CN115725636A - Pichia pastoris mutant strain with high lipase yield - Google Patents
Pichia pastoris mutant strain with high lipase yield Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of genetic engineering, and particularly relates to a Pichia pastoris mutant strain with high lipase yield and application thereof. The mutant bacteria are obtained by an ultraviolet mutagenesis method, the yield of lipase is obviously improved, and the mutant bacteria are named as pichia pastoris ZT-42 (Pichia pastorisZT-42), with a preservation number of CCTCC NO: m20221118. The mutant strain is fermented in a shake flaskThe lipase activity in the supernatant reaches 19610U/ml, is improved by 127 percent compared with that of the original strain, and can be widely applied to the production of lipase, thereby being beneficial to reducing the production cost of the lipase and promoting the popularization and application of the lipase in the industrial field.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a Pichia pastoris mutant strain with high lipase yield and application thereof in lipase production.
Background
Lipase (also called triacylglycerol acyl hydrolase) belongs to carboxyl ester hydrolase, is a classification of esterase, and has the ability to hydrolyze Triglyceride (TG) into glycerol and Fatty Acid (FA). Lipases are widely available in nature, and animals, plants and microorganisms have the ability to produce fatty acids, wherein lipases produced by bacteria, fungi and yeast have high activity, and microorganisms multiply rapidly, thus having broader pH and temperature ranges than lipases of animal and plant origin. Compared with lipase from animal and plant sources, the lipase produced by microorganisms has lower cost, is easier to separate and purify, is more convenient to produce and the like, thereby having more research value.
Currently, the research on lipase mainly focuses on fatty triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), monoacylglycerol lipase (MGL), and these 3 enzymes have a definite function in lipid metabolism and decomposition in body fat and non-fat tissues. During hydrolysis of TG, these 3 enzymes exhibit "sequential" catalytic functions, i.e. ATGL first hydrolyzes TG into Diacylglycerol (DAG) and 1 molecule of FA; then converting DAG into Monoacylglycerol (MAG) and 1 molecule FA by continuous catalysis of HSL; finally, MGL plays a catalytic role to decompose MAG into glycerol and FA; to ensure the homeostasis of hydrolysis in the body, DAG and MAG may also generate TAG again through esterification process of diacylglycerol acyltransferase (DGAT) and monoacylglycerol acyltransferase (MGAT).
Lipase is one of important industrial enzyme preparations, and is widely applied to industries such as paper making, oil processing, food, medicine, daily chemicals and the like. 1. Cleaning agent industry: it is the largest commercial application field of lipase as an additive of household or industrial detergents. 2. Food industry: the lipase is used for producing the new-structure grease and the derivatives thereof which have both functionality and health care curative effect, thereby increasing the wide applicability of the grease and improving the economic benefit and the development potential of the grease industry. 3. Fiber and paper industry: the addition of the lipase simplifies the refining process of the kapok fiber, does not damage the kapok fiber, removes the resin (triglyceride and wax) in the paper pulp, and avoids the situation of serious fracture in the paper making process. 4. Biological energy: the molecular weight range of natural grease, kitchen waste oil and the like is relatively close to that of diesel oil, the main component of the grease is ester formed by fatty acid and glycerol, the grease is hydrolyzed by lipase, and the biodiesel can be obtained through a series of chemical conversions. 5. Pharmaceutical industry: by utilizing the unique stereoselectivity of lipase, stereoisomerides with optical activity can be effectively distinguished to synthesize various drugs. 6. Wine making: in the production of white spirit, ester compounds can be formed through lipase catalysis to increase the flavor of the white spirit, and starch embedded by fat is opened to accelerate the fermentation speed.
Lipases are one of the important enzymes in production and life, and have been studied in many ways by more and more researchers. In order to meet the large demands of the market, it is of great significance to search for lipase strains with high and stable yield.
Disclosure of Invention
The invention aims to provide a pichia pastoris mutant strain and application thereof in lipase production. The mutant strain is a lipase high-yield strain obtained by screening through an ultraviolet mutagenesis method, and can be widely applied to the production of the lipase.
One aspect of the present invention relates to a recombinant expression vector carrying a lipase gene.
The nucleotide sequence of the lipase gene is SEQ ID NO:1, the coded amino acid sequence is SEQ ID NO:2.
the invention relates to a pichia pastoris engineering bacterium which carries the recombinant expression vector.
The invention also relates to a pichia pastoris mutant strain, which is obtained by taking the pichia pastoris engineering strain as a starting strain and adopting an ultraviolet mutagenesis method.
The mutant strain is pichia pastoris ZT-42 (Pichia pastorisZT-42), which has been deposited in the center for type culture collection in china at the university of wuhan, 2022, 7 months and 15 days, with the deposition number being CCTCC NO: m20221118.
The invention also relates to a method for producing lipase by fermentation, which takes the pichia pastoris mutant strain as a fermentation strain.
Advantageous effects
The invention firstly constructs engineering bacteria Pichia pastoris ZT of recombinant expression lipase gene, and the highest lipase activity in shake flask fermentation supernatant reaches 8650U/ml; then, pichia pastoris ZT is used as a starting strain, and a mutant strain pichia pastoris ZT-42 is obtained by ultraviolet mutagenesis screening, so that the expression quantity of lipase can be greatly improved, the lipase activity in the shake flask fermentation supernatant is as high as 19610U/ml, the lipase activity is improved by 127 percent compared with that of the starting strain, and an unexpected technical effect is achieved.
The mutant strain can be used as a fermentation production strain of lipase, is beneficial to reducing the production cost of the lipase and promotes the wide application of the lipase in the industrial field.
Detailed Description
The present invention uses conventional techniques and methods used IN the fields of genetic engineering and MOLECULAR BIOLOGY, such as those 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, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the field on the basis of the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention.
Strain and carrier: pichia pastoris GS115, vector pPIC9k, G418 were purchased from Invitrogen.
Reagent: DNA polymerase was purchased from Takara, T4 ligase, restriction enzyme from Fermentas, plasmid extraction kit and gel purification recovery kit from Omega. The other reagents are all domestic analytical purifiers.
The enzyme activity determination method of the lipase in the embodiment of the invention comprises the following steps:
(1) Definition of enzyme activity: the enzyme amount required for hydrolyzing a substrate to generate 1 mu mol of titratable fatty acid in 1min under the conditions of 40 ℃ and pH value of 7.5 is an enzyme activity unit U.
(2) Substrate solution: weighing 40g of polyvinyl alcohol (PVA: polymerization degree 1750 +/-50), adding 800ml of water, soaking for 4-6h, heating in a boiling water bath, stirring until the PVA is completely dissolved, cooling under stirring, metering to 1000ml, filtering with clean 6-8 layers of gauze, and taking the filtrate for later use. Weighing above filtrate 150ml, adding oleum Olivarum 50ml, and processing with high speed homogenizer for 10min (divided into 4 times, with interval of 5min, each time for 2-3 min) to obtain milky PVA emulsion. It is prepared as before use.
(3) The determination method comprises the following steps: taking two 100ml triangular bottles, respectively adding 4ml of substrate solution and 5ml of pH 7.5 phosphate buffer solution into a blank bottle (A) and a sample bottle (B), adding 15ml of 95% ethanol into the blank bottle (A), and preheating in a water bath at 40 ℃ for 5min; adding 1ml of enzyme solution to be detected into the blank bottle (A) and the sample bottle (B), mixing uniformly immediately, timing, reacting accurately for 15min, adding 15ml of 95% ethanol into the sample bottle (B) by using a sample separating pipettor immediately, stopping the reaction, and taking out; pouring the reaction solution into a 50ml beaker, adding 5ml of pure water into a triangular flask, uniformly shaking, pouring into the 50ml beaker, and adding 2 drops of phenolphthalein indicator; adding 0.05mol/L sodium hydroxide solution into a beaker under the stirring condition by using a pH meter for correcting the over-alkaline condition, and titrating to a pH value of 9.92; titration was carried out until the pH no longer changed at 20s to the end point and the volume of sodium hydroxide consumed standard solution was recorded.
The enzyme activity calculation formula is as follows: x =
。
X-alkaline lipase activity, U/ml;
v1-consumption of sodium hydroxide standard solution volume, ml, when titrating a sample;
v2-consumption of sodium hydroxide standard solution volume, ml, at blank titration;
c is the concentration of the sodium hydroxide standard solution, mol/L;
50-0.05 mol/L sodium hydroxide solution 1ml is equivalent to fatty acid 50 mu mol;
n is enzyme solution dilution times;
0.05-conversion coefficient of sodium hydroxide standard solution concentration;
15-time scaling factor.
The present invention will be further described with reference to the following detailed description.
Example 1 cloning of genes
The amino acid sequence of lipase gene (GenBank number is AOE 45082.1) is taken as the basis, the amino acid sequence of the lipase is analyzed, the signal peptide of the lipase is removed, the codon optimization is carried out on the lipase according to the codon preference of pichia pastoris, and the whole gene synthesis is carried out by Huada gene company. The applicant named the lipase gene ZT, the nucleotide sequence of which is SEQ ID NO. 1, and the encoded amino acid sequence of which is SEQ ID NO. 2.
The PCR reaction is adopted to clone the lipase gene ZT fragment, and the primers and the reaction conditions are as follows:
primer 1 (F): GCGCGAATTCTCTCTCCAATTAGAAGAGAAGTTTCTC (underlined)EcoThe cleavage site of R I);
primer 1 (R): TAAAGCGGCCGCTTACAAAACAAGTACCAAACCCA (underlined)NotCleavage site of I).
The PCR conditions were: denaturation at 94 deg.C for 5min; then, the mixture is denatured at 94 ℃ for 30s, renatured at 56 ℃ for 30s, extended at 72 ℃ for 1min, and after 35 cycles, the mixture is kept at 72 ℃ for 10min. The overall length of the ZT gene is 825bp.
Example 2 construction of Pichia engineering bacteria
1. Construction of recombinant expression vectors
The cloned mature protein coding sequence of the lipase ZT gene is subjected to restriction endonucleaseEcoR is andNotafter double enzyme digestion, the double enzyme digestion is inserted into the multiple cloning site of a pichia pastoris expression vector pPIC9K, the multiple cloning site is positioned at the downstream of an alpha-factor signal peptide sequence to obtain a recombinant plasmid pPIC9K-ZT, and the plasmid is sequenced and identified. Sequencing verified that the correct transformants were transferred to LB + Amp liquid medium (0.5% yeast extract, 1% peptone, 1% NaCl, 100. Mu.g/mL ampicillin, pH 7.0), cultured overnight at 37 ℃ and the plasmids, i.e., recombinant yeast expression vector pPIC9K-ZT, were upgraded.
2. Transformation, screening and shake flask fermentation
Recombinant yeast expression vector pPIC9K-ZT for useSalI, linearization is carried out, a linearization product is purified by a column purification kit, and then pichia pastoris GS115 is transformed by an electroporation method and coated on an MD plate. In MD plate (1.34% YNB, 4X 10) -5 % biotin, 1% glycerol and 2% agarose) as engineering strains of pichia pastoris, and then screening multiple copies of transformants on YPD plates (1% yeast extract, 2% peptone and 2% glucose) containing different concentrations of geneticin G418.
Single clones on the high concentration plate were picked up and inoculated into BMGY medium (2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10, respectively -5 % biotin, 1% glycerol), shaking-culturing at 30 deg.C and 220rpm for 24 hr, transferring into BMMY medium (2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 -5 % biotin, 0.5% methanol) was added to the culture medium at 30 ℃ and 220rpm with shaking, and 0.5% methanol was added every 24 hours. After the induction expression is carried out for 4d, centrifuging and taking supernatant fluid to carry out lipase enzyme activity determination.
The result shows that the pichia pastoris engineering constructed by the invention is fermented under the condition of a shake flask, and the highest enzyme activity of lipase in the fermented supernatant reaches 8650U/ml. The transformant with the highest enzyme activity level is named as pichia pastoris ZT (Pichia pastoris ZT）。
Example 3 mutagenesis screening of Lipase-producing strains
Mutation breeding is to induce organisms to generate mutation under artificial conditions by using physical and chemical factors, select them and culture them into new varieties of animals, plants and microorganisms. Specific methods include physical mutagenesis and chemical mutagenesis.
The advantages and disadvantages of mutation breeding are that good varieties can be obtained in a short time, and some characters are greatly changed. However, the frequency of the beneficial mutation is still low, and the direction and the nature of the mutation are difficult to control.
The mutation caused by ultraviolet mutagenesis has strong randomness, and the effect generated by mutation is random and is difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, the screening workload is large, and the possibility that effective positive mutations cannot be obtained exists. However, UV mutagenesis requires simple equipment, is low in cost, and can obtain a large number of mutants in a short time, so that it is still a common mutagenesis breeding method.
The applicant takes pichia pastoris ZT as an original strain, and performs genetic modification on the pichia pastoris ZT by an ultraviolet mutagenesis method, so that the yield of lipase is further improved.
Inoculating Pichia pastoris ZT to YPD plate, culturing at 30 deg.C for 2-3 days, washing with sterile water to obtain suspension, and diluting to 1 × 10 6 Irradiating with ultraviolet lamp (40W) for 2-10min at a distance of about 22cm to achieve a lethality of above 90%, coating the plate, and culturing at 30 deg.C for 48h.
About 100 single colonies of mutant bacteria were obtained in the first round of UV mutagenesis, and each single colony was inoculated to a 96-well plate containing 200. Mu.l of BMGY liquid medium, subjected to shaking culture at 30 ℃ and 250rpm for 1 day, centrifuged to remove the upper layer medium, added with 200. Mu.l of BMMY medium, subjected to shaking culture at 30 ℃ and 250rpm for 2 days, and added with 0.5% methanol per day. After 2 days of induction expression, centrifuging to remove thalli and obtaining supernatant; and detecting the lipase activity in the fermentation supernatant.
The results show that none of the mutant bacteria obtained by the first round of ultraviolet mutagenesis screeningThe lipase activity in the mutant strain fermentation supernatant is higher than that of the original strain. The applicant further carries out 52 rounds of mutagenesis screening according to the method, and finally obtains 1 mutant strain with lipase yield obviously higher than that of the original strain, namely named pichia pastoris ZT-42 (R) ((R))Pichia pastoris ZT-42）。
The lipase activity of the mutant strain in the fermentation supernatant under the shake flask fermentation condition is as high as 19610U/ml, which is 127% higher than that of the original strain, and unexpected technical effects are achieved.
In conclusion, the pichia pastoris mutant provided by the invention can be used as a fermentation strain to be applied to the production of lipase, is favorable for reducing the production cost of the lipase and promotes the wide application of the lipase in the industrial field.
Claims (6)
1. A recombinant expression vector, which is characterized in that the recombinant expression vector carries a lipase gene.
2. The recombinant expression vector of claim 1, wherein the nucleotide sequence of the lipase gene is SEQ ID NO:1, the coded amino acid sequence is SEQ ID NO:2.
3. a Pichia pastoris engineering bacterium, which is characterized in that the Pichia pastoris engineering bacterium carries the recombinant expression vector of claim 1 or 2.
4. A Pichia pastoris mutant strain, which is characterized in that the mutant strain is obtained by taking the Pichia pastoris engineering strain of claim 3 as a spawn and adopting an ultraviolet mutagenesis method.
5. The Pichia pastoris mutant bacterium according to claim 4, wherein the mutant bacterium is Pichia pastoris ZT-42 (R) (A: (B) ())Pichia pastorisZT-42), which has been preserved in the chinese type culture collection of the university of wuhan, china in 2022, 7 months and 15 days, with the preservation number of CCTCC NO: m20221118.
6. A method for producing lipase, characterized in that the Pichia pastoris mutant strain of claim 4 or 5 is used as a fermentation strain.
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