batch fermentation mode, the work of phospholipase B fermenting enzyme reaches 43.2U.mL ^-1Wherein fermention medium is: final concentration percentage composition (g/mL) 0.01～5% soybean phospholipid, 0.1～10% starch, 0.01～5% extractum carnis, 0.01～5% peptone, 0.01～3%NaCl, 0.01～2%MgSO ₄7H ₂O transfers pH3.0～11.0.Be cloned into the phospholipase B gene by SDS-PAGE and from Pseudomonas fluorescens, determined that further bacterial strain BIT-18 only produces phospholipase B, fat-free enzymic activity.

Beneficial effect

Phospholipase gene B of the present invention shows activity at low temperatures; and the activity of fat-free enzyme; two fatty acyl group groups in can the complete phosphatide of hydrolyzation catalysis; have a wide range of applications in commercial runs such as oil and fat refining and decorated phospholipid emulsifying agent, preparation method of the present invention is simple, quality product is high, production cost is low.

Description of drawings

Fig. 1 is the standard substance gas chromatogram of Uniphat A60;

Fig. 2 is the standard substance gas chromatogram of Witconol 2301;

Fig. 3 identifies figure for the gas-chromatography that Pseudomonas fluorescens BIT-18 expresses Phospholipid hydrolase;

Fig. 4 is the phospholipase B temperature variation curve;

Fig. 5 is phospholipase B pH value change curve;

Fig. 6 is the agarose gel electrophoresis of the phospholipase B gene conservative fragments of pcr amplification Pseudomonas fluorescens BIT-18; Wherein M represents DL2000Marker, the single primer PLB-1PCR product of 1 expression, two primer PLB-1 of 2 expressions and PLB-2PCR product, 3 single primer PLB-2PCR products;

Fig. 7 is the agarose gel electrophoresis of the phospholipase B full-length gene of pcr amplification Pseudomonas fluorescens BIT-18; Wherein M represents DL2000Marker, 1 expression phospholipase B full-length gene PCR product;

Fig. 8 is intestinal bacteria recombinant plasmid pET28a (+)-plb single endonuclease digestion and double digestion figure; Wherein M represents 1kbMarker, 1 expression EcoRI and Hind III double digestion, 2 expression EcoRI single endonuclease digestions, 3 expression HindIII single endonuclease digestions;

Fig. 9 is the SDS-PAGE at the phospholipase B of expression in escherichia coli; Wherein M represents albumen Marker, the phospholipase B that 1 and 6 expression Pseudomonas fluorescens BIT-18 express, full cell before 2 expression recombination bacillus coli BL21 (DE3) induce, 3 expression recombination bacillus coli BL21 (DE3) induce the full cell in back, 4: the pericentral siphon part of expression recombination bacillus coli BL21 (DE3), the supernatant part of 5 expression recombination bacillus coli BL21 (DE3).

Embodiment

Embodiment 1

One, produces the screening of Phospholipid hydrolase bacterial strain

1, the separation screening of bacterial classification

Pick up from Shihezi, Xinjiang Uygur Autonomous Regions, Datong City, Shanxi Province, Yuncheng, Zhengzhou City, Henan Province, comprising the soil sample that reaches soybean and vegetable seed planting base around soybean oil, rapeseed oil, the Oleum Gossypii semen job shop, be following 10cm place, the face of land and gather totally 18 parts.Soil sampling 10g puts into the triangular flask that fills the 90mL sterilized water and have granulated glass sphere, leaves standstill behind the room temperature vibration 12h.Draw the 1mL supernatant liquid and be inoculated in 49mL enrichment medium (soybean phospholipid 4.0g/L, KN0 ₃1.0g/L, K ₂HPO ₄1.0g/L, NaCl 0.5g/L, MgSO ₄7H ₂O 0.5g/L) in, 30 ℃ of shake-flask culture 24h, the enrichment culture that takes a morsel then liquid coat primary dcreening operation substratum (soybean phospholipid 4.0g/L, peptone 10.0g/L, K ₂HPO ₄1.0g/L, NaCl 0.5g/L, MgSO ₄7H ₂O 0.5g/L, agar 18.0g/L) on the flat board, separate that to obtain can be sole carbon source growth and the single bacterium colony that produces the hydrolysis circle with the soybean phospholipid, hydrolysis loop diameter and colony diameter ratio (R) are big more to show that then the vigor of Phospholipid hydrolase is big more.Adopt the isolation medium screening to obtain the microorganism that Phospholipid hydrolase is produced in 136 strains, wherein have the hydrolysis circle of 4 strain bacterium bigger.

The 4 strain bacterium that dull and stereotyped primary dcreening operation is obtained are inoculated in LB phosphatide flat board (adopting phenol red respectively and rhodamine dyeing), cultivate 72h for 30 ℃, and hydrolysis loop diameter and colony diameter ratio (R) are big more to show that then the vigor of Phospholipid hydrolase is big more.Select the stronger bacterial strain of enzymatic productivity and carry out the inclined-plane preservation.Single strain shake flask fermentation (glucose 10.0g/L, extractum carnis 7.5g/L, peptone 7.5g/L, NaCl 3.0g/L, MgS0 ₄7H ₂01.0g/L, soybean phospholipid 4g/L) and the multiple sieve of test shows that bacterial strain phospholipase activity that BIT-18 produces is the highest, enzyme activity reaches 25U.mL ^-1, can be used as the original strain of further research.

2. strain identification

(1) Physiology and biochemistry detects

Morphological specificity is carried out with reference to the method in " uncle Jie Shi systematic bacteriology identification handbook " (" Bergey ' s Manual of Systematic Bacteriology ") the 9th edition and " the bacterial system identification handbook commonly used ", and physiological and biochemical property adopts bacterium biochemical test assessing instrument and supporting Gram-negative bacteria to detect test card to carry out physiological and biochemical property and identify sieving bacterial strain again.The biological characteristics of the microorganism strains BIT-18 of generation Phospholipid hydrolase is as shown in table 1:

The physiological and biochemical property of table 1 bacterial strain BIT-18

Annotate :+: utilize and maybe should react positive;-: do not utilize maybe this reaction negative

(2) molecular biology identification

1. the extraction of bacterial strain BIT-18 genomic dna

Extract bacterial strain BIT-18 genomic dna with ordinary method well known by persons skilled in the art.Get the 1mL culture in 1.5mL EP pipe, room temperature 10, the centrifugal 5min of 000rpm abandons supernatant, and precipitation is suspended among the 1mL TE (pH8.0) again; The N,O-Diacetylmuramidase that adds 6 μ l 50mg/mL, 37 ℃ of effect 2h.Add 2mol/LNaCl 50 μ l again, 10%SDS 110 μ l, 50 ℃ of effects 3h or 37 ℃ spend the night; Bacterium liquid is all assigned to two 1.5mLEP pipes, adds isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), and mixing, room temperature is placed 5-10min.14, the centrifugal 10min of 000rpm, twice of extracting.Add isopyknic chloroform, mix gently, 14,4 ℃ of centrifugal 3min of 000rpm, supernatant suck in another dry centrifuge tube; Add sodium-acetate (10mmol/L) solution of 1/10 volume, mixing adds 0.6 times of Virahol that volume is cold, and mixing is placed 30min, 14, the centrifugal 10min of 000rpm for-20 ℃; Precipitate the washing with alcohol twice with 75%, vacuum-drying is until no ethanol flavor; Take out after (cold) do, be dissolved in 40～50 μ l TE or ddH ₂Among the O, get 5 μ l electrophoresis, it is standby to remain-20 ℃ of preservations.

2. 16S rDNA sequential analysis

The universal primer 16SL of employing 16S rDNA (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 16SR (5 '-AAGGAGGTGATCCAGCCGCA-3 '), with the BIT-18 strain gene group DNA is template, carry out pcr amplification, the sequence that obtains in 16S rDNA sequence and the GenBank database is carried out the homology comparison, the result shows that bacterial strain BIT-18 and Pseudomonas fluorescens form cluster, its homology reaches more than 99%, and its 16S rDNA gene order is as follows:

1 ATTAGAGTTT GATCCTGGCT CAGATTGAAC GCTGGCGGCA GGCCTAACAC ATGCAAGTCG

61?AGCGGATGAA AGGAGCTTGC TCCTGGATTC AGCGGCGGAC GGGTGAGTAA TGCCTAGGAA

121TCTGCCTGGT AGTGGGGGAC AACGTTTCGA AAGGAACGCT AATACCGCAT ACGTCCTACG

181GGAGAAAGCA GGGGACCTTC GGGCCTTGCG CTATCAGATG AGCCTAGGTC GGATTAGCTA

241GTTGGTGAGG TAATGGCTCA CCAAGGCGAC GATCCGTAAC TGGTCTGAGA GGATGATCAG

301TCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG GGAATATTGG

361ACAATGGGCG AAAGCCTGAT CCAGCCATGC CGCGTGTGTG AAGAAGGTCT TCGGATTGTA

421AAGCACTTTA AGTTGGGAGG AAGGGTTGTA GATTAATACT CTGCAATTTT GACGTTACCG

481ACAGAATAAG CACCGGCTAA CTCTGTGCCA GCAGCCGCGG TAATACAGAG GGTGCAAGCG

541TTAATCGGAA TTACTGGGCG TAAAGCGCGC GTAGGTGGTT TGTTAAGTTG GATGTGAAAT

601CCCCGGGCTC AACCTGGGAA CTGCATTCAA AACTGACAAG CTAGAGTATG GTAGAGGGTG

661?GTGGAATTTC CTGTGTAGCG GTGAAATGCG TAGATATAGG AAGGAACACC AGTGGCGAAG

721?GCGACCACCT GGACTGATAC TGACACTGAG GTGCGAAAGC GTGGGGAGCA AACAGGATTA

781?GATACCCTGG TAGTCCACGC CGTAAACGAT GTCAACTAGC CGTTGGGAGC CTTGAGCTCT

841?TAGTGGCGCA GCTAACGCAT TAAGTTGACC GCCTGGGGAG TACGGCCGCA AGGTTAAAAC

901?TCAAATGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT TCGAAGCAAC

961?GCGAAGAACC TTACCAGGCC TTGACATCCA ATGAACTTTC CAGAGATGGA TTGGTGCCTT

1021CGGGAGCATT GAGACAGGTG CTGCATGGCT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT

1081TAAGTCCCGT AACGAGCGCA ACCCTTGTCC TTAGTTACCA GCACGTTATG GTGGGCACTC

1141TAAGGAGACT GCCGGTGACA AACCGGAGGA AGGTGGGGAT GACGTCAAGT CATCATGGCC

1201CTTACGGCCT GGGCTACACA CGTGCTACAA TGGTCGGTAC AAAGGGTTGC CAAGCCGCGA

1261GGTGGAGCTA ATCCCATAAA ACCGATCGTA GTCCGGATCG CAGTCTGCAA CTCGACTGCG

1321TGAAGTCGGA ATCGCTAGTA ATCGCGAATC AGAATGTCGC GGTGAATACG TTCCCGGGCC

1381TTGTACACAC CGCCCGTCAC ACCATGGGAG TGGGTTGCAC CAGAAGTAGC TAGTCTAACC

1441TTCGGGAGGA CGGTTACCAC GGTGTGATTC ATGACTGGGG?TGAAGTCGTA ACAAGGTAGC

1501CGTAGGGGAA CCTGCGGCTG GATCACCTCC TTAATC

Can determine from microscopic features bacterium, physiological and biochemical property and the molecular characterization of bacterial strain BIT-18, produce Phospholipid hydrolase bacterial strain BIT-18, belong to Pseudomonas fluorescens system, because of with its called after Pseudomonas fluorescens BIT-18, the number of landing in the GenBank database is GU367870.

Two, Pseudomonas fluorescens BIT-18 produces determining of Phospholipid hydrolase kind

By the kind of lipid acid in gas Chromatographic Determination and Phospholipid hydrolase and the phosphatide standard substance reaction product, thereby judge its kind.

The pre-treatment of testing sample.25mg PC standard substance (1-palmityl-2-oleoyl phosphatidic acid) are dissolved in the 0.5mL ether; Get 0.1mL dissolving back standard substance and 2.9mL enzyme liquid in 25 ℃, 150rpm reacts 3h; Add 6mL 5% hydrochloric acid-methanol solution in 95 ℃, reaction 2h; Add the 6mL hexane, vibration shakes up, and the centrifugal 15min of 1500rpm collects supernatant liquor, repeats twice; Eliminate hexane with the nitrogen remover, with the dissolving of 100 μ L anhydrous methanols, be used for next step gas chromatographic analysis then.

Gas-chromatography (Shimadzu, GC-2010) condition: the FFAP capillary column (0.32mm * 25m); Fid detector; Adopt temperature programming, 180 ℃ keep 0.5min after, be warming up to 250 ℃ with the speed of 10 ℃/min, keep 7min; Injector temperature and detector temperature are respectively 250 ℃ and 275 ℃; Sample size, 4 μ L.The result as Figure 1-3, wherein Fig. 1 and Fig. 2 represent palmitinic acid and oleic standard substance gas chromatogram respectively, Fig. 3 represents the gas chromatogram of testing sample.As can be seen from Figure 3, Pseudomonas fluorescens BIT-18 produces Phospholipid hydrolase hydrolysis PC standard substance and produces palmitinic acid and oleic acid simultaneously, Sn-1 position acyl group and the Sn-2 position acyl group of this Phospholipid hydrolase hydrolysis simultaneously PC is described, thereby can judges that it is a phospholipase B.

Three, the vitality test of the phospholipase B gene of Pseudomonas fluorescens BIT-18 generation

The NaOH potentiometric titration is adopted in the phospholipase B determination of activity.Get a certain amount of soybean phospholipid, 0.5%PVA is dissolved in the damping fluid of predetermined pH value, with high-shear homogenizer homogeneous 3min under the condition of 10000r/min, obtains substrate solution.Get the triangular flask of 4 100mL, 2 as blank bottle, 2 as sample bottle, respectively add substrate solution 9mL, in blank bottle, add 95% ethanol 20mL again, preheating 5min in the water-bath of preset temperature, the diluent 1mL (diluting) that in each bottle, adds enzyme liquid predetermined dilution multiple then with damping fluid, mixing timing immediately, the condition of 180r/min vibration accurate response certain hour in the water-bath of preset temperature, in sample bottle, add 95% ethanol 20mL termination reaction immediately, take out, carry out titration with 0.05mol/L NaOH standardized solution, record standard alkali lye average consumption, calculate phospholipase activity, its calculation formula

$X=\frac{(V-V_0)\×c\×50}{0.05\×t}=\frac{{10}^3\×(V-V_0)\×c}{t}$

In the formula: the phospholipase activity of X-sample, U/mL;

The NaOH standardized solution volume that consumes during V-titration sample, mL;

V ₀The NaOH standardized solution volume that consumes when-titration is blank, mL;

The concentration of c-NaOH standardized solution, mol/L;

50-0.05mol/L NaOH standardized solution 1.00mL is equivalent to lipid acid 50 μ mol;

T-measures the reaction times that enzyme is lived.

Lipase activity is measured with reference to GB GB/T1803-93 (2002) and is adopted the NaOH volumetry.

Calculation formula according to phospholipase activity can obtain the change curve of phospholipase activity with temperature and pH, and as shown in Figure 5 and Figure 6, the result shows 25～30 ℃ of phospholipase B optimum temperatures, optimal pH 6.5.

Embodiment 2

One, the clone of the phospholipase B gene of Pseudomonas fluorescens BIT-18

1.PCR amplification phospholipase B gene conservative fragments

Pair of degenerate primers P1-F (5 '-GVSAACAACGGCGGCTACGC-3 ') and P2-R (5 '-GCCARYTCCAYTGCGGRTGC-3 ') have been designed according to the conserved sequence of the bacterium phospholipase B of having announced on the NCBI, with Pseudomonas fluorescens BIT-18 genomic dna is template, carries out pcr amplification.The PCR reaction conditions is: 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 40s, 56 ℃ of annealing 30s, 72 ℃ are extended 2min, and 35 circulations are extended 10min for back 72 ℃.The PCR reaction solution separates through 1% agarose gel electrophoresis, as shown in Figure 6.The result shows that the size that increases is about the dna fragmentation of 450bp.

The dna fragmentation that the size that pcr amplification is gone out is about 450bp is connected on the pMD19-T carrier, is coated on and contains the LB solid medium that final concentration is 50 μ g/mL Amp transforming good intestinal bacteria, cultivates 16-18h down for 37 ℃.Single bacterium colony of picking white in containing the LB solid medium of Amp with the method screening positive clone of bacterium colony PCR, again by identifying through EcoR I and Hind III double digestion, is sent to the order-checking of Beijing big gene company limited of six directions China at last.Sequencing result shows, amplification obtains the conservative fragments of 426bp, to guard the segment sequencing result and in NCBI, carry out the BLAST comparison, in the result of comparison, find, (the GenBank number of landing: homology CP000094.2) is the highest, reaches 91% for a unknown zymoprotein gene among conservative segment and the Pseudomonas fluorescens Pf0-1.

2.PCR amplification phospholipase B full-length gene

With the agnoprotein gene order as a pair of Auele Specific Primer P2-F of stencil design (5 '-ATAGCGCCGGGACGAAAATG-3 ') and P2-R (5 '-AGCTCGCCGTGCAGCATCA-3 '), with Pseudomonas fluorescens BIT-18 genomic dna is template, carries out pcr amplification.The PCR reaction conditions is: 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 40s, 59 ℃ of annealing 30s, 72 ℃ are extended 2min, and 35 circulations are extended 10min for back 72 ℃.The PCR reaction solution separates through 1% agarose gel electrophoresis, as shown in Figure 7.The result shows that the size that increases is about the dna fragmentation of 1300bp.

The dna fragmentation that the size that pcr amplification is gone out is about 1300bp is connected on the pMD19-T carrier, is coated on and contains the LB solid medium that final concentration is 50 μ g/mL Amp transforming good intestinal bacteria, cultivates 16-18h down for 37 ℃.Single bacterium colony of picking white in containing the LB solid medium of Amp with the method screening positive clone of bacterium colony PCR, again by identifying through EcoR I and Hind III double digestion, is sent to the order-checking of Beijing big gene company limited of six directions China at last.Sequencing result shows that amplification obtains the nucleotide sequence of the phospholipase B gene complete of 1272bp, referring to phospholipase B nucleotide sequence SEQ NO.1.

Two, the abduction delivering of phospholipase B gene in intestinal bacteria

1. intestinal bacteria construction of recombinant plasmid

Genomic dna with Pseudomonas fluorescens BIT-18 is a template, employing primer P3-F (5 '-CCGGAATTCATGAAAAAAGTCATGCTCAA-3 ', contain EcoR I restriction enzyme site) and primer P3-R (5 '-CCCAAGCTTTCAGAAGCGGTAGGTCGCGC-3 ', contain Hind III restriction enzyme site), pcr amplification contains the dna fragmentation of plb gene.Reaction conditions is: 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 40s, 62.4 ℃ of annealing 30s, 72 ℃ are extended 2min, and 30 circulations are extended 10min for back 72 ℃.Reclaim the PCR product with EcoR I/Hind III double digestion, endonuclease bamhi reclaims behind the purifying and is connected acquisition recombinant plasmid pET28a (+)-plb through the pET28a (+) of EcoR I/HindIII double digestion carrier.Recombinant plasmid transformed e. coli bl21 (DE3) extracts plasmid, and carries out enzyme and cut evaluation, as shown in Figure 8.The result shows acquisition positive colony BL21/pET28a (+)-plb.

2. the abduction delivering of phospholipase B gene in intestinal bacteria

Recombination bacillus coli is containing 50 μ gmL ^-1In the LB liquid nutrient medium of kantlex, and the shaking table cultivation (37 ℃, 170rpm) spend the night, transferring with 1% inoculum size contains 50 μ gmL in 50mL ^-1In the LB substratum of kantlex, and the shaking table cultivation (37 ℃, 170rpm) to 0.6, adding inductor IPTG again is 0.8mmol/L to final concentration, in 22 ℃, behind the inducing culture 5h, (4 ℃, 10000rpm 8min) collects thalline and supernatant liquor to frozen centrifugation under the 170rpm.Supernatant liquor is used for enzyme activity determination, and thalline is suspended from the acetate buffer solution of 50mmol/L (pH 7.0), the ice-bath ultrasonic fragmentation.Thalline after the fragmentation supernatant liquor of centrifugal acquisition again is used for enzyme process determination of activity and SDS-PAGE analysis.Wherein the supernatant liquor enzyme is lived and is 3U.mg ^-1, periplasmic enzyme is lived and is 84.5U.mg ^-1, the result shows that the expression product overwhelming majority of phospholipase B gene in intestinal bacteria is arranged in the cell pericentral siphon, has only on a small quantity in supernatant liquor.

All soluble proteinss carry out the SDS-PAGE electrophoretic analysis, and as shown in Figure 9, the result shows that supernatant liquor does not have tangible protein band, and pericentral siphon partly has the obvious expression protein band.Recombination bacillus coli BL21/pET28a (+)-plb induces in the position that molecular weight is about 50kDa through IPTG and special protein band occurred, behind the fusion rotein of deduction 4kDa, molecular weight is about 45.8kDa, the Phospholipid hydrolase size of expressing with Pseudomonas fluorescens BIT-18 is consistent, also consistent with original theoretical value size with the supposition of phospholipase B aminoacid sequence, illustrate to have phospholipase B albumen in the expression product, and the contrast bacterium that contains pET28a (+) empty plasmid after inducing, IPTG does not have special band on same position.Above electrophoretic analysis result and enzyme activity determination result prove that all Pseudomonas fluorescens BIT-18 phospholipase B gene can express effectively in intestinal bacteria.

Three, produce the nucleotide sequence of phospholipase B engineering bacteria

Producing the engineering bacteria of phospholipase B, is prokaryotic cell prokaryocyte or the eukaryotic cell that contains one of following nucleotide sequences:

(1) the SEQ NO.1 in the sequence table;

(2) polynucleotide of SEQ NO.2 protein sequence in the code sequence tabulation;

(3) with sequence table in the dna sequence dna that limits of SEQ NO.1 have 80% above homology, and the identical function protein DNA sequence of encoding;

(4) under the rigorous condition of moderate can with the nucleotide sequence of the nucleotide sequence hybridization of SEQ NO.1 in the sequence table.

Wherein said protokaryon or eukaryotic cell be intestinal bacteria and yeast cell preferably, also can be other various animal and plant cellss.Can select various suitable expression vector well known to those skilled in the art for use, as various plasmids, phage and the virus vector etc. of market sale.Can directly the phospholipase gene sequence be directly connected in expression regulation sequence downstream in the expression vector, form the phospholipase B expression vector.The host cell that comprises isolated nucleic acid molecule of the present invention can be the conventional prokaryotic host cell that uses, yeast, Mammals, insect cell etc.

Claims (10)

1. phospholipase B, its two fatty acyl group groups in can hydrolytic phosphatide is characterized in that: a strain system that derives from Pseudomonas fluorescens.

2. a kind of phospholipase B according to claim 1, its characteristic is: the strain of Pseudomonas fluorescens is BIT-18, its GenBank number is GU367870.

3. a kind of phospholipase B according to claim 1, its characteristic is: be made up of 423 amino acid, the zymoprotein theoretical molecular is 45.8kDa.

4. a kind of phospholipase B according to claim 3, its characteristic is: 423 amino acid comprise the aminoacid sequence that at least 90% homology is arranged with aminoacid sequence shown in the sequence SEQ NO.2, and two fatty acyl group groups in can hydrolytic phosphatide.

5. a kind of phospholipase B according to claim 3, its characteristic is: the N terminal amino acid sequence that 423 amino acid comprises is a sequence shown in the 1-423 position of sequence SEQ NO.2.

6. a kind of phospholipase B according to claim 3, its characteristic is: 423 pairing nucleotides sequences of amino acid are classified SEQ NO.1 as, and total length is 1272bp, 1～69bp coding phosphor lipase signal peptide wherein, 70～1272bp coding phosphor lipase mature peptide.

7. the production method of a phospholipase B is characterized in that: cultivate the Pseudomonas fluorescens strain system that can produce phospholipase B in suitable fermention medium; By optimization to fermention medium and fermentation parameter, select cheap common nutritive ingredient for use, adopt conventional single batch fermentation mode, and centrifugal subsequently recovery phospholipase B; Wherein fermention medium is: final concentration percentage composition g/mL, 0.01～5% soybean phospholipid, 0.1～10% starch, 0.01～5% extractum carnis, 0.01～5% peptone, 0.01～3%NaCl, 0.01～2%MgSO ₄7H ₂O transfers pH3.0～11.0.

8. the production method of a phospholipase B is characterized in that step is: the dna sequence dna that separates the coding phospholipase B from the Pseudomonas fluorescens strain system that can produce phospholipase B; On suitable carriers, this dna fragmentation is connected suitable expression signal; Transform suitable heterologous host organism with this carrier; Under the condition that can cause phospholipase B to be expressed, cultivate, and from substratum, reclaim phospholipase B through the host transformed organism.

9. the production method of a kind of phospholipase B according to claim 8, it is characterized in that: the heterologous host object is a prokaryotic cell prokaryocyte.

10. the production method of a kind of phospholipase B according to claim 9, it is characterized in that: prokaryotic cell prokaryocyte is intestinal bacteria.

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