CN107488644B - Lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability and gene and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability, a gene and application thereof, wherein an amino acid sequence is shown as SEQ ID NO. 4. The residual enzyme activity of the mutant TTL-Gly60Glu/Ser61Asn after being subjected to water bath at 80 ℃ for 5min is 25.1 percent, and is improved to 1.37 times of that of the parent lipase.

Description

Lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability and gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability, and a gene and application thereof.

Background

Lipase (EC 3.1.1.3) is known as triacylglycerol hydrolase, belongs to the alpha/beta-sheet enzyme family, and is a serine hydrolase. The lipase can not only catalyze the hydrolysis of natural substrate grease on an oil-water interface and release glyceride or glycerol with less ester bonds and fatty acid, but also catalyze reactions such as acidolysis, transesterification, ester synthesis and the like in a non-aqueous phase system. The lipase for industrial feed is mainly derived from microorganisms, and the lipase secreted by the microorganisms has a wide action pH value and action temperature. With the continuous popularization of enzyme catalysis technology, the application conditions of enzyme preparations are more and more strict, such as high temperature, strong acid and strong alkali and other special conditions. Lipases are widely used as an important enzyme species in the fields of food processing, feed, washing, medicine, and the like. The processes of oil and fat processing and feed granulation are generally performed at relatively high temperatures, and lipases with poor thermostability are subject to denaturation in the above processes, so that the development of lipases with high thermostability has been the object of academic and industrial efforts.

In order to obtain a better enzyme preparation, on one hand, a proper enzyme gene can be screened from the nature, and on the other hand, the existing industrialized enzyme protein is modified through a molecular biology technology so as to adapt to the requirements of different industries. The two major strategies for modifying enzyme protein are currently available, one of which is irrational design, and the enzyme protein which better meets the action condition is selected according to the specific modification purpose by modifying the gene of the enzyme through random mutation. The strategy has the advantages that the structure and the action mechanism of the enzyme do not need to be deeply researched, but the screening is completed by extremely large workload, and the efficiency is low.

A scientist is reported in documents to separate a thermophilic eupodium strain from a volcanic region in south of Tony, the secreted lipase of the thermophilic eupodium strain has better heat resistance, and 50 percent of enzyme activity is remained after 1 hour of treatment at 70 ℃. Pichia pastoris has simple culture conditions, is easy for industrial production, efficiently secretes and expresses exogenous proteins, and successfully expresses a plurality of exogenous lipases. The invention utilizes pichia pastoris to efficiently secrete and express the lipase from the gymnosperm thermophilum, and obtains the lipase mutant with obviously improved thermal stability by using the gymnosperm thermophilum lipase as a template and through a site-directed mutagenesis technology.

Disclosure of Invention

The invention aims to provide a lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability.

It is still another object of the present invention to provide a gene encoding the above mutant.

The invention further aims to provide application of the lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability.

nucleotide sequence SEQ ID NO.1 of wild lipase

TCTCCAGTCAGACGTGAGGTTTCTCAGGACTTGTTCGACCAGTTTAATTTGTTCGCTCAGTACTCTGCTGCTGCCTACTGTGCCAAGAATAACGATGCCCCAGCTGGTGCTAACGTTACCTGTAGAGGTTCCATCTGCCCAGAGGTTGAGAAGGCTGACGCCACCTTCTTGTACTCTTTCGAGGACTCTGGAGTTGGTGACGTTACCGGTTTCTTGGCTTTGGACAACACCAACAGATTGATCGTCTTGTCTTTCCGTGGTTCCCGTTCCCTTGAGAATTGGATCGGTAACATTAATTTGGATTTGAAGGGTATCGACGACATCTGCTCTGGTTGCAAGGGACACGATGGTTTCACCTCCTCTTGGAGATCCGTTGCCAACACCTTGACCCAACAGGTTCAGAACGCCGTTAGAGAGCATCCAGACTACCGTGTCGTCTTCACTGGTCACTCTTTGGGTGGTGCTTTGGCTACCGTTGCTGGTGCCTCTTTGAGAGGTAACGGTTACGACATCGACGTTTTCTCCTACGGTGCTCCTCGTGTTGGTAACAGAGCCTTCGCTGTCTTCTTGACCGCTCAGACCGGTGGTACCTTGTACAGAATCACCCATACCAACGATATCGTCCCACGTTTGCCACCAAGAGAGCTTGGATACTCCCACTCTTCCCCAGAGTACTGGATCACCTCCGGTACTTTGGTTCCAGTTACCAAGAACGATATTGTTAAGGTTGAGGGAATTGACTCCACCGACGGTAACAACCAGCCAAATACCCCAGACATTGCTGCCCACTTGTGGTACTTCGGTTTGATTGGTACTTGTTTGTAA

Amino acid sequence SEQ ID NO.2 of wild lipase

SPVRREVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGANVTCRGSICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGTLVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL

Nucleotide sequence SEQ ID NO.3 of lipase TTL mutant TTL-Gly60Glu/Ser61Asn gene

TCTCCAGTCAGACGTGAGGTTTCTCAGGACTTGTTCGACCAGTTTAATTTGTTCGCTCAGTACTCTGCTGCTGCCTACTGTGCCAAGAATAACGATGCCCCAGCTGGTGCTAACGTTACCTGTAGAGAAAATATCTGCCCAGAGGTTGAGAAGGCTGACGCCACCTTCTTGTACTCTTTCGAGGACTCTGGAGTTGGTGACGTTACCGGTTTCTTGGCTTTGGACAACACCAACAGATTGATCGTCTTGTCTTTCCGTGGTTCCCGTTCCCTTGAGAATTGGATCGGTAACATTAATTTGGATTTGAAGGGTATCGACGACATCTGCTCTGGTTGCAAGGGACACGATGGTTTCACCTCCTCTTGGAGATCCGTTGCCAACACCTTGACCCAACAGGTTCAGAACGCCGTTAGAGAGCATCCAGACTACCGTGTCGTCTTCACTGGTCACTCTTTGGGTGGTGCTTTGGCTACCGTTGCTGGTGCCTCTTTGAGAGGTAACGGTTACGACATCGACGTTTTCTCCTACGGTGCTCCTCGTGTTGGTAACAGAGCCTTCGCTGTCTTCTTGACCGCTCAGACCGGTGGTACCTTGTACAGAATCACCCATACCAACGATATCGTCCCACGTTTGCCACCAAGAGAGCTTGGATACTCCCACTCTTCCCCAGAGTACTGGATCACCTCCGGTACTTTGGTTCCAGTTACCAAGAACGATATTGTTAAGGTTGAGGGAATTGACTCCACCGACGGTAACAACCAGCCAAATACCCCAGACATTGCTGCCCACTTGTGGTACTTCGGTTTGATTGGTACTTGTTTGTAA

Amino acid sequence SEQ ID NO.4 of lipase TTL mutant TTL-Gly60Glu/Ser61Asn

SPVRREVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGANVTCRENICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGTLVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL

According to the technical scheme of the invention, the parent lipase TTL is subjected to site-directed mutagenesis to obtain the lipase mutant. The expression vectors for expressing the lipase mutants are pPICZ alpha A and pPIC 9K; the host cells used for transformation of the expression vector are pichia pastoris X33 and GS 115. The lipase mutant is as follows: TTL-Gly60Glu/Ser61 Asn.

The residual enzyme activity of the parent lipase TTL is 18.3 percent after being soaked in water at 80 ℃ for 5 min. The invention utilizes the molecular biology technology to perform site-specific mutagenesis on the lipase TTL to obtain the lipase mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability. The residual enzyme activity of the mutant TTL-Gly60Glu/Ser61Asn after being subjected to water bath at 80 ℃ for 5min is 25.1 percent, and is improved to 1.37 times of that of the parent lipase.

Drawings

FIG. 1 shows fermentation diagram of Pichia pastoris X33 recombinant strain 50L tank containing pPICz alpha A-TTL.

FIG. 2 protein electrophoresis of enzyme solutions at various time periods of fermentation.

FIG. 3 is a diagram showing the enzymatic properties of lipase TTL.

FIG. 4 is a diagram showing the enzymatic properties of the lipase mutant TTL-Gly60Glu/Ser61 Asn.

Detailed Description

In order to increase the industrial application value of the lipase, the lipase gene TTL is expressed in pichia pastoris, and after the three-dimensional structure of the lipase is further researched, the thermal stability of the lipase is improved by aiming at the amino acid mutation of the key characteristics of the lipase. The method of modifying lipase and the improved lipase obtained by the method of the present invention will be described in detail below.

the molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available.

Experimental materials and reagents:

1. Bacterial strains and vectors

Coli strain Topl0, Pichia pastoris X33, GS115, vector pPICz. alpha.A, Ppic9K, Zeocin were purchased from Invitrogen.

2. Enzyme and kit

PCR enzyme, restriction enzyme, plasmid extraction and gel purification kits were purchased from Shanghai Biotech.

Example 1 high expression of Lipase TTL in Pichia pastoris X33

1. Lipase gene TTL is used as a target gene, the full length of wild lipase gene is 825 bases (SEQ ID No.1), 274 amino acids (SEQ ID No.2) are coded by the wild lipase gene, restriction enzymes EcoRI and XbaI are used for enzyme digestion, the wild lipase gene is constructed into a pPICz alpha A vector to obtain an expression vector pPICz alpha A-TTL, the pPICz alpha A-TTL is converted into a Top 10 competent cell, and the positive clone is obtained after screening by an antibiotic Zeocin. And linearizing the recombinant expression vector by SacI, and performing electric shock transformation on the linearized recombinant vector to obtain a pichia pastoris recombinant strain transformant. And (3) performing high-density fermentation culture on the recombinant bacterium single bacterial strain. The enzyme activity is measured by timing sampling in the fermentation process, the expression condition of the lipase is shown in figure 1, and the enzyme activity of the lipase is 31500U/mL at 189h of fermentation. The lipase in the fermentation liquor is subjected to fractional precipitation by ammonium sulfate, full dialysis and purification by ion exchange column chromatography, and the purified protein liquid is subjected to 12% SDS-PAGE detection, and the result is shown in figure 2.

2. The lipase activity is determined by adopting an olive oil emulsion hydrolytic titration method, and the definition of the enzyme activity is as follows: the enzyme amount consumed by the lipase for hydrolyzing the olive oil emulsion to generate 1 mu mol/min of fatty acid under the conditions of 40 ℃ and pH 7.0 is 1 lipase activity unit. The lipase heat treatment adopts a water bath method, enzyme liquid is properly diluted and then placed in a glass test tube, and the temperature is kept for 5min in water bath pots (70-90 ℃) with different temperatures.

Example 2 determination of enzymatic Properties of Lipase TTL

1. Determination of optimum reaction temperature and thermal stability of Lipase TTL

Respectively measuring the activity of lipase at 30-80 deg.C and 10 deg.C intervals, and measuring the relative activity at different temperatures by using the activity at 50 deg.C as reference. As a result, as shown in FIG. 3(a), the optimum temperature for the action of lipase was 50 ℃. And (3) placing the lipase liquid in a glass test tube, carrying out heat treatment for 5min at different temperatures (70-90 ℃), measuring the residual lipase activity, and comparing the enzyme activity after heat treatment with the untreated enzyme activity as a reference to obtain the residual relative enzyme activity at the temperature. The results are shown in FIG. 3(b), and the lipase residual activity after heat treatment at 80 ℃ for 5min was 18.3%.

2. Determination of optimum reaction pH and pH stability of Lipase TTL

And respectively measuring the enzyme activity of the lipase in buffer solution systems with different pH values (3.0-9.0), and calculating the relative enzyme activity under different pH values by taking the enzyme activity with the pH value of 7.0 as a reference. As a result, as shown in FIG. 3(c), the optimum pH for the action of lipase was 7.0. And respectively adding diluted enzyme solution into the buffer solution systems with different pH values, processing for 2h at room temperature, measuring the activity of residual lipase, and calculating the residual relative enzyme activity at the pH value by taking the untreated enzyme activity as a reference. As shown in FIG. 3(d), the enzyme has a wide pH stability range and is stable in enzyme activity within a pH range of 5.0 to 9.0.

Example 3 Lipase mutant with improved thermostability TTL-Gly60Glu/Ser61Asn

1. And (3) carrying out PCR amplification by taking the plasmid pPICZ alpha A-TTL as a template and respectively taking the primers F1 and PIC-R and the primers R1 and PIC-F, and then carrying out fusion PCR on the obtained 2 DNA fragments to obtain the recombinant vector pPICZ alpha A-TTL-G60ES 61N. And (3) transforming the recombinant vector into a Top 10 competent cell, and screening by an antibiotic Zeocin to obtain a positive clone. And linearizing the recombinant expression vector by SacI, and performing electric shock transformation on the linearized recombinant vector to obtain a pichia pastoris recombinant strain transformant. The total length of the mutated lipase gene is 825 basic groups (shown as SEQ ID NO. 3), and the mutated lipase gene encodes 274 amino acids (shown as SEQ ID NO. 4).

The primers used were as follows:

F1:5’-CTGTAGAGAAAATATCTGCCCAGAGGTT-3’

R1:5’-CTCTGGGCAGATATTTTCTCTACAGGTAAC-3’

PIC-F：5’-CTTGCTTGAGAAGGTTTTGGGACGC-3’

PIC-R：5’-CTTGGAGCGAACGACCTACACCGAA-3’

2. Determination of optimum reaction temperature and thermal stability of recombinant Lipase

Respectively measuring the activity of lipase at 30-80 deg.C and 10 deg.C intervals, and measuring the relative activity at different temperatures by using the activity at 50 deg.C as reference. As a result, as shown in FIG. 4(a), the optimum temperature for the action of lipase was 50 ℃. And (3) carrying out heat treatment on the lipase liquid at different temperatures (70-90 ℃) for 5min, measuring the activity of residual lipase, and calculating to obtain the residual relative enzyme activity at the temperature by taking the untreated enzyme activity as a reference. The results are shown in FIG. 4(b), and the lipase residual activity after heat treatment at 80 ℃ for 5min is 25.1%, which is increased to 1.37 times of that of the parent lipase.

3. Determination of optimum reaction pH and pH stability of recombinant Lipase

And respectively measuring the enzyme activity of the lipase in buffer solution systems with different pH values (3.0-9.0), and measuring the relative enzyme activity under different pH values by taking the enzyme activity with the pH value of 7.0 as a reference. The results are shown in FIG. 4 (c): the optimum pH for the action of the lipase was 7.0. And respectively adding diluted enzyme liquid into the buffer solution systems with different pH values, processing for 2 hours at room temperature, measuring the activity of residual lipase, and comparing the enzyme activity of the processed enzyme liquid with that of the unprocessed enzyme liquid by taking the untreated enzyme activity as a reference to obtain the residual relative enzyme activity under the pH value. As shown in FIG. 4(d), the enzyme had a wide pH stability range and was stable in enzyme activity within a pH range of 5.0 to 9.0.

Claims (5)

1. The lipase TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability is characterized in that the amino acid sequence of the lipase TTL mutant TTL-Gly60Glu/Ser61Asn is shown in SEQ ID NO. 4.

2. A gene encoding the improved thermal stability lipase TTL mutant according to claim 1 which is TTL-Gly60Glu/Ser61 Asn.

3. The TTL mutant gene according to claim 2, wherein the nucleotide sequence is represented by SEQ ID No. 3.

4. Use of the TTL mutant TTL-Gly60Glu/Ser61Asn with improved thermal stability according to claim 1 for hydrolyzing fats and oils in vitro.

5. Use of the lipase TTL mutant gene with improved thermostability according to claim 2 in food processing, feed or washing.