CN111909915A

CN111909915A - Lipase gene, lipase and application thereof

Info

Publication number: CN111909915A
Application number: CN202010738968.XA
Authority: CN
Inventors: 党蕊娟; 李娜; 王建荣; 祝木金; 曹革
Original assignee: Shenzhen Raink Plant Nutrition Technology Co ltd
Current assignee: Shenzhen Raink Plant Nutrition Technology Co ltd
Priority date: 2020-07-28
Filing date: 2020-07-28
Publication date: 2020-11-10

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a lipase gene, lipase and application thereof. The lipase gene Lrl-op of the invention has the GC content of 51 percent and the codon adaptation index of 0.84, and the nucleotide sequence is shown as SEQ ID NO. 1. The lipase gene Lrl-op is obtained by comprehensively optimizing a genome sequence of aureobasidium polyclonum according to the preference of pichia pastoris codons. The lipase LRL is a 1, 3-site specific lipase, and the amino acid sequence of the lipase LRL is shown in SEQ ID NO. 4. The invention not only enriches the variety and source approaches of lipase, but also has higher lipase activity of LRL, better thermal stability and pH stability, and can be used for decomposing glyceride and/or modifying grease.

Description

Lipase gene, lipase and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a lipase gene Lrl-op, a primer for amplifying the lipase gene Lrl-op, a lipase LRL, a corresponding recombinant expression vector, a recombinant expression strain and application of the lipase LRL.

Background

Lipase is a generic name of a class of enzymes which catalyze the decomposition or synthesis of glyceride on an oil-water interface, can catalyze the hydrolysis of natural substrate grease, release glyceride with less ester bonds or glycerol and fatty acid, and can catalyze reactions such as acidolysis, transesterification, ester synthesis and ester exchange. Because the lipase has unique enzymological characteristics, the lipase has wide application potential in the fields of grease modification, biodiesel, feed, paper making and the like.

Lipases can be classified into 1, 3-position-specific and non-specific lipases, depending on their hydrolysis properties on triglycerides. The 1, 3-position specific lipase has wide application potential in the field of structural lipids, and can be used for preparing various structural lipids. The current research on 1,3 position specific lipases is mainly focused on rhizopus lipases, such as: rhizopus oryzae lipase, Rhizopus radiculosus lipase, Rhizomucor miehei lipase, etc., and relatively few studies on other 1,3 position-specific lipases. Because of the effectiveness of 1, 3-position specific lipases in the field of oil and fat modification, if 1, 3-position specific lipases from different sources can be screened widely, the role of such lipases in the field of oil and fat modification can be further exerted, and therefore 1, 3-position specific lipases from other microorganisms need to be searched.

Disclosure of Invention

The invention aims to provide a lipase gene Lrl-op derived from aureobasidium polyclonum, a primer for amplifying the lipase gene Lrl-op, a lipase LRL, a corresponding recombinant expression vector, a recombinant expression strain and application of the lipase LRL, and aims to solve the problems that the existing research on 1, 3-position specific lipase mainly focuses on rhizopus lipase, the research on other-source lipase is less, the source of the 1, 3-position specific lipase is not wide enough and the like.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides a lipase gene Lrl-op, wherein the GC content of the lipase gene Lrl-op is 51%, the codon adaptation index is 0.84, and the nucleotide sequence of the lipase gene Lrl-op is shown as SEQ ID NO. 1.

The invention also provides a primer for amplifying the lipase gene Lrl-op, which comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3.

In another aspect, the invention provides a lipase LRL, wherein the lipase LRL is a 1, 3-site specific lipase, and the amino acid sequence of the lipase LRL is shown in SEQ ID NO. 4.

In still another aspect, the present invention provides a recombinant expression vector comprising the lipase gene Lrl-op as described above.

In still another aspect, the present invention provides a recombinant expression strain comprising the recombinant expression vector described above.

In a final aspect of the invention, the invention provides an application of the lipase LRL in glyceride decomposition and/or oil modification.

The lipase gene Lrl-op provided by the invention is obtained by comprehensively optimizing a aureobasidium polyclonum genome sequence (NCBI accession number: LK023313.1) according to the preference of pichia pastoris codons, so that the GC content in the nucleotide sequence of the lipase gene Lrl-op reaches 51%, the codon adaptation index is improved from 0.78 to 0.84, the sequence stability and the codon adaptation are obviously improved, the efficient expression in pichia pastoris expression strains is realized, and the production cost of lipase is reduced.

The primer provided by the invention is designed according to the nucleotide sequence of the lipase gene Lrl-op, is used for amplifying the specific primer of the lipase gene Lrl-op, and is beneficial to avoiding non-specific amplification during amplification of the lipase gene Lrl-op, thereby realizing large-scale specific amplification of the lipase gene Lrl-op.

The recombinant expression vector provided by the invention comprises the lipase gene Lrl-op, and the sequence of the lipase gene Lrl-op has better stability and codon adaptability, so that the recombinant expression vector provided by the invention also has better stability, is beneficial to stable and efficient expression of the lipase gene Lrl-op, and plays an important role in reducing the production cost of the lipase.

The recombinant expression strain provided by the invention comprises the recombinant expression vector, and therefore, can be used for expressing the lipase LRL. Moreover, on the basis that the sequence of the lipase gene Lrl-op has better stability and codon adaptability, the obtained recombinant expression strain can stably and efficiently express the lipase LRL and plays an important role in reducing the production cost of the lipase.

Through analyzing the position specificity of the lipase LRL provided by the invention, the lipase LRL is not hydrolyzed into a 2-position butyrate ester bond of tributyrin, so that the lipase LRL is a 1, 3-position specific lipase. The lipase LRL provided by the invention is a lipase from aureobasidium polyclonum, not only enriches the types and source ways of the lipase, but also has higher enzyme activity, better thermal stability and pH stability and good application prospect.

The lipase LRL is a 1, 3-site specific lipase, so that the lipase LRL can be used for decomposing glyceride and/or modifying oil and fat, and has the advantages of high efficiency and low cost.

Drawings

FIG. 1 is a schematic diagram of a process for constructing a recombinant expression vector pPICZ alpha A-lrl-op according to one embodiment of the present invention;

FIG. 2 is a high density fermentation curve of the recombinant expression strain in a 5L fermentor, according to one embodiment of the present invention;

FIG. 3 is an SDS-PAGE protein electrophoresis of the LRL lipase provided in one embodiment of the present invention;

FIG. 4 is a graph showing the optimal reaction temperature and thermal stability of a lipase LRL according to one embodiment of the present invention;

FIG. 5 is a graph showing the optimum reaction pH and pH stability of a lipase LRL according to one embodiment of the present invention;

FIG. 6 is a high performance liquid chromatogram of a substrate for LRL hydrolysis of lipase provided in one embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the description of the present invention, it should be understood that the content of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the content among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the concentration described in the examples of the present invention may be pg/mL, ng/μ L, μ g/mL, g/L, kg/L, or other units of mass known in the biological field.

In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.

It should be noted that the molecular biology experimental methods not specifically described in the examples of the present invention are performed by referring to the specific methods listed in the molecular cloning experimental manual (third edition) j. sambrook, or according to the kit and the product specification; the reagents and biomaterials, if not specifically indicated, are commercially available.

The embodiment of the invention provides a lipase gene Lrl-op, wherein the GC content of the lipase gene Lrl-op is 51%, the codon adaptation index is 0.84, and the nucleotide sequence of the lipase gene Lrl-op is shown as SEQ ID NO. 1.

Nucleotide sequence of Lrl-op (SEQ ID NO: 1):

ATGCACTCCCACTTTGTCGTCTTGTTGTTGGCCGTCTTCATCTGCATGTGCTCTGTCTCTGGAGTCCCTTTGCAAATCGACCCTAGAGACGACAAGTCCTACGTCCCAGAGCAATACCCTTTGAAGGTCAACGGTCCTTTGCCAGAAGGTGTCTCTGTCATCCAGGGTTACTGCGAGAACTGCACTATGTACCCTGAGGAGAACTCCGTTTCCGCTTTCTCCTCCTCTTCTACCCAAGACTACCGTATTGCCTCCGAAGCCGAGATTAAGGCTCACACCTTCTACACCGCTTTGTCCGCTAACGCCTACTGCAGAACTGTCATTCCAGGTGGTAGATGGTCTTGCCCACATTGTGGTGTTGCTTCTAATTTGCAAATTACCAAAACTTTCTCCACCTTGATCACCGATACCAACGTCTTGGTTGCCGTCGGTGAGAAGGAAAAGACCATCTACGTCGTCTTCCGTGGTACTTCCTCCATTCGTAACGCTATCGCCGACATTGTCTTTGTCCCAGTTAATTACCCACCAGTCAACGGAGCCAAGGTTCACAAGGGTTTCCTTGACTCCTACAACGAGGTCCAGGACAAGTTGGTTGCCGAGGTTAAGGCCCAGTTGGACAGACACCCTGGTTACAAGATCGTCGTCACTGGTCACTCCCTTGGTGGTGCTACCGCTGTTTTGTCCGCCTTGGACTTGTACCACCACGGTCACGCTAACATCGAGATTTACACTCAAGGTCAGCCAAGAATCGGTACCCCTGCTTTCGCTAACTACGTTATTGGTACCAAGATCCCATACCAGAGATTGGTCCACGAGCGTGATATCGTCCCTCACTTGCCACCTGGTGCTTTTGGATTCTTGCACGCCGGTGAGGAGTTCTGGATTATGAAGGACTCTTCCTTGAGAGTCTGCCCAAACGGTATTGAGACCGACAACTGCTCCAATTCCATCGTTCCATTCACCTCCGTCATCGACCACTTGTCCTACTTGGACATGAACACCGGTTTGTGTTTGTAA。

the lipase gene Lrl-op provided by the embodiment of the invention is characterized in that according to the preference of pichia pastoris codon, the nucleotide sequence of the gene Lrl-op of aureobasidium polyclonum (NCBI accession number: LK023313.1) is comprehensively optimized, the GC content in the nucleotide sequence of the lipase gene Lrl-op is up to 51%, the codon adaptation index is improved from 0.78 to 0.84, the sequence stability and the codon adaptation are obviously improved, the efficient expression in a pichia pastoris expression strain is realized, and the production cost of the lipase is reduced.

Correspondingly, in order to amplify the lipase gene Lrl-op, the embodiment of the invention also provides a primer for amplifying the lipase gene Lrl-op, which comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3.

Lrl-op forward primer (SEQ ID NO: 2):

agtcgaattcGTCCCT TTGCAAATCGACCCT；

lrl-op reverse primer (SEQ ID NO: 3):

ttctctagaTTACAAACACAAACCGGTGTT。

Correspondingly, the embodiment of the invention also provides a recombinant expression vector, which comprises the lipase gene Lrl-op.

The recombinant expression vector provided by the embodiment of the invention comprises the lipase gene Lrl-op, and the sequence of the lipase gene Lrl-op has better stability and codon adaptability, so that the recombinant expression vector provided by the embodiment of the invention also has better stability, is beneficial to stable and efficient expression of the lipase gene Lrl-op, and plays an important role in reducing the production cost of the lipase.

Because the lipase gene Lrl-op provided by the embodiment of the invention is optimized according to the preference of pichia pastoris codons, and pichia pastoris has the advantages of high integration stability of expressed genes, convenient and economic culture and suitability for industrial scale enlargement, in some embodiments, a pichia pastoris recombinant expression vector is selected, which is beneficial to improving the expression stability and the expression quantity of the lipase gene Lrl-op. Specifically, the expression vector pPICZ α A can be selected for the construction of recombinant expression vectors, and accordingly, the resulting recombinant expression vector is designated pPICZ α A-lrl-op 1.

The construction method of recombinant expression vector is that on the basis of basic skeleton of vector, expression element is added to make the target gene capable of expressing. The present invention is not particularly limited with respect to the specific construction method of the recombinant expression vector.

It is understood that the copy number of the lipase gene Lrl-op in the recombinant expression vector may be one, two, or more. By increasing the copy number of the lipase gene Lrl-op, the expression level of the gene can be increased, and the expression level of the corresponding lipase LRL can be further improved.

Correspondingly, the embodiment of the invention also provides a recombinant expression strain, which comprises the recombinant expression vector.

The recombinant expression strain provided by the embodiment of the invention comprises the recombinant expression vector, and therefore, can be used for expressing the lipase LRL. Moreover, on the basis that the sequence of the lipase gene Lrl-op has better stability and codon adaptability, the obtained recombinant expression strain can stably and efficiently express the lipase LRL and plays an important role in reducing the production cost of the lipase.

Because the lipase gene Lrl-op provided by the embodiment of the invention is optimized according to the preference of pichia pastoris codons, and the pichia pastoris eukaryotic expression system is a widely used eukaryotic expression system at present, the pichia pastoris eukaryotic expression system has the advantages of good biological safety, stable genetic element, capability of realizing high-density fermentation, high expression level, easiness in separating and purifying proteins, suitability for industrial large-scale production and the like, in some embodiments, a pichia pastoris expression strain is selected as a recombinant expression strain. Specifically, pichia pastoris X33 can be selected as the recombinant expression strain.

The recombinant expression strain is obtained by transferring a recombinant expression vector into a host cell, the target gene is replicated along with the propagation of the host cell, and correspondingly, the obtained recombinant expression strain can express corresponding protein. The present invention is not particularly limited with respect to the specific construction method of the recombinant expression strain.

By culturing the recombinant expression strain constructed as described above, the corresponding protein can be obtained from the strain culture. Correspondingly, the embodiment of the invention also provides a lipase LRL, wherein the lipase LRL is a 1, 3-site specific lipase, and the amino acid sequence of the lipase LRL is shown as SEQ ID NO. 4.

Amino acid sequence of lipase LRL (SEQ ID NO: 4):

MHSHFVVLLLAVFICMCSVSGVPLQIDPRDDKSYVPEQYPLKVNGPLPEGVSVIQGYCENCTMYPEENSVSAFSSSSTQDYRIASEAEIKAHTFYTALSANAYCRTVIPGGRWSCPHCGVASNLQITKTFSTLITDTNVLVAVGEKEKTIYVVFRGTSSIRNAIADIVFVPVNYPPVNGAKVHKGFLDSYNEVQDKLVAEVKAQLDRHPGYKIVVTGHSLGGATAVLSALDLYHHGHANIEIYTQGQPRIGTPAFANYVIGTKIPYQRLVHERDIVPHLPPGAFGFLHAGEEFWIMKDSSLRVCPNGIETDNCSNSIVPFTSVIDHLSYLDMNTGLCL。

through the analysis of the position specificity of the lipase LRL provided by the embodiment of the invention, the lipase LRL is not hydrolyzed into a butyrate ester bond at the 2-position of tributyrin, so that the lipase LRL provided by the embodiment of the invention is a lipase with the specificity at the 1, 3-position. The lipase LRL provided by the embodiment of the invention is a lipase from aureobasidium polycephalum, not only enriches the types and source ways of the lipase, but also has higher enzyme activity, better thermal stability and pH stability, low production cost and good application prospect.

Because the lipase LRL provided by the embodiment of the invention is a 1, 3-site specific lipase, correspondingly, the lipase LRL provided by the embodiment of the invention can be used for decomposing glyceride and/or modifying oil and fat.

The lipase LRL provided by the embodiment of the invention is used for decomposing glyceride and/or modifying oil, and has the advantages of high efficiency and low cost when being used for decomposing glyceride and/or modifying oil correspondingly under the conditions of high enzyme activity, good thermal stability and pH stability and low production cost of the lipase LRL.

Different enzymes have different catalytic activities at different reaction temperatures, and when the enzymes are at the optimal reaction temperature, the enzymatic reaction speed is the highest, so that the reaction efficiency is improved. In some embodiments, when the lipase LRL provided by the embodiments of the present invention is used for glyceride decomposition and/or lipid modification, the reaction temperature is preferably 40 ℃ to 60 ℃, in the temperature range, the relative enzyme activity of the lipase LRL is more than 75%, and most preferably the reaction temperature is 50 ℃, which is the optimal reaction temperature of the lipase LRL provided by the embodiments of the present invention, through experimental tests.

Different enzymes may also exhibit different catalytic activities in different pH environments. When the reaction pH is at the optimum reaction pH, the enzymatic reaction speed is the greatest, which is favorable for improving the reaction efficiency. In some embodiments, when the lipase LRL provided in the embodiments of the present invention is used for glyceride decomposition and/or lipid modification, the reaction pH is preferably 6 to 9, in which the relative enzyme activities of the lipase LRL are all greater than 75%, and most preferably the reaction pH is 8, which is the optimum reaction pH of the lipase LRL provided in the embodiments of the present invention, as tested experimentally.

In order to make the details and operations of the above-described embodiments of the present invention clearly understood by those skilled in the art and to make the progress of the lipase gene Lrl-op, the lipase Lrl and the applications thereof obvious in the embodiments of the present invention, the above-described embodiments are illustrated by examples.

Example 1

And (3) analyzing the sequence of the lipase LRL gene and optimizing and designing a codon to obtain lipase Lrl-op:

comparing and analyzing the amino acid conserved sequence (ERDIVPHLPPGAFGFLHAGEE) of the rhizomucor miehei lipase in NCBI database to obtain a partial amino acid sequence (IPYQRLVHERDIVPHLPPGAFGFLHAGEEFWIMKDSSLRVCP); the accession number of the genomic sequence of M.ramorum at NCBI database was obtained from the partial amino acid sequence (LK 023313.1). By analyzing the genomic sequence of the aureobasidium polyclonum, the DNA sequence within the range of 1295503bp-1296956bp is the complete sequence of the lipase gene Lrl. The total length of the DNA of the lipase gene Lrl is 1363bp, and the lipase gene Lrl comprises 7 exons and 6 introns, and the sequence of the lipase gene Lrl is shown as SEQ ID NO. 5. Sequence alignment analysis shows that the similarity of the protein encoded by the lipase gene Lrl and the Rhizomucor miehei lipase is 64 percent at most, and the similarity is 53 percent and 50 percent respectively for the synchalalastum racemosum lipase and the Rhizopus miehei lipase. The signal peptide prediction software SignalP-5.0Server prediction analysis finds that the first 21 amino acids of the protein coded by the lipase gene Lrl are the signal peptide.

Nucleotide sequence of Lipase Gene Lrl (SEQ ID NO: 5):

ATGCATTCTCATTTTGTAGTCTTATTGCTAGCAGTATTCATCTGCATGTGCTCTGTATCGGGTGTGCCACTGCAAATTGATCCACGCGATGACAAGAGCTATGTTCCTGAACAATATCCTTTGAAGGTGAATGGTCCTTTGCCAGAAGGTGTAAGCGTGATCCAAGGCTATTGTGAAAACTGTACCATGTATCCTGAAGAAAATAGTGTATCGGCATTCTCGTCATCATCCACACAAGATTATCGTATTGCAAGCGAGGCAGAGATTAAGGCACACACATTTTACACAGCATTGTCAGCCAATGCATACTGCAGAACTGTCATTCCTGGTGGTCGATGGAGCTGTCCCCACTGTGGTGTTGCATCCAATTTGCAAATTACCAAGACTTTCAGCACCTTAATCACTGATACTAATGTCTTGGTGGCTGTTGGCGAAAAGGAGAAGACCATCTATGTAGTTTTTCGTGGTACAAGCTCAATTCGCAACGCCATTGCTGTAAGTTCACCCCTTACAAACATGACACTTTGTTGCTCATCCGACTCATTCTTTCTTACAGGACATTGTTTTTGTACCAGTGAATTATCCACCTGTTAATGGAGCCAAAGTACACAAAGGTATGTGATGATGTGGTGTCATTTATATATAAGAATGCTCAATATGCTCATTTACTATCTAGGATTTCTTGATAGCTATAACGAAGTCCAGGATAAACTTGTTGCTGAAGTCAAGGCACAACTTGATCGTCATCCAGGATACAAGATCGTCGTCACTGGGTAAATACCTGAAAAGACATGGATGGCACGTGACTAAATCTGTGTCATTGGTAGACATTCCTTGGGAGGTGCAACAGCTGTTCTCAGTGCACTTGACCTTTATCACCATGGCCATGCCAATATCGAAATCTATACTCAAGGTCAGCCACGTATAGGTACTCCAGCATTTGCAAACTATGTGATTGGCACCAAGATTCCATACCAACGTCTTGTCCATGAGCGTGACAGTAAGTGTACCTTGCACGACATGTTCGTTTTCCCCGACGTACTAAAGTATTGTATAGTTGTTCCTCACCTTCCACCTGGTGCATTTGGTTTCTTGCATGCTGGTGAAGAGTTTTGGATCATGAAAGATAGCTCGTTGCGTAAGTAGTGTCATTGAAAAGGTTGAAGCTATAATACTGACTATATTGGGTAGGCGTATGTCCAAATGGCATTGAAACTGACAACTGCAGCAACTCCATTGTTCCCTTCACTAGTGTCATTGACCATTTAAGGTGAATAGTAGCTTTATTCATGTCATTCATCCATGTAAACTAACACTTGTCGTATCTAGCTATCTTGACATGAACACTGGTCTCTGTTTATAA。

according to the codon preference of pichia pastoris, the lipase gene Lrl is optimally designed, the GC content of the lipase gene Lrl is adjusted from 44% to 51%, and the adaptation index of the lipase gene Lrl is improved from 0.78 to 0.84. The optimized lipase gene is named as Lrl-op, the total length is 1017bp, 338 amino acids are coded, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence of the coded protein lipase LRL is shown as SEQ ID NO. 4.

Example 2

The construction process of the recombinant expression vector is as follows:

designing a pair of primers (the nucleotide sequence of the forward primer is shown as SEQ ID NO:2, the nucleotide sequence of the reverse primer is shown as SEQ ID NO:3) according to the sequence of the lipase gene Lrl-op for amplifying the mature peptide gene Lrl-op; obtaining mature peptide gene Lrl-op through PCR amplification, respectively carrying out overnight enzyme digestion on an expression vector pPICZ alpha A and the mature peptide gene Lrl-op by using restriction endonucleases EcoRI and XbaI, purifying and recovering the overnight enzyme digestion expression vector pPICZ alpha A and the mature peptide gene Lrl-op, and then carrying out ligation reaction; transferring the ligation reaction product into escherichia coli Top10 by adopting a heat shock method, and verifying a recombinant transformant by adopting a bacterial liquid PCR; and (3) inoculating the successfully verified transformant into an LBZ liquid culture medium, extracting plasmids, performing sequencing verification, and finally obtaining a recombinant expression vector pPICZ alpha A-lrl-op (the construction flow is shown in figure 1).

Example 3

Constructing recombinant expression engineering bacteria:

after the recombinant expression vector pPICZ alpha A-lrl-op obtained in example 2 was linearized with a restriction enzyme SacI, it was transferred into Pichia pastoris X33 by the electrotransformation method, thus obtaining recombinant transformants. The positive transformant is obtained by adopting a 24-well plate method, and the specific steps are as follows: the recombinant transformants on the YPDZ plate were picked up one by one with a toothpick into 24-well plates containing 1.8mL of BMGY medium per well, cultured overnight at 30 ℃ and 200rpm for 24 hours, centrifuged at 4000rpm to remove the supernatant, added with 1.6mL of BMMY medium, cultured at 30 ℃ and 200rpm for 24 hours, and the lipase activity of the recombinant transformants was measured. The lipase activity is determined according to the national standard GB/T23535-2009, wherein the enzyme activity is defined as the enzyme quantity required by the reaction with a substrate to generate 1 mu mol of fatty acid per minute under certain reaction conditions is defined as 1 enzyme activity unit and is expressed by U.

96 positive transformants were obtained by the above method, and three of the positive transformants (designated as L3, L11 and L65, respectively) had the highest enzyme activities of 21U/mL, 33U/mL and 23U/mL, respectively.

Example 4

And (3) carrying out shake flask fermentation culture on the positive transformant:

the three positive transformants (i.e. the recombinant pichia pastoris engineering bacteria) obtained in the example 3 are respectively inoculated into a 50mL centrifuge tube containing 5mL BMGY culture medium, the culture is carried out for 24 hours under the conditions of 30 ℃ and 220rpm, the cultured recombinant pichia pastoris engineering bacteria are inoculated into a 500mL triangular flask containing 100mL BMMY culture medium according to the inoculation amount of 1% (v/v), the shake flask culture is carried out under the conditions of 30 ℃ and 220rpm, 0.75% (v/v) methanol is added every 24 hours for induction, and simultaneously, sampling is carried out for the determination of lipase activity. Enzyme activity determination shows that the recombinant Pichia pastoris engineering bacteria reach the maximum after 96 hours of induction culture, wherein the highest fermentation enzyme activity of L11 is 75U/mL, and the highest fermentation enzyme activity is L65(53U/mL) and the highest fermentation enzyme activity is L11 (45U/mL).

Example 5

High-density fermentation culture of the recombinant pichia pastoris engineering bacteria L11:

taking recombinant pichia pastoris engineering bacteria L11 as an example for high-density fermentation culture, the specific process is as follows: inoculating a single colony of the recombinant Pichia pastoris engineering bacterium L11 into a 250mL triangular flask containing 50mL YPG medium, and carrying out shaking overnight culture at 30 ℃ and 200 rpm; inoculating the overnight cultured recombinant Pichia pastoris engineering bacteria L11 into a 500mL triangular flask containing 100mL YPG medium according to the inoculation amount of 1% (v/v), and performing shaking overnight culture at 30 ℃ and 200rpm until the OD 600 is more than 10; the recombinant pichia pastoris engineering bacteria cultured twice overnight are inoculated into a 5L fermentation tank containing 2L of BSM culture medium according to the inoculation amount of 10% (v/v). The culture conditions of the recombinant pichia pastoris engineering bacteria in a 5L fermentation tank are as follows: the temperature was 30 ℃, the pH was 5.0, the stirring speed was 500rpm, and the air flow rate was 40L/min. In the initial stage of culture, cells were grown using glycerol as a carbon source. When the wet weight of the cells reaches a certain amount (about 170 g/L), the glycerol feeding is stopped, and the induction with methanol is started after the glycerol is completely absorbed by the cells (the dissolved oxygen rises rapidly). The amount of methanol added was adjusted according to the dissolved oxygen. During the culture, samples were taken every 24 hours to determine the wet weight, enzyme activity and total protein concentration of the cells, and the results are shown in FIG. 2.

As can be seen from FIG. 2, the lipase activity was gradually increased with the increase of the fermentation time, and when the induction culture was carried out for 144 hours, the fermentation enzyme activity was maximized (7089U/mL), the total protein concentration was maximized at 3.08g/L, and the wet weight of the cells was maximized after 168 hours of induction (435 g/L).

Example 6

Purification of lipase LRL:

centrifuging fermentation liquor of a 5L fermentation tank in example 5, and taking supernatant for purification and recovery; carrying out ultrafiltration concentration on the supernatant enzyme solution by using a 10kDa ultrafiltration tube; purifying with Ni-IDA protein purification kit, and determining enzyme activity and total protein content at each step in the purification process. The specific enzyme activity of the purified lipase LRL was 4612U/mg. The electrophoresis results of the purified SDS-PAGE proteins are shown in FIG. 3.

As can be seen from FIG. 3, the lipase LRL showed two protein bands, one of which (about 35kDa) was the glycosylation-modified lipase LRL.

Example 7

The enzyme activity of the lipase LRL at 20-70 ℃ was measured at pH 7.5 (same as example 3), the enzyme activity at the highest temperature was measured as 100%, and the relative enzyme activities at other temperatures were calculated, the results are shown in FIG. 4.

As shown in FIG. 4, the lipase LRL had an optimum reaction temperature of 50 ℃ and had a relative enzyme activity of more than 75% at a temperature of … … ℃.

Example 8

The enzyme activity of the lipase LRL at the pH of 4-9 was measured at 50 ℃ to determine the enzyme activity at the highest pH of the enzyme activity as 100%, and the relative enzyme activities at other pHs were calculated, the results are shown in FIG. 5.

As can be seen from FIG. 5, the optimum reaction pH of the lipase LRL was 8, and the relative enzyme activities were all greater than 75% in the range of pH 6-9.

Example 9

The position specificity of tributyrin was determined by analyzing the hydrolysis of tributyrin by lipase LRL under the following conditions: 1g triolein, 150uL, pH7, 0.1M phosphate buffer, 10. mu.L lipase LRL, reaction at 50 deg.C and 500rpm for 30min, sampling, and performing high performance liquid chromatography, with the detection results shown in FIG. 6.

As can be seen from fig. 6, 5 sample peaks appear in the high performance liquid chromatogram, corresponding substance components are found one by one according to the liquid chromatogram of the laboratory standard, and the contents of the components are calculated, and the experimental results are shown in table 1. As is apparent from Table 1, the content of Tributyrin (TAG) was 49.87%, the content of fatty acid (FFA) was 4.79%, the content of 2, 3-diglyceride (2,3-DAG) was 26.24%, the content of 1, 2-diglyceride (1,2-DAG) was 12.9%, and the content of mono-dry ester (MAG) was 6.19% by hydrolysis reaction for 30 minutes. Since 1, 3-diglyceride (1,3-DAG) was not present in the liquid phase diagram, it was suggested that lipase LRL did not hydrolyze the butyrate ester bond at the 2-position of tributyrin, and thus it was a 1, 3-position specific lipase.

TABLE 1 analysis of substrate composition of glycerol tributyrate hydrolyzed by lipase LRL

	Name (R)	Time to peak (min)	Content (%)
				1	TAG	3.101	49.87
2	FFA	3.663	4.79
				3	2,3-DAG	4.405	26.24
4	1,2-DAG	5.466	12.90
				5	MAG	25.660	6.19

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shenzhen Runkang plant Nutrition technology Limited

<120> lipase gene, lipase and use thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1017

<212> DNA

<213> Aureobasidium pullulans (lichtheima ramosa)

<400> 1

atgcactccc actttgtcgt cttgttgttg gccgtcttca tctgcatgtg ctctgtctct 60

ggagtccctt tgcaaatcga ccctagagac gacaagtcct acgtcccaga gcaataccct 120

ttgaaggtca acggtccttt gccagaaggt gtctctgtca tccagggtta ctgcgagaac 180

tgcactatgt accctgagga gaactccgtt tccgctttct cctcctcttc tacccaagac 240

taccgtattg cctccgaagc cgagattaag gctcacacct tctacaccgc tttgtccgct 300

aacgcctact gcagaactgt cattccaggt ggtagatggt cttgcccaca ttgtggtgtt 360

gcttctaatt tgcaaattac caaaactttc tccaccttga tcaccgatac caacgtcttg 420

gttgccgtcg gtgagaagga aaagaccatc tacgtcgtct tccgtggtac ttcctccatt 480

cgtaacgcta tcgccgacat tgtctttgtc ccagttaatt acccaccagt caacggagcc 540

aaggttcaca agggtttcct tgactcctac aacgaggtcc aggacaagtt ggttgccgag 600

gttaaggccc agttggacag acaccctggt tacaagatcg tcgtcactgg tcactccctt 660

ggtggtgcta ccgctgtttt gtccgccttg gacttgtacc accacggtca cgctaacatc 720

gagatttaca ctcaaggtca gccaagaatc ggtacccctg ctttcgctaa ctacgttatt 780

ggtaccaaga tcccatacca gagattggtc cacgagcgtg atatcgtccc tcacttgcca 840

cctggtgctt ttggattctt gcacgccggt gaggagttct ggattatgaa ggactcttcc 900

ttgagagtct gcccaaacgg tattgagacc gacaactgct ccaattccat cgttccattc 960

acctccgtca tcgaccactt gtcctacttg gacatgaaca ccggtttgtg tttgtaa 1017

<210> 2

<211> 31

<212> DNA

<213> primers (Primer)

<400> 2

agtcgaattc gtccctttgc aaatcgaccc t 31

<210> 3

<211> 30

<212> DNA

<213> primers (Primer)

<400> 3

ttctctagat tacaaacaca aaccggtgtt 30

<210> 4

<211> 338

<212> PRT

<213> Protein (Protein)

<400> 4

Met His Ser His Phe Val Val Leu Leu Leu Ala Val Phe Ile Cys Met

1 5 10 15

Cys Ser Val Ser Gly Val Pro Leu Gln Ile Asp Pro Arg Asp Asp Lys

20 25 30

Ser Tyr Val Pro Glu Gln Tyr Pro Leu Lys Val Asn Gly Pro Leu Pro

35 40 45

Glu Gly Val Ser Val Ile Gln Gly Tyr Cys Glu Asn Cys Thr Met Tyr

50 55 60

Pro Glu Glu Asn Ser Val Ser Ala Phe Ser Ser Ser Ser Thr Gln Asp

65 70 75 80

Tyr Arg Ile Ala Ser Glu Ala Glu Ile Lys Ala His Thr Phe Tyr Thr

85 90 95

Ala Leu Ser Ala Asn Ala Tyr Cys Arg Thr Val Ile Pro Gly Gly Arg

100 105 110

Trp Ser Cys Pro His Cys Gly Val Ala Ser Asn Leu Gln Ile Thr Lys

115 120 125

Thr Phe Ser Thr Leu Ile Thr Asp Thr Asn Val Leu Val Ala Val Gly

130 135 140

Glu Lys Glu Lys Thr Ile Tyr Val Val Phe Arg Gly Thr Ser Ser Ile

145 150 155 160

Arg Asn Ala Ile Ala Asp Ile Val Phe Val Pro Val Asn Tyr Pro Pro

165 170 175

Val Asn Gly Ala Lys Val His Lys Gly Phe Leu Asp Ser Tyr Asn Glu

180 185 190

Val Gln Asp Lys Leu Val Ala Glu Val Lys Ala Gln Leu Asp Arg His

195 200 205

Pro Gly Tyr Lys Ile Val Val Thr Gly His Ser Leu Gly Gly Ala Thr

210 215 220

Ala Val Leu Ser Ala Leu Asp Leu Tyr His His Gly His Ala Asn Ile

225 230 235 240

Glu Ile Tyr Thr Gln Gly Gln Pro Arg Ile Gly Thr Pro Ala Phe Ala

245 250 255

Asn Tyr Val Ile Gly Thr Lys Ile Pro Tyr Gln Arg Leu Val His Glu

260 265 270

Arg Asp Ile Val Pro His Leu Pro Pro Gly Ala Phe Gly Phe Leu His

275 280 285

Ala Gly Glu Glu Phe Trp Ile Met Lys Asp Ser Ser Leu Arg Val Cys

290 295 300

Pro Asn Gly Ile Glu Thr Asp Asn Cys Ser Asn Ser Ile Val Pro Phe

305 310 315 320

Thr Ser Val Ile Asp His Leu Ser Tyr Leu Asp Met Asn Thr Gly Leu

325 330 335

Cys Leu

<210> 6

<211> 1363

<212> DNA

<213> Aureobasidium pullulans (lichtheima ramosa)

<400> 6

atgcattctc attttgtagt cttattgcta gcagtattca tctgcatgtg ctctgtatcg 60

ggtgtgccac tgcaaattga tccacgcgat gacaagagct atgttcctga acaatatcct 120

ttgaaggtga atggtccttt gccagaaggt gtaagcgtga tccaaggcta ttgtgaaaac 180

tgtaccatgt atcctgaaga aaatagtgta tcggcattct cgtcatcatc cacacaagat 240

tatcgtattg caagcgaggc agagattaag gcacacacat tttacacagc attgtcagcc 300

aatgcatact gcagaactgt cattcctggt ggtcgatgga gctgtcccca ctgtggtgtt 360

gcatccaatt tgcaaattac caagactttc agcaccttaa tcactgatac taatgtcttg 420

gtggctgttg gcgaaaagga gaagaccatc tatgtagttt ttcgtggtac aagctcaatt 480

cgcaacgcca ttgctgtaag ttcacccctt acaaacatga cactttgttg ctcatccgac 540

tcattctttc ttacaggaca ttgtttttgt accagtgaat tatccacctg ttaatggagc 600

caaagtacac aaaggtatgt gatgatgtgg tgtcatttat atataagaat gctcaatatg 660

ctcatttact atctaggatt tcttgatagc tataacgaag tccaggataa acttgttgct 720

gaagtcaagg cacaacttga tcgtcatcca ggatacaaga tcgtcgtcac tgggtaaata 780

cctgaaaaga catggatggc acgtgactaa atctgtgtca ttggtagaca ttccttggga 840

ggtgcaacag ctgttctcag tgcacttgac ctttatcacc atggccatgc caatatcgaa 900

atctatactc aaggtcagcc acgtataggt actccagcat ttgcaaacta tgtgattggc 960

accaagattc cataccaacg tcttgtccat gagcgtgaca gtaagtgtac cttgcacgac 1020

atgttcgttt tccccgacgt actaaagtat tgtatagttg ttcctcacct tccacctggt 1080

gcatttggtt tcttgcatgc tggtgaagag ttttggatca tgaaagatag ctcgttgcgt 1140

aagtagtgtc attgaaaagg ttgaagctat aatactgact atattgggta ggcgtatgtc 1200

caaatggcat tgaaactgac aactgcagca actccattgt tcccttcact agtgtcattg 1260

accatttaag gtgaatagta gctttattca tgtcattcat ccatgtaaac taacacttgt 1320

cgtatctagc tatcttgaca tgaacactgg tctctgttta taa 1363

Claims

1. A lipase gene Lrl-op is characterized in that the GC content of the lipase gene Lrl-op is 51%, the codon adaptation index is 0.84, and the nucleotide sequence of the lipase gene Lrl-op is shown as SEQ ID NO. 1.

2. A primer for amplifying lipase gene Lrl-op is characterized by comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3.

3. A lipase LRL is characterized in that the lipase LRL is a 1, 3-position specific lipase, and the amino acid sequence of the lipase LRL is shown as SEQ ID NO. 4.

4. A recombinant expression vector comprising the lipase gene Lrl-op of claim 1.

5. The recombinant expression vector according to claim 4, wherein the recombinant expression vector is a Pichia pastoris recombinant expression vector.

6. A recombinant expression strain comprising the recombinant expression vector of claim 6.

7. The recombinant expression strain of claim 6, wherein the recombinant expression strain is a Pichia pastoris recombinant expression strain.

8. Use of the lipase LRL of claim 3 for the degradation of glycerides and/or lipid modifications.

9. Use according to claim 8, wherein the reaction temperature of the lipase LRL is 40 ℃ to 60 ℃ in the reaction for the degradation of glycerides and/or lipid-modifying agents.

10. Use according to claim 8, wherein the lipase LRL has a reaction pH of 6-9 in the glyceride and/or lipid splitting and/or lipid modifying reaction.