CN107916256B

CN107916256B - Phospholipase

Info

Publication number: CN107916256B
Application number: CN201711280927.5A
Authority: CN
Inventors: 王艺璇
Original assignee: Individual
Current assignee: Beijing Zhongnongda High-tech Co.,Ltd.
Priority date: 2017-12-07
Filing date: 2017-12-07
Publication date: 2021-06-11
Anticipated expiration: 2037-12-07
Also published as: CN107916256A

Abstract

The invention relates to the technical field of genetic engineering, in particular to phospholipase and application thereof. The phospholipase has the optimum action pH value of 8.5 and the optimum action temperature of 45 ℃, is kept stand in a water bath at 4-50 ℃ for 1 hour, has stable and almost unchanged enzyme activity, can still keep more than 80% of the enzyme activity after being kept stand in the water bath at 55-65 ℃ for 1 hour, can still keep more than 50% of the enzyme activity after being kept stand in the water bath at 70 ℃ for 1 hour, and has obvious heat-resistant effect. The phospholipase can be widely applied to enzymatic degumming of vegetable oil, the residual phosphorus in the degummed oil is only 13.29ppm, the degumming effect is good, and the application prospect is wide.

Description

Phospholipase

Technical Field

The invention relates to the technical field of genetic engineering, in particular to phospholipase and application thereof.

Background

Phospholipases are enzymes responsible for the metabolism and biosynthesis of phospholipids in the body, and can catalyze various hydrolysis reactions of glycerophospholipids, and can be classified into 5 types according to their phospholipid hydrolysis positions: phospholipase a1, a2, B, C, D.

The biological functions of phospholipases can be classified into three categories: maintenance and repair of cell membrane structures; regulation of intracellular metabolic mechanisms and signaling, and digestion of phospholipids in vivo. For example, phospholipase A2 (PLA 2) is abundantly present in snake venom, bee venom, scorpion venom, animal pancreas and plant tissues, and can mediate the production of lipid mediators with the functions of phospholipid transport, membrane repair, extracellular hydrolysis and neuron transfer factor. Phospholipase C (PLC) plays a role of a second messenger in the life activities of organisms, and is widely existed in various prokaryotes and eukaryotes, but has slight difference on molecular structure.

Phospholipase has important physiological function in organism, and has high application value, and can be widely used in scientific research, medicine, feed improvement and food industry. For example, in the aspect of medicine, PLA2 is applied to the production of anti-inflammatory drugs, PLC is applied to the development of anti-tumor drugs and the like; in the aspect of feed improvement, the phospholipase is added into the feed to hydrolyze glycerophospholipid, so that the utilization efficiency of the feed can be improved, and the growth of animals can be promoted; in the aspect of food industry, phospholipase A1 (PLA 1), phospholipase A2 (PLA 2) and phospholipase C (PLC) can be widely applied to degumming of oil and fat, and meanwhile, the phospholipase can enable dough to form a colloidal complex and reduce starch retrogradation, so that the phospholipase is also widely applied to the baking industry. Phospholipase d (pld) is also widely used for glycerophospholipid modification.

Phospholipases are ubiquitous in animals, plants and microorganisms. The phospholipase from microorganisms has the characteristics of various varieties, mostly exogenetic expression, single subunit protein, easiness in large-scale rapid preparation, low cost and the like, and becomes the most important way for the current food industry application. Common microorganisms producing phospholipase are mainly Pseudomonas alcaligenes (Pseudomonas alcaligenes), Vibrio harveyi (Vibrio harveyi), Serratia marcescens (Serratiamarcens), Streptomyces (Streptomyces), Aspergillus oryzae (Aspergillus oryzae) and the like.

However, the phospholipase production capacity of the existing wild strains is often limited, and the requirements of industrial production cannot be met. Therefore, in order to adapt to extreme environments of high temperature, high pressure, high acidity, high ion concentration and excessive heavy metal ions in the industrial production process, new genes of phospholipase with improved performance need to be further screened.

Disclosure of Invention

The invention aims to provide a compound of aspergillus (A), (B), (C) and (C)Aspergillus sp.) The phospholipase and the recombinant expression engineering strain thereof. According to the invention, the expression vector containing the phospholipase gene of aspergillus is constructed and transformed into pichia pastoris, so that a pichia pastoris engineering strain is constructed, and the phospholipase can be efficiently secreted and expressed.

The invention provides a phospholipase which is characterized in that the phospholipase is

(a) An enzyme having the amino acid sequence of SEQ ID NO. 1;

(b) an enzyme having a phospholipase activity, which is obtained by substituting, deleting or adding one or more amino acids to the amino acids in (a).

The gene for coding the phospholipase has one coding nucleotide sequence of SEQ ID NO. 2.

In another aspect, the invention provides a vegetable oil degumming method.

The method specifically comprises the following steps: heating 200g vegetable oil to 70-80 deg.C under stirring, adding 0.16g 45% citric acid solution, shearing the mixture for 1min, stirring with a magnetic stirrer at 70-75 deg.C for 30min, cooling the oil until the oil temperature is 55-65 deg.C, adding 8% sodium hydroxide solution, shearing and mixing the mixture for 30s, maintaining the temperature at 50-55 deg.C, adding 5mL of phosphorus-containing lipase, mixing and shearing for 1min, stirring at 50-55 deg.C for 5 hr, centrifuging the enzyme-treated oil, and collecting the separated oil and wet gum, wherein the obtained oil is degummed vegetable oil.

The vegetable oil comprises one of peanut oil, soybean oil, corn oil, sunflower seed oil, sesame oil and rapeseed oil.

Advantageous effects

The Pichia pastoris engineering strain constructed by the invention can efficiently express aspergillus-derived phospholipase PD in vitro by recombination, and the shake flask fermentation enzyme activity is as high as 470U/mL, thereby making up the defects of the prior art and realizing the in vitro secretory expression of aspergillus phospholipase. Moreover, the phospholipase PD has the optimum action pH value of 8.5 and the optimum action temperature of 45 ℃, is kept stand in a water bath at 4-50 ℃ for 1 hour, the enzyme activity is relatively stable and almost unchanged, more than 80% of the enzyme activity can be still kept after the phospholipase PD is kept stand in the water bath at 55-65 ℃ for 1 hour, more than 50% of the enzyme activity can be still kept after the PD is kept stand in the water bath at 70 ℃ for 1 hour, and the heat-resistant effect is obvious. The phospholipase can be widely applied to enzymatic degumming of vegetable oil, the residual phosphorus in the degummed oil is only 13.29ppm, the degumming effect is good, and the application prospect is wide.

Detailed Description

The present invention uses conventional techniques and methods used IN the fields of genetic engineering and MOLECULAR BIOLOGY, such as the methods described IN MOLECULAR CLONING, A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, it is not intended that the invention be limited to any particular methodology, protocols, and reagents described, as these may vary.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 3nd Ed. (Singleton et al, 2006) AND COLLINS DICTIONARY BIOLOGY (Hale et al, 2003) provide the skilled person with a general explanation OF many OF the terms used in this invention.

In the following examples of the present invention, the detection of phospholipase activity was performed by the molybdenum blue method, unless otherwise specified.

The molybdenum blue method is a method for detecting the enzyme activity of phospholipase by detecting inorganic phosphorus released by hydrolysis reaction, and comprises the following specific processes:

reagent:

10% (w/v) ascorbic acid: 2g of the extract was dissolved in 20mL of water, and the solution was warmed slightly to promote dissolution and stored at 4 ℃ (fresh preparation).

2.5% (w/v) ammonium molybdate solution: 2.5g of ammonium molybdate is dissolved in 100mL of 30 percent sulfuric acid solution and placed at normal temperature;

1% mixed phospholipid: dissolving 0.5g soybean powder phospholipid in 50mL deionized water, rotating uniformly in a beaker, storing, and storing at 4 deg.C (fresh preparation);

500mM Tris-Cl (pH7.5): taking 100mL of 1M Tris-Cl mother liquor, adding water to 150mL, adjusting the pH value to 7.5, metering the volume to 200mL, and storing at normal temperature;

100mM CaCL 2: weighing 0.555g of CaCL2, dissolving in 50mL of water, and storing at 4 ℃;

1M Tris-Cl (pH9.0): weighing 24.228g of Tris, dissolving in 180mL of water, adjusting the pH value to 9.0, metering the volume to 200mL, and storing at normal temperature;

500mM MgCl 2: weighing 9.521g of MgCl2, dissolving in 200mL of water, and storing at normal temperature;

500U/mL alkaline phosphatase (AP, from Takara): diluting by 20 times to obtain 500U/mL at 10U/μ L, and storing at 4 deg.C.

Making a standard curve of the method for detecting inorganic phosphorus by using phosphorus-molybdenum blue:

0.1431g of dried dipotassium phosphate reagent powder is taken to be dissolved in deionized water, the volume is determined to be 100mL, and 1mg/mL PO is prepared₄ ^3-A dipotassium hydrogen phosphate solution. From 1mg/mL PO₄ ^3-0, 2, 5, 8, 10, 15, 20 and 50 mu L of dipotassium hydrogen phosphate solution are respectively taken out, deionized water is added to complement to 940 mu L, 20 mu L of 10% (w/v) ascorbic acid solution is added, after shaking for 30s, 40 mu L of 2.5% (w/v) ammonium molybdate solution (the solvent is 30% sulfuric acid solution) is added, finally the volume is fixed to 1mL, and the final PO is obtained₄ ^3-The concentration was 0, 2, 5, 8, 10, 15, 20, 50. mu.g/mL. Each concentration point is made 3 times in parallelAnd (5) compounding samples. And (3) after water bath at 37 ℃ for 10min, carrying out light absorption value detection under the condition of light absorbance of 700nm, and carrying out linear fitting on the obtained data to obtain a standard curve for detecting the phosphomolybdic blue.

EXAMPLE 1 cloning of the Gene

Applicant first extracted Aspergillus (Aspergillus sp.) Genomic total DNA of WLP (the strain was selected from the fallen leaf surface of mountain laoshan mountain forest land, Qingdao, Shandong province, by mussaja jones in 2017). Then, the total genomic DNA is used as a template, and the upstream and downstream primers are used for amplification.

PCR amplification conditions were 95 ℃ for 4 min; 30S at 94 ℃; 40S at 55 ℃ and 1min at 72 ℃ for 30 cycles; 7min at 72 ℃. And recovering the PCR amplification product by using the gel recovery kit, and sending the PCR amplification product to a Beijing Huada gene research center for sequencing analysis.

The result shows that the nucleotide sequence of the amplification product is SEQ ID NO. 2, and the coded amino acid sequence is SEQ ID NO. 1. Through NCBI Blast comparison, the sequence shown in SEQ ID NO 1 and Aspergillus fumigatus (A), (B) and (C)Aspergillus fumigatus) The similarity of the phospholipase amino acid sequences is only 68%, therefore, the invention obtains a new allele named PD.

EXAMPLE 2 construction of expression vectors

And respectively connecting the recovered amplification products to a pMD18-T vector to obtain a cloning vector pMD-PD. Using plasmid pMD-PD as a template, designing a primer for PCR amplification, wherein the amplification condition is 95 ℃ for 4 min; 30 cycles of 94 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 1.5 min; 7min at 72 ℃. Recovering the amplification product from the gel, and performingEco RI and Not I double digestion. Similarly, the expression of plasmid pPIC9K was also performedEco RI and Not I double digestion. The double enzyme digestion product, i.e., the cloned gene, was ligated with the expression vector overnight at 4 ℃ using T4 ligase. Finally, the ligation product was introduced into E.coli BL 21. The corresponding positive clone expression plasmid was designated pPIC-PD.

Example 3 construction of recombinant expression engineering bacteria

For expression of plasmid pPIC-PDSal I enzyme cutting electrophoresis identification, ethanol precipitation concentration, DNA concentration determination, and plasmid fragment dilution at 3 ug/ul concentration for storage. Preparation of Pichia pastoris GS115 electrotransformation InductionThe cells in the form of cells were finally resuspended in 1mL of precooled electrophoresis buffer (containing 1mM MgCl)₂10mM HEPES, 250mM sucrose, pH 7.8). Adding 5 mu L of linearized recombinant plasmid into 80 mu L of competent cells; electrotransformation (conditions 1500V, 200. omega., 25. mu.F); finally, the plates were plated on MM (MM medium fraction: 1.34% YNB, 4X 10)^-5% biotin, 0.5% methanol), selecting one positive transformant, and naming the transformant as Pichia pastoris PD (A), (B), (C), and (C)Pichia pastoris PD）。

Example 4 fermentation and enzyme Activity measurement

The Pichia pastoris engineering bacterium PD constructed in example 3 was inoculated into 5ml of BMGY (1% yeast extract, 2% protein orange, 1.34% YNB, 4X 10^-5% biotin, l% glycerol), culturing at 30 deg.C overnight, centrifuging to collect thallus, adding thallus into 50ml BMMY induction culture medium (1% yeast extract, 2% protein, 1.34% YNB, 4 × 10^-5% biotin and 0.5% methanol), adding 50 mu L of methanol every 12 hours, carrying out induced culture for 7 days, centrifuging fermentation liquor, taking supernate and carrying out SDS-PAGE electrophoresis detection, wherein a protein band is found at the position of 27kDa and is consistent with the predicted molecular weight.

0.5ml of the fermentation supernatant, 2.0ml of 0.1M Tris-HCl buffer (pH 8.0), and 2.5ml of 2% phosphatidylcholine (obtained from Allantin reagent Co., Ltd. using phosphatidylcholine having a purity of more than 98%), were mixed, and reacted in a water bath at 37 ℃ and 150rpm for 24 hours with shaking.

After the reaction, 1ml of the mixture was sampled, n-hexane was added in an equal volume to extract the mixture, the mixture was shaken and mixed, centrifuged at 12000rpm for 2 minutes, and the upper organic phase fraction was taken out and added to a new tube. And taking the lower-layer aqueous phase, repeatedly extracting and centrifuging, collecting and combining the two n-hexane extract liquids, uncovering, and placing in a fume hood to completely volatilize the organic phase. Add 15ul of isopropanol to each tube and dissolve well. Take 5ul spot chromatography plate. TLC detection was carried out (for TLC detection methods see Toida J., Arikawa Y., Kondou K., Fukuzawa M., Purification and characterization of triacylglycerol lipase from Aspergillus oryzae.1998.Bioscience Biotechnology Biochemistry,62(4): 759-.

The TLC detection result shows that diglyceride appears in the hydrolysate, so that the protein coded by the novel gene PD provided by the invention is judged to be phospholipase.

In a 200. mu.L reaction system containing soybean powder phospholipid 0.5% (w/v), a buffer system (25mM Tris-Cl, pH7.5), 5mM CaCl2, 20. mu.L (3. mu.g/ml) of phospholipase PD was added, the reaction was carried out for 30min, 200. mu.L of chloroform was added thereto and the mixture was shaken and mixed for 30s, and centrifugation was carried out at 12,000rpm for 1min, 80. mu.L of the supernatant was added to the phospholipase reaction system to give a final volume of 200. mu.L, which system further contained 50mM Tris-Cl (pH9.0), 10mM MgCl2, and CIAP 10U/. mu.L. The reaction was carried out in a 37 ℃ water bath for 30 min. 740. mu.L of deionized water was added to each, followed by 20. mu.L of 10% (w/v) ascorbic acid and 40. mu.L of 2.5% (w/v) ammonium molybdate solution. Color development was carried out at 37 ℃ for 10 min. And taking the developed solution to detect the absorbance at 700 nm. And (4) analyzing and regressing the data by combining a standard curve detected by phosphomolybdic blue, and multiplying the data by the dilution factor to obtain the activity value.

The measurement results show that: the enzyme activity of the phospholipase in the fermentation supernatant is 470U/mL, so that the pichia pastoris engineering strain PD constructed by the invention can actually secrete and express the phospholipase PD in vitro.

Example 5 phospholipase enzymatic Properties analysis

1. pH optimum assay

Diluting the fermentation supernatant with buffer solutions with pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, measuring the enzyme activity of the fermentation supernatant at 35 ℃, calculating the relative enzyme activity by taking the highest enzyme activity as 100%, and making a pH-relative enzyme activity curve. The results show that: the optimum action pH value of the phospholipase PD is 8.5.

Optimum action temperature analysis

The enzyme activity was measured at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and pH5.5, the relative enzyme activity was calculated with the highest enzyme activity as 100%, and a temperature-relative enzyme activity curve was constructed. The results show that: the optimal action temperature of the phospholipase PD is 45 ℃.

Temperature stability

And (3) packaging 500ul of the fermented supernatant into small parts, respectively placing the small parts in water bath pots with the temperature of 4 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 70 ℃ for heat preservation for 1h, detecting the enzyme activity, and dividing the enzyme activity of the sample in water bath with the temperature of 4 ℃ into 100% and the enzyme activity of other temperature points by the enzyme activity, thereby obtaining the relative enzyme activity value.

The result shows that the phospholipase PD provided by the invention is stable in enzyme activity and almost unchanged after standing for 1 hour in a water bath at 4-45 ℃, the enzyme activity of more than 83% can be still maintained after standing for 1 hour in the water bath at 50-60 ℃, the activity of more than 50% can be still maintained after standing for 1 hour in the water bath at 70 ℃, and the heat-resistant effect is obvious.

Example 6 use of phospholipase in degumming vegetable oil

The enzymatic degumming is that phosphatide in crude oil is hydrolyzed by phospholipase in oil and fat refining to generate oil-soluble diglyceride and water-soluble phosphate group, and the diglyceride is dissolved in the oil to become a part of edible oil, so that the yield of the degummed oil can be improved. In addition, the hydrolysis destroys the hydrophile and lipophile of phospholipid molecules, thereby reducing the formation of colloid for combining a large amount of oil in the degumming process and improving the yield of the oil on the other hand. Therefore, the enzymatic degumming has the advantages of reducing the production cost, increasing the oil yield and the like.

The applicant carried out enzymatic degumming using the phospholipase PD prepared in example 4:

heating 200g of crude soybean oil to 70-80 ℃ under stirring, adding 0.16g of 45% citric acid solution, shearing the mixture for 1 minute, stirring the mixture for 30 minutes at 70-75 ℃ by using a magnetic stirrer, cooling the oil until the oil temperature is 55-65 ℃, then adding 0.5g of 8% sodium hydroxide solution, shearing and mixing the mixture for 30 seconds, maintaining the temperature at 50-55 ℃, adding 5mL of fermentation supernatant containing the phospholipase, then mixing and shearing the mixture for 1 minute, stirring the mixture for reaction for 5 hours at 50-55 ℃, then centrifuging the enzyme-treated oil, collecting separated oil and wet gum, wherein the residual phosphorus in the degummed oil is only 13.29ppm, thereby showing that the phospholipase PD provided by the invention has better degumming effect.

Sequence listing

<110> Jaundice painting

<120> a phospholipase

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 241

<212> PRT

<213> Aspergillus (Aspergillus sp.)

<400> 1

Ser Ala Pro Asn Arg Ala Ser Lys Pro Gly Pro Gly Leu Arg Ile Leu

1 5 10 15

Thr Ala Thr Val Ile Lys Thr His Gly Leu Gly Asp Arg Lys Pro Leu

20 25 30

Ala Gln Asn Trp Arg Arg Arg Gly Met Tyr Gly Lys Val Ala Phe Met

35 40 45

Phe Pro Lys Ala Pro Ile Ile Pro Ile Arg Val Ile Phe Glu Met Thr

50 55 60

Met Leu Arg Asp His Asn Phe Lys Arg Arg Gly Arg Asp Leu Asp Trp

65 70 75 80

Lys Ser Ala Ile Arg His Gln Asp Glu Arg Gly Phe Arg Arg Cys Arg

85 90 95

Asp Tyr Phe Arg Thr Leu Asn Arg Lys Gln Ile Cys Arg Ser Ile Glu

100 105 110

Pro Ser Arg Ile Val Leu Gly Gly Phe Ser Gln Gly Ala Asn Val Phe

115 120 125

Val Phe Ser Gly Ile Thr Arg Lys Glu Lys Leu Gly Gly Val Phe Asp

130 135 140

Leu Val Ser Asn Leu Val Val Asn Lys Tyr Leu Lys Asp Asp Ile Glu

145 150 155 160

Glu Asn Trp Pro Asn Lys Lys Lys Pro Leu Phe Leu Ala His Gly Phe

165 170 175

Lys Asp Glu Val Gly Leu Phe Asp Phe Gly Glu Leu Leu Ala Asn Lys

180 185 190

Met Lys Glu Ile Gly Leu Glu Asp Ala Thr Phe Lys Ser Tyr Pro Asn

195 200 205

Leu Gly Pro Phe Ala Asp Pro Val Glu Ile Glu Val Trp Ala Arg Phe

210 215 220

Pro Gln Lys Val Ile Pro Pro Glu Asn Asp Gly Gln Ala Ser Ala Gly

225 230 235 240

Leu

<210> 2

<211> 726

<212> DNA

<213> Aspergillus (Aspergillus sp.)

<400> 2

agtgctccaa atcgcgcatc gaaacccggg ccggggctta gaatactcac tgcaacggta 60

atcaagaccc atggactggg tgacaggaag ccccttgctc agaactggcg tcgccggggg 120

atgtacggta aggttgcttt tatgttccct aaagcgccta taatcccgat cagggtgatc 180

tttgagatga cgatgcttag agatcacaat tttaagaggc gtggtcgcga tctcgattgg 240

aaatcagcca ttcggcacca agacgaacgg ggtttccgtc gatgtcggga ctacttcagg 300

acgttgaata ggaaacaaat ttgtaggagt atcgagccct cccggattgt tctgggtggt 360

ttctcccaag gggctaatgt gtttgtcttt tctggtatta ctcgtaagga gaagctcggt 420

ggtgtcttcg atttggtcag caatctggtg gtcaataaat atctgaagga cgatattgaa 480

gagaattggc cgaataagaa aaagcctttg ttcctcgctc atggctttaa agatgaagtc 540

gggctgttcg acttcggtga acttttggcg aacaagatga aagagatcgg cttggaggat 600

gccactttca aatcttatcc taacttgggc cccttcgccg atccagtaga gattgaggtt 660

tgggcgcgat ttcctcagaa agtcattcct ccagaaaacg acgggcaggc ttccgccgga 720

ttatga 726

Claims

1. The phospholipase is characterized in that the amino acid sequence of the phospholipase is SEQ ID NO. 1.

2. The gene encoding the phospholipase of claim 1, wherein one of the encoding nucleotide sequences is SEQ ID NO 2.

3. Use of the phospholipase of claim 1 in degumming of vegetable oils.

4. The use according to claim 3, wherein the vegetable oil comprises any one of peanut oil, soybean oil, corn oil, sunflower oil, sesame oil, rapeseed oil.