CN112608912A

CN112608912A - Lipase mutant D170A with improved catalytic activity and application thereof

Info

Publication number: CN112608912A
Application number: CN202011501645.5A
Authority: CN
Inventors: 黄遵锡; 韩楠玉; 唐敏源; 姜占宝
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2019-06-17
Filing date: 2019-06-17
Publication date: 2021-04-06
Anticipated expiration: 2039-06-17
Also published as: CN112592907A; CN112608912B; CN110129301B; CN110129301A; CN112574974B; CN112574974A; CN112592907B

Abstract

The divisional application discloses a lipase mutant D170A with improved catalytic activity and application thereof, wherein the amino acid sequence of the lipase mutant D170A is shown as SEQ ID NO.4, the lipase mutant D170A is obtained by changing aspartic acid at position 170 in the thermomyces lanuginosus lipase amino acid sequence SEQ ID NO.1 into alanine, and the coding gene is shown as SEQ ID NO. 8. The mutant has improved catalytic activity.

Description

Lipase mutant D170A with improved catalytic activity and application thereof

The application is divisional application with application number 201910523658.3, application date 2019, 6.17 and invention name 'lipase mutant with improved catalytic activity and application thereof'.

Technical Field

The invention belongs to the technical field of enzyme engineering, and relates to a lipase mutant, in particular to a lipase mutant D170A with improved catalytic activity and application thereof.

Background

Lipase (EC 3.1.1.3) is called triacylglycerol acyl hydrolase, widely exists in organism, and belongs to carboxyl ester hydrolase. The lipase can catalyze the reactions of ester exchange, esterification, alcoholysis, acidolysis and the like, and can also catalyze the hydrolysis of the glyceride bond of the grease. Lipases are widely used in the industries of detergents, foods, fats and oils, leather, medicines, and the like. The lipase comes from animals, plants and microorganisms, wherein the lipase from the microorganisms has the advantages of various types, short growth period and wide temperature and pH action range, so that the lipase from the microorganisms can be more easily applied to industrial production.

Thermomyces lanuginosus is a fungus with wide distribution and higher upper limit of growth temperature, and can produce lipase, protease and the like with good thermal stability and important industrial application value. Compared with lipases from other microbial sources, the Thermomyces lanuginosus lipase has better thermal stability, but still cannot meet the industrial requirements; in addition, the catalytic activity of Thermomyces lanuginosus lipase is still influenced by factors such as temperature, organic solvent in the reaction medium, substrate concentration and the like. Therefore, increasing the catalytic activity or thermal stability of Thermomyces lanuginosus lipase would allow for a wider range of industrial applications of Thermomyces lanuginosus lipase. Therefore, the invention makes the Thermomyces lanuginosus lipase gene become a lipase with improved catalytic activity by modifying the Thermomyces lanuginosus lipase gene.

Disclosure of Invention

The invention utilizes a computer rational design method to carry out site-directed mutagenesis of charged amino acid on the aschersonia lanuginosa lipase gene and express the charged amino acid in a eukaryotic exogenous protein expression system pichia pastoris so as to improve the catalytic activity of the aschersonia lanuginosa lipase.

The invention is realized by the following technical scheme:

a lipase mutant with improved catalytic activity is obtained by site-directed mutagenesis of an amino acid sequence of Thermomyces lanuginosus lipase, wherein the amino acid sequence of the Thermomyces lanuginosus lipase is shown as SEQ ID No. 1.

The lipase mutant is obtained by changing 116 th aspartic acid in the Thermomyces lanuginosus lipase amino acid sequence SEQ ID NO.1 into lysine.

The amino acid sequence of the lipase mutant is shown as SEQ ID NO. 2.

The catalytic activity of the lipase mutant is improved by 35.5 percent.

The lipase mutant is obtained by changing the 163 th aspartic acid in the Thermomyces lanuginosus lipase amino acid sequence SEQ ID NO.1 into phenylalanine.

The amino acid sequence of the lipase mutant is shown as SEQ ID NO. 3.

The catalytic activity of the lipase mutant is improved by 17.0 percent.

The lipase mutant is obtained by changing aspartic acid at position 170 in an amino acid sequence SEQ ID NO.1 of thermomyces lanuginosus lipase into alanine.

The amino acid sequence of the lipase mutant is shown as SEQ ID NO. 4.

The catalytic activity of the lipase mutant is improved by 44.2 percent.

The lipase mutant is obtained by changing aspartic acid at the 259 th site in a Thermomyces lanuginosus lipase amino acid sequence SEQ ID NO.1 into tyrosine.

The amino acid sequence of the lipase mutant is shown as SEQ ID NO. 5.

The catalytic activity of the lipase mutant is improved by 25.7 percent.

In another aspect of the present invention, the amino acid sequences of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are modified, deleted or added with one or more amino acids to obtain an amino acid sequence, and a sequence with only 90% homology is also within the protection scope of the present invention.

The nucleotide sequence of the lipase mutant coding gene is shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

In another aspect of the invention, the nucleotide sequence of the encoding gene SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or the complementary sequence thereof or which differs from the nucleotide sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or the complementary sequence thereof due to the degeneracy of the genetic code is also within the scope of the invention.

In another aspect of the invention, the invention also includes a plasmid carrying a lipase mutant with the gene sequence SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO. 9.

In another aspect of the invention, the invention also provides an engineering bacterium of a gene of a lipase mutant, wherein the engineering bacterium contains a vector with genes shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

The engineering bacteria are obtained by cloning genes shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 onto an expression vector and then carrying out cell transformation.

The expression vector of the coding gene SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 is selected from pPIC9K, pPIC9, pPICZaA \ B \ C, pPICZA \ B \ C or pGAPZaA \ B \ C.

In another aspect of the present invention, the use of the lipase mutant provided by the present invention in feed additives is also within the scope of the present invention.

The invention has the beneficial effects that:

the invention provides a lipase mutant, wherein the enzyme activities of mutants D116K, D163F, D170A and D259Y are respectively improved by 35.5%, 17.0% and 44.2% compared with the wild type.

Drawings

FIG. 1 is a flow chart of a method for constructing a lipase mutant according to an embodiment of the present invention;

FIG. 2 is a graph showing the determination of the optimum pH according to the example of the present invention;

FIG. 3 is a graph of pH stability provided by an embodiment of the present invention;

FIG. 4 is a graph of the optimum temperature profile provided by an embodiment of the present invention;

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Through protein sequence analysis and computer software simulation of Thermomyces lanuginosus lipase, the mutation sites of the Thermomyces lanuginosus lipase are determined to be that aspartic acid at position 116 is changed into lysine, aspartic acid at position 163 is changed into phenylalanine, aspartic acid at position 170 is changed into alanine, and aspartic acid at position 259 is changed into tyrosine. The specific implementation scheme is as follows: a novel lipase gene is obtained by taking a Thermomyces lanuginosus lipase gene as a template through a site-directed mutagenesis method, the mutated gene is linked with vectors such as pPIC9, pPICZaA \ B \ C, pPICZA \ B \ C, PGAPZaA \ B \ C and the like to construct recombinant plasmids, the recombinant plasmids are transferred into corresponding host bacteria (GS115 or X33, SMD1168 and PICHIAPINK) for heterologous expression, and the lipase mutant can be obtained through fermentation. The mutant has improved catalytic activity, ideal heat-resistant property and suitability for high-temperature resistant granulation, and is suitable for industrial production.

1 materials of the experiment

1.1 strains and vectors

(1) The gene source strain: thermomyces lanuginosus (Thermomyces lanuginosus), stored in the laboratory;

(2) expression host bacteria and vectors: pichia pastoris (Pichia pastoris) GS115, vector pPIC9K, available from Novagen, Germany;

(3) cloning host bacteria: DMT competent cells, purchased from TransGen Biotech, Beijing;

(4) original plasmid: the lipase TLL was ligated to the vector pPIC9K and stored as a laboratory construct.

1.2 Primary reagents

Site-directed Mutagenesis kit Fast Mutagenesis System (Beijing TransGen Biotech Co.); plasmid Mini Kit I (Omega company); gel extraction kit (Omega corporation); restriction endonucleases (EcoRI, NotI); p-nitrophenol palmitate (p-NP) (Sigma).

1.3 Experimental instruments

GD100 molecular water bath (Grant corporation); legend micro21 high speed centrifuge (Thermo corporation, USA); MK-20 metal baths (Ouchun, Hangzhou Co.); ETC811PCR instrument (tosporus, suzhou); PowerB nucleic acid electrophoresis apparatus (Cavoy, Beijing); universal Hood II gel imager (Bio-Rad, USA); SpectraMax Plus384 microplate reader (Molecular Devices, USA); DZKW-4 electronic thermostat water bath (Beijing Zhongxing company).

1.4 Main Medium

LB culture medium: 0.5% (w/v) yeast extract, 1.0% (w/v) tryptone, 1.0% (w/v) NaCl, dissolved using double distilled water; YEPD medium: 1.0% (w/v) yeast extract, 2.0% (w/v) peptone, 2.0% (w/v) glucose, dissolved with double distilled water.

EXAMPLE 1 preparation of lipase mutants

As shown in FIG. 1, site-directed mutagenesis was performed using a site-directed mutagenesis kit and the following primers using pPIC9K-TLL recombinant plasmid as a template. After amplification, 10. mu.L of the LPCR product was subjected to agarose gel electrophoresis, and after the band size was verified to be correct, 1. mu.L of DMT enzyme was added to the PCR product, mixed well, and digested at 37 ℃ for 1 hour. Then, conversion is carried out: adding 3 μ L of the digested product into 50 μ L DMT competent cells, ice-bathing for 30min, heat-shocking in 42 deg.C molecular water bath for 45s, ice-bathing for 2min, adding 500 μ LLB culture medium into the product, incubating in 37 deg.C shaking table at 180rpm for 1 hr, and applying 200 μ L bacterial solution to kan⁺Resistant LB plates were incubated overnight in an incubator at 37 ℃. Randomly picking single colony on the plate for positive clone verification the next day, sequencing and comparing the positive bacteria, and comparing the sequencing result with the template sequenceIn this regard, it was determined whether the mutation was successful. After sequencing and mutation verification are successful, extracting recombinant plasmids of the mutants, performing linearization treatment by using restriction endonuclease, and transferring the linearized recombinant vectors into pichia pastoris by electric shock to obtain pichia pastoris recombinant strain transformants. And (3) fermenting the recombinant strain to obtain fermentation liquor for measuring the lipase activity.

Forward-D116K：GGATGCAGAGGACATAAGGGTTTCACTT；

Reverse-D116K：GGACGAAGTGAAACCCTTATGTCCTCTG；

Forward-D163F：ACTGTTGCCGGAGCATTCCTGAGAGGAA；

Reverse-D163F：ACCATTTCCTCTCAGGAATGCTCCGGCA；

Forward-D170A：AGAGGAAATGGTTATGCTATCGACGTGT；

Reverse-D170A：TGAAAACACGTCGATAGCATAACCATTT；

Forward-D259Y：CAGCCTAACATTCCTTACATCCCTGCCC；

Reverse-D259Y：TAGGTGGGCAGGGATGTAAGGAATGTTA。

Example 2 determination of enzyme Activity and enzymatic Properties of Lipase mutants

1. Determination of enzyme Activity of Lipase mutant

The enzyme activity unit is defined as: the enzyme amount required for hydrolyzing the substrate p-NP every minute under a certain condition to generate 1 mu moL of p-nitrophenol is expressed as U as one enzyme activity unit. P-nitrophenol method: sucking 420 mu L of 50 mMTris-HCl buffer solution with the pH value of 9.0 into a centrifuge tube, adding 30 mu L of 10mM substrate p-NP, fully mixing uniformly, preheating for 2min at 37 ℃, adding 50 mu L of diluted enzyme solution, reacting for 5min, adding 50 mu L of 10% SDS to terminate the reaction, finally adding 500 mu L of 0.5M sodium carbonate for color development, and measuring the OD value of the product by using an enzyme-labeling instrument at the wavelength of 405 nm. The results of the mutant enzyme activity assay are shown in table 1: compared with the wild type, the enzyme activities of the mutants D116K, D163F, D170A and D259Y are respectively improved by 35.5 percent, 17.0 percent and 44.2 percent.

TABLE 1 determination of the enzyme Activity of the mutants

2. Determination of enzymatic Properties of Lipase mutants

And (3) optimum pH determination: the enzyme activities of the lipases were measured in buffer systems of different pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0), respectively, the enzyme solutions were diluted to an appropriate fold, and the optimum pH of the lipase mutants was determined by the p-nitrophenol method at 37 ℃. As shown in FIG. 2, the optimum pH values of both Thermomyces lanuginosus lipase and mutants D116K, D163F, D170A and D259Y were 9.0.

And (3) measuring the pH stability: the enzyme solution was diluted with buffers of different pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0), the diluted enzyme solution was allowed to stand in a water bath at 37 ℃ for 1 hour, and the enzyme activity was immediately measured by the p-nitrophenol method at the optimum pH at 37 ℃. The enzyme solution of the control group was an enzyme solution that was not tolerated. As shown in FIG. 3, the Thermomyces lanuginosus lipase and the 4 mutants D116K, D163F, D170A, and D259Y were all stable at pH 8.0-12.0.

Optimum temperature measurement: under the condition of optimum pH, the reaction system is placed at different temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃) for reaction. As shown in FIG. 4, the optimum temperatures of Thermomyces lanuginosus lipase and D116K were 55 ℃, D163F was 60 ℃ and D170A and D259Y were 50 ℃.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> university of Yunnan Master

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Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala

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Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr

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Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val

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Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys

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Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr

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Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys

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

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Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala

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Gly Ala Asp Leu Arg Gly Asn Gly Tyr Ala Ile Asp Val Phe Ser Tyr

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Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val

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tgcacgggaa atgcctgccc agaggtagag aaggccgatg caacgtttct ctactcgttt 180

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tcctggagat ctgtagccga tacgttaaga cagaaggtgg aggatgctgt gagagagcat 420

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cctgcccacc tatggtactt cggtttaatt ggaacatgtc tttag 825

<210> 9

<211> 825

<212> DNA

<213> encoding Gene (Coding gene)

<400> 9

agtcctatta gaagagaggt ctcgcaggat ctgtttaacc agttcaatct ctttgcacag 60

tattctgcag ccgcatactg cggaaaaaac aatgatgccc cagctggtac aaacattacg 120

tgcacgggaa atgcctgccc agaggtagag aaggccgatg caacgtttct ctactcgttt 180

gaagactctg gagtgggaga tgtcaccgga ttccttgctc tcgacaacac gaacaaattg 240

atcgtcctct ctttcagagg atctagatcc attgagaact ggatcggaaa tcttaacttc 300

gacttgaaag agatcaatga catttgctcc ggatgacgag gacatgacgg tttcacttcg 360

tcctggagat ctgtagccga tacgttaaga cagaaggtgg aggatgctgt gagagagcat 420

ccagactata gagtggtgtt taccggacat agcttgggtg gtgcattggc aactgttgcc 480

ggagcagacc tgagaggaaa tggttatgat atcgacgtgt tttcatatgg agcccctaga 540

gtcggaaaca gagcttttgc agagttcctg accgtacaga ccggaggaac actctacaga 600

attacccaca ccaatgatat tgtccctaga ctccctccac gcgagttcgg ttacagacat 660

tctagcccag agtactggat caaatctgga acccttgtcc cagtcaccag aaacgatatc 720

gtgaagattg aaggaatcga tgccaccgga ggaaacaacc agcctaacat tccttacatc 780

cctgcccacc tatggtactt cggtttaatt ggaacatgtc tttag 825

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggatgcagag gacataaggg tttcactt 28

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggacgaagtg aaacccttat gtcctctg 28

<210> 12

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

actgttgccg gagcattcct gagaggaa 28

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

accatttcct ctcaggaatg ctccggca 28

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

agaggaaatg gttatgctat cgacgtgt 28

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tgaaaacacg tcgatagcat aaccattt 28

<210> 16

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cagcctaaca ttccttacat ccctgccc 28

<210> 17

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

taggtgggca gggatgtaag gaatgtta 28

Claims

1. A lipase mutant D170A with improved catalytic activity is characterized in that the amino acid sequence of the lipase mutant D170A is shown as SEQ ID No. 4.

2. A DNA molecule encoding the lipase mutant D170A of claim 1.

3. The DNA molecule of claim 2, wherein the nucleotide sequence of said DNA molecule is set forth in SEQ ID No. 8.

4. An engineering bacterium, which is characterized by comprising a vector with a gene shown as SEQ ID NO. 8.

5. The engineering bacterium of claim 4, wherein the vector is pPIC9K, pPIC9, pPICZaA \ B \ C, pPICZA \ B \ C or PGAPZaA \ B \ C.

6. The lipase mutant D170A as claimed in claim 1, which is used in feed additives.