CN112626052B - Polygalacturonase mutant with improved thermal stability and application thereof - Google Patents

Polygalacturonase mutant with improved thermal stability and application thereof Download PDF

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CN112626052B
CN112626052B CN202011430468.6A CN202011430468A CN112626052B CN 112626052 B CN112626052 B CN 112626052B CN 202011430468 A CN202011430468 A CN 202011430468A CN 112626052 B CN112626052 B CN 112626052B
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刘文龙
涂涛
佟新伟
王兴吉
王克芬
张�杰
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Shandong Lonct Enzymes Co ltd
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Abstract

The invention belongs to the technical field of feed biology, and particularly relates to a polygalacturonase mutant and a coding gene and application thereof. Is a mutant E247C/S276C obtained by point mutation by using polygalacturonase derived from Talaromyces leycettanus JCM12802 as a female parent. Compared with the wild polygalacturonase, the polygalacturonase mutant has the advantages that the polygalacturonase is enhanced in heat stability, about 21.78% of activity is still kept after 30min treatment at 70 ℃, and the activity of the wild enzyme is disappeared. In addition, the specific activity of the wild type is 50483U/mg, the specific activity of the mutant is 46270U/mg, and the mutant can be applied to the fields of energy, food and feed.

Description

Polygalacturonase mutant with improved thermal stability and application thereof
The technical field is as follows:
the invention belongs to the technical field of feed biology, and particularly relates to a polygalacturonase mutant and a coding gene and application thereof.
Background art:
polygalacturonases (PGs) hydrolyze the alpha-1, 4-glycosidic linkages linking galacturonic acid residues, and play a key role in the degradation of pectin. Wherein the PG of fungal origin has a high activity, generally having an optimum activity at a weakly acidic pH and an optimum temperature of 30 to 50 ℃. But its use in the food and feed industries is limited due to its low thermal stability. Then, the development of a new high temperature-resistant polygalacturonase is under way. Currently, the most widely studied is the modification of enzyme molecules by protein engineering to improve the thermostability of enzymes. Strategies to improve protein thermostability by rational design generally include the following: optimizing the surface charge of the protein, introducing disulfide bonds, realizing proline effect, designing free energy for protein development, designing temperature factors (B factors) and the like.
The invention mainly achieves the purpose of improving the thermal stability of the enzyme by introducing disulfide bonds into the structure of polygalacturonase, analyzes the three-dimensional structure of the enzyme protein by a computer, adds disulfide bonds at different sites, and finds that the thermal stability of the enzyme is obviously improved after the disulfide bonds are introduced at key sites.
The invention content is as follows:
an object of the invention is to provide a mutant E247C/S276C obtained by point mutation using polygalacturonase derived from Talaromyces leycettanus JCM12802 as a parent.
It is still another object of the present invention to provide a gene encoding the above mutant, as shown in SEQ ID NO. 3.
The invention also aims to provide the amino acid sequence of the mutant, which is shown as SEQ ID NO. 2.
It is still another object of the present invention to provide a recombinant vector comprising the above mutant gene.
It is still another object of the present invention to provide a recombinant strain comprising the above mutant gene.
According to a particular embodiment of the invention, the wild-type polygalacturonase enzyme having the amino acid sequence shown in SEQ ID No.1 is subjected to site-directed mutagenesis.
SEQ ID NO.1
MHTIQPLLTYGLAVGAVLSSAAPTAVEKRASCTFTDAASAMASKTACSTITLN NIAVPAGTTLDLTGLTSGTRVIFEGTTTFGYQEWSGPLVSISGTDITVQGASGSV LDGDGARWWDGQGSNGGKTKPKFFYAHSLDSSSITGITIKNSPVQVFSIQSNN LSLTDITVDDADGDTQGGHNTDAFDIGSSTYITITNANVHNQDDCIAVNSGENI IFTGGTCTGGHGLSIGSVGGRSDNTVKNVTIEHSTVTNSQNGVRIKTVYGATG SVSEVTYSNIQMSGITNYGIVIEQDYENGSPTGTPTNGVPITDLTLNTVTGSVSS GATEIYILCGSGSCSSWTWTGVSITGGSKSTKCENVPSGVSC
The invention carries out mutation at 247 and 276 sites on wild polygalacturonase with an amino acid sequence shown as SEQ ID NO.1, and respectively mutates glutamic acid and serine into cysteine, thereby obtaining a polygalacturonase mutant E247C/S276C.
The polygalacturonase mutant with high thermal stability has an amino acid sequence shown as SEQ ID No.2 and consists of 365 amino acids.
SEQ ID NO.2：
MHTIQPLLTYGLAVGAVLSSAAPTAVEKRASCTFTDAASAMASKTACSTITLN NIAVPAGTTLDLTGLTSGTRVIFEGTTTFGYQEWSGPLVSISGTDITVQGASGSV LDGDGARWWDGQGSNGGKTKPKFFYAHSLDSSSITGITIKNSPVQVFSIQSNN LSLTDITVDDADGDTQGGHNTDAFDIGSSTYITITNANVHNQDDCIAVNSGENI IFTGGTCTGGHGLSIGSVGGRSDNTVKNVTICHSTVTNSQNGVRIKTVYGATG SVSEVTYCNIQMSGITNYGIVIEQDYENGSPTGTPTNGVPITDLTLNTVTGSVS SGATEIYILCGSGSCSSWTWTGVSITGGSKSTKCENVPSGVSC
The invention also provides a gene for coding the polygalacturonase mutant with high thermal stability, and the nucleotide sequence is shown as SEQ ID NO.3 and is 1098bp (including a terminator).
SEQ ID NO.3：
ATGCATACGATCCAACCTCTTCTAACCTATGGGCTGGCCGTGGGAGCTG TCCTTTCCTCAGCGGCCCCAACTGCTGTCGAGAAGCGTGCCAGCTGCACCT TTACCGATGCTGCTTCTGCCATGGCAAGCAAGACAGCCTGCTCGACTATCA CGCTGAACAACATTGCCGTTCCTGCTGGGACCACCTTGGACCTGACGGGCT TGACATCCGGCACCAGGGTCATCTTCGAAGGAACAACCACCTTTGGATACC AGGAATGGAGCGGTCCCCTGGTTTCTATCTCCGGCACCGATATTACCGTTCA GGGTGCTTCGGGCTCCGTGCTTGACGGTGACGGTGCCCGCTGGTGGGATG GACAGGGCAGCAATGGCGGCAAGACCAAGCCCAAGTTCTTCTACGCCCAT AGCTTGGACTCTTCGTCCATCACTGGCATTACTATCAAGAACTCCCCTGTTC AAGTCTTCAGCATCCAGTCCAACAATTTGAGCCTGACGGATATCACCGTCG ATGACGCCGATGGCGACACCCAAGGCGGCCACAATACCGACGCCTTTGATA TCGGTAGCTCCACTTATATCACGATCACGAACGCTAATGTTCACAATCAGGA TGACTGCATTGCAGTCAACTCAGGGGAGAACATCATCTTCACTGGCGGCAC CTGCACCGGCGGCCACGGTCTCTCCATCGGCTCTGTCGGCGGCCGCTCAGA CAACACCGTCAAGAACGTCACCATCTGTCACTCCACCGTGACCAACTCCC AGAATGGCGTGCGTATCAAGACCGTGTACGGCGCGACCGGCTCCGTCTCCG AAGTCACTTACTGTAACATCCAAATGTCTGGAATCACGAACTATGGCATCG TGATCGAGCAGGACTACGAGAACGGCAGCCCAACTGGTACCCCGACAAAC GGTGTCCCTATTACAGATCTCACTCTCAATACTGTGACTGGTAGCGTTTCGA GTGGTGCTACGGAGATTTACATTCTCTGCGGATCTGGAAGCTGCTCTAGTTG GACTTGGACGGGTGTTTCAATTACTGGTGGCTCGAAGAGCACTAAATGTGA GAATGTGCCTTCTGGAGTTTCTTGCTAG
The invention also provides a recombinant vector containing the polygalacturonase mutant gene;
preferably, the expression vector of the recombinant vector is specifically pPIC9 plasmid.
The invention also provides a recombinant strain containing the polygalacturonase mutant gene;
preferably, the recombinant strain employs a host of GS 115.
The invention also provides a method for preparing polygalacturonase with high specific activity, the recombinant strain is taken as a fermentation strain, and the fermentation method comprises the following steps:
seed culture medium: 1 to 1.5 percent of yeast powder; peptone 1.8-2%, glucose 1.7-2%, pH5.0-5.5;
seed culture conditions: the culture temperature is as follows: 29-30 ℃, rotation speed: 200rpm-250rpm, incubation time: 30h-34 h;
fermentation medium: 3 to 5 percent of glycerin, 2 to 3 percent of monoammonium phosphate, 0.1 to 0.2 percent of monopotassium phosphate, 2 to 3 percent of magnesium sulfate, 0.8 to 1.2 percent of potassium sulfate, 0.1 to 0.2 percent of calcium sulfate, 0.2 to 0.4 percent of potassium hydroxide and the balance of water;
fermentation culture conditions: inoculation amount: 0.5-1%, culture temperature: 29-30 ℃, rotation speed: 200rpm-350rpm, incubation time: 48-50 h.
Compared with the wild polygalacturonase, the polygalacturonase mutant has the advantages that the polygalacturonase is enhanced in heat stability, about 21.78% of activity is still kept after 30min treatment at 70 ℃, and the activity of the wild enzyme is disappeared. In addition, the specific activity of the wild type is 50483U/mg, and the specific activity of the mutant is 46270U/mg.
The invention also provides application of the polygalacturonase mutant with high thermal stability, and the polygalacturonase mutant can be applied to the fields of energy, food and feed.
Has the advantages that:
the invention overcomes the defects of the prior art and provides a polygalacturonase mutant with high thermal stability and suitable for application in the fields of energy, food, feed and the like. Therefore, the polygalacturonase mutant provided by the invention can well meet the application requirements in the fields of energy, food, feed and the like, and has a very wide application prospect.
Description of the drawings:
FIG. 1 is a graph of the optimum temperature profile;
fig. 2 is a temperature stability graph.
The specific implementation mode is as follows:
the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
Test materials and reagents
1. Bacterial strain and carrier: the expression host Pichia pastoris GS115 of the invention and the expression plasmid vector pPIC 9.
2. Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, and the ligase was purchased from Invitrogen. Others are made in China (all can be purchased from common biochemical agents).
3. Culture medium:
coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH natural);
pichia pastoris medium YPD (Yeast Extract 1%, Trytone 2%, Glucose 2% pH natural);
BMGY (Yeast Extract 1%, Trytone 2%, YNB 10%, biotin 0.1%, pH Natural);
BMMY (Yeast Extract 1%, Trytone 2%, methanol 0.5%, YNB 10%, biotin 0.1%, pH Natural).
Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
Example 1 preparation of recombinant Strain GS115(pPIC9-PG3427)
1. Amplification of the nucleic acid sequence of polygalacturonase wild-type PG3427 PG3427
The PCR method is adopted to amplify the pg3427 gene and the vector pPIC9 nucleic acid fragment, the two are connected through a recombination kit to obtain a recombinant plasmid pPIC9-pg3427, and Pichia pastoris GS115 is transformed to obtain a recombinant Pichia pastoris strain GS115(pPIC9-pg 3427).
The gene sequence of the wild type of pg3427 is as follows (SEQ ID NO. 4):
ATGCATACGATCCAACCTCTTCTAACCTATGGGCTGGCCGTGGGAGCTG TCCTTTCCTCAGCGGCCCCAACTGCTGTCGAGAAGCGTGCCAGCTGCACCT TTACCGATGCTGCTTCTGCCATGGCAAGCAAGACAGCCTGCTCGACTATCA CGCTGAACAACATTGCCGTTCCTGCTGGGACCACCTTGGACCTGACGGGCT TGACATCCGGCACCAGGGTCATCTTCGAAGGAACAACCACCTTTGGATACC AGGAATGGAGCGGTCCCCTGGTTTCTATCTCCGGCACCGATATTACCGTTCA GGGTGCTTCGGGCTCCGTGCTTGACGGTGACGGTGCCCGCTGGTGGGATG GACAGGGCAGCAATGGCGGCAAGACCAAGCCCAAGTTCTTCTACGCCCAT AGCTTGGACTCTTCGTCCATCACTGGCATTACTATCAAGAACTCCCCTGTTC AAGTCTTCAGCATCCAGTCCAACAATTTGAGCCTGACGGATATCACCGTCG ATGACGCCGATGGCGACACCCAAGGCGGCCACAATACCGACGCCTTTGATA TCGGTAGCTCCACTTATATCACGATCACGAACGCTAATGTTCACAATCAGGA TGACTGCATTGCAGTCAACTCAGGGGAGAACATCATCTTCACTGGCGGCAC CTGCACCGGCGGCCACGGTCTCTCCATCGGCTCTGTCGGCGGCCGCTCAGA CAACACCGTCAAGAACGTCACCATCGAGCACTCCACCGTGACCAACTCCC AGAATGGCGTGCGTATCAAGACCGTGTACGGCGCGACCGGCTCCGTCTCCG AAGTCACTTACTCAAACATCCAAATGTCTGGAATCACGAACTATGGCATCG TGATCGAGCAGGACTACGAGAACGGCAGCCCAACTGGTACCCCGACAAAC GGTGTCCCTATTACAGATCTCACTCTCAATACTGTGACTGGTAGCGTTTCGA GTGGTGCTACGGAGATTTACATTCTCTGCGGATCTGGAAGCTGCTCTAGTTG GACTTGGACGGGTGTTTCAATTACTGGTGGCTCGAAGAGCACTAAATGTGA GAATGTGCCTTCTGGAGTTTCTTGCTAG
the primers used for PCR were as follows:
TABLE 1 wild-type polygalacturonase-specific primers
Figure BDA0002826496320000051
Wherein pg3427-pPIC9-F and pg3427-pPIC9-R are used for amplifying the gene coding sequence of polygalacturonase wild type pg 3427; the vector pPIC9 was obtained by culturing the preserved strain in a culture flask and then extracting.
After the amplification is finished, carrying out nucleic acid electrophoresis detection on the PCR product and the extracted plasmid, wherein the sizes of the bands of pg3427 and the vector pPIC9 are 1098bp and 8088bp respectively, and using the vectorEcoR IAnd Not I, carrying out enzyme digestion, and respectively recovering and purifying the PCR product and the enzyme digestion product.
2. Construction of recombinant Strain GS115(pPIC9-pg3427)
The recovered pg3427 and pPIC9 gene fragments were subjected to recombinant ligation by using a kit recombinase, and the recombinant product was transformed into E.coli Trans I competent cells and plated on LB (containing 100. mu.g/mL Ampicillin) for screening.
After the sequencing is correct, the recombinant plasmid pPIC9-pg3427 is cut by Bgl II restriction enzyme, the product is recovered and transformed into pichia pastoris competent cell GS115 by electric shock for induction expression, and the recombinant expression strain GS115(pPIC9-pg3427) is obtained.
Example 2 preparation of recombinant Strain GS115(pPIC9-pg3427-E247C/S276C)
1. Construction of recombinant plasmid pPIC9-pg3427-E247C/S276C
The optimized mutation sites are designed to respectively mutate glutamic acid and serine at positions 247 and 276 into cysteine, mutation sites are introduced on the plasmid pPIC9-pg3427 by a method of a point mutation kit, and sequencing verification is carried out on the mutation sites to obtain the polygalacturonase mutant plasmid pPIC9-pg 3427-E247C/S276C. The primers used are shown in table 2:
TABLE 2 mutant polygalacturonase-specific primers
Figure BDA0002826496320000061
Wherein, firstly, primers E247C-F and E247C-R are used for constructing a mutant plasmid pPIC9-pg3427-E247C, after the sequence to be tested is correct, the primers S276C-F and S276C-R are used for constructing a mutant plasmid pPIC9-pg 3427-E247C/S276C.
2. Construction of recombinant Strain GS115(pPIC9-pg3427-E247C/S276C)
The recombinant plasmid pPIC9-pg3427-E247C/S276C is subjected to enzyme digestion by Bgl II, and a product is recovered and transformed into pichia pastoris competent cells GS115 by electric shock for induced expression to obtain a recombinant expression strain GS115(pPIC9-pg 3427-E247C/S276C).
Example 3, obtention of polygalacturonase wild type PG3427 and mutant E247C/S276C
1. Inducible expression of proteins PG3427 and E247C/S276C
The obtained recombinant expression strains GS115(pPIC9-pg3427) and GS115(pPIC9-pg3427-E247C/S276C) are inoculated into YPD culture medium for seed culture, after culture is carried out at 200rpm and 30 ℃ for 48h, the strains are transferred into BMGY culture medium at the inoculation amount of 1 percent, culture is carried out at 200rpm and 30 ℃ for 48h, and after sufficient strain enrichment, the strains are collected and added into BMMY culture medium containing 1 percent of methanol for induced expression. The specific activity of the wild type is 50483U/mg, and the specific activity of the mutant is 46270U/mg.
The method for measuring enzyme activity uses a DNS method, and comprises the following specific steps: under the given conditions of pH and temperature, 1mL of reaction system comprises 100 μ L of appropriate diluted enzyme solution and 900 μ L of substrate, the reaction is carried out for 10min, 1.5mL of DNS is added to stop the reaction, and the reaction is boiled in boiling water for 5 min. After cooling, the OD was measured at 540 nm.
1 enzyme activity unit (U) is defined as the amount of enzyme required to break down polygalacturonic acid to 1. mu. mol D- (+) -galacturonic acid per minute under given conditions.
2. Purification of proteins PG3427 and E247C/S276C
Centrifuging the bacteria solution after induction expression at 12000rpm for 10min, collecting supernatant, concentrating, dialyzing with 10mM disodium hydrogen phosphate solution (pH is adjusted to 6.5 by citric acid), performing ion exchange chromatography on the enzyme solution after dialysis, wherein the solution A is 10mM disodium hydrogen phosphate solution (pH is adjusted to 6.5 by citric acid), the solution B is solution A, adding 1M NaCl, purifying protein, collecting eluent, and performing SDS-PAGE analysis, wherein a single protein band appears at about 40kDa and is higher than the theoretical molecular weight of the enzyme protein, namely 35.2 kDa. While in the N glycosylation prediction analysis, E247C/S276C was found to have 2 potential glycosylation sites. After the Endo H deglycosylation treatment, the molecular weight of the enzyme is obviously reduced and is consistent with the theoretical molecular weight. This shows that the purification effect of the enzyme protein reaches electrophoretic purity, and the enzyme protein has higher glycosylation modification after efficient expression of pichia pastoris.
3. Variation of thermostability of the mutant and wild type
The purified mutants and wild type were subjected to thermostability determination as follows: the thermostability of the mutant and wild type was determined by treatment of the buffer liquid system at 0.1mol/L citrate-disodium hydrogen phosphate buffer (pH 3.5) at 60 ℃ and 70 ℃ for various periods of time (2, 5, 10, 20 and 30min, respectively), followed by residual enzyme activity determination at 70 ℃. As shown in FIG. 1, the optimum temperature was 70 ℃ for both the mutant and the wild type, and there was no significant difference. As shown in FIG. 2, after wild type is treated at 70 ℃ for 30min, the residual enzyme activity is only 6.77% of that before treatment; after the mutant is treated at 70 ℃ for 30min, the residual enzyme activity is equivalent to 21.78% before treatment, and the improvement range is obvious compared with that of the wild type.
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 patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
SEQUENCE LISTING
<110> Shandonglongket enzyme preparations Co., Ltd
<120> polygalacturonase mutant with improved thermal stability and application thereof
<130> 1
<160> 4
<170> PatentIn version 3.5
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<213> Talaromyces leycettanus JCM12802
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atgcatacga tccaacctct tctaacctat gggctggccg tgggagctgt cctttcctca 60
gcggccccaa ctgctgtcga gaagcgtgcc agctgcacct ttaccgatgc tgcttctgcc 120
atggcaagca agacagcctg ctcgactatc acgctgaaca acattgccgt tcctgctggg 180
accaccttgg acctgacggg cttgacatcc ggcaccaggg tcatcttcga aggaacaacc 240
acctttggat accaggaatg gagcggtccc ctggtttcta tctccggcac cgatattacc 300
gttcagggtg cttcgggctc cgtgcttgac ggtgacggtg cccgctggtg ggatggacag 360
ggcagcaatg gcggcaagac caagcccaag ttcttctacg cccatagctt ggactcttcg 420
tccatcactg gcattactat caagaactcc cctgttcaag tcttcagcat ccagtccaac 480
aatttgagcc tgacggatat caccgtcgat gacgccgatg gcgacaccca aggcggccac 540
aataccgacg cctttgatat cggtagctcc acttatatca cgatcacgaa cgctaatgtt 600
cacaatcagg atgactgcat tgcagtcaac tcaggggaga acatcatctt cactggcggc 660
acctgcaccg gcggccacgg tctctccatc ggctctgtcg gcggccgctc agacaacacc 720
gtcaagaacg tcaccatctg tcactccacc gtgaccaact cccagaatgg cgtgcgtatc 780
aagaccgtgt acggcgcgac cggctccgtc tccgaagtca cttactgtaa catccaaatg 840
tctggaatca cgaactatgg catcgtgatc gagcaggact acgagaacgg cagcccaact 900
ggtaccccga caaacggtgt ccctattaca gatctcactc tcaatactgt gactggtagc 960
gtttcgagtg gtgctacgga gatttacatt ctctgcggat ctggaagctg ctctagttgg 1020
acttggacgg gtgtttcaat tactggtggc tcgaagagca ctaaatgtga gaatgtgcct 1080
tctggagttt cttgctag 1098
<210> 4
<211> 1098
<212> DNA
<213> Talaromyces leycettanus JCM12802
<400> 4
atgcatacga tccaacctct tctaacctat gggctggccg tgggagctgt cctttcctca 60
gcggccccaa ctgctgtcga gaagcgtgcc agctgcacct ttaccgatgc tgcttctgcc 120
atggcaagca agacagcctg ctcgactatc acgctgaaca acattgccgt tcctgctggg 180
accaccttgg acctgacggg cttgacatcc ggcaccaggg tcatcttcga aggaacaacc 240
acctttggat accaggaatg gagcggtccc ctggtttcta tctccggcac cgatattacc 300
gttcagggtg cttcgggctc cgtgcttgac ggtgacggtg cccgctggtg ggatggacag 360
ggcagcaatg gcggcaagac caagcccaag ttcttctacg cccatagctt ggactcttcg 420
tccatcactg gcattactat caagaactcc cctgttcaag tcttcagcat ccagtccaac 480
aatttgagcc tgacggatat caccgtcgat gacgccgatg gcgacaccca aggcggccac 540
aataccgacg cctttgatat cggtagctcc acttatatca cgatcacgaa cgctaatgtt 600
cacaatcagg atgactgcat tgcagtcaac tcaggggaga acatcatctt cactggcggc 660
acctgcaccg gcggccacgg tctctccatc ggctctgtcg gcggccgctc agacaacacc 720
gtcaagaacg tcaccatcga gcactccacc gtgaccaact cccagaatgg cgtgcgtatc 780
aagaccgtgt acggcgcgac cggctccgtc tccgaagtca cttactcaaa catccaaatg 840
tctggaatca cgaactatgg catcgtgatc gagcaggact acgagaacgg cagcccaact 900
ggtaccccga caaacggtgt ccctattaca gatctcactc tcaatactgt gactggtagc 960
gtttcgagtg gtgctacgga gatttacatt ctctgcggat ctggaagctg ctctagttgg 1020
acttggacgg gtgtttcaat tactggtggc tcgaagagca ctaaatgtga gaatgtgcct 1080
tctggagttt cttgctag 1098

Claims (9)

1. A polygalacturonase mutant is characterized in that the mutant is E247C/S276C, and the amino acid sequence is shown in a sequence table SEQ ID NO. 2.
2. A gene encoding the polygalacturonase mutant according to claim 1.
3. The encoding gene of claim 2, which is represented by SEQ ID No.3 of the sequence Listing.
4. A recombinant vector or a recombinant strain comprising a gene encoding the polygalacturonase mutant of claim 1.
5. The recombinant vector according to claim 4, wherein the expression vector used is the pPIC9 plasmid.
6. The recombinant strain of claim 4, wherein the host used is Pichia pastoris GS 115.
7. Use of the recombinant vector or recombinant strain of claim 4 for the production of polygalacturonase.
8. The use of claim 7, wherein the recombinant strain is used for fermentative production of polygalacturonase by the following method:
the strains are inoculated according to the following weight percentage: 0.5-1% of the culture medium is inoculated into a fermentation culture medium, and the culture is carried out for 48-50 h under the conditions that the culture temperature is 29-30 ℃ and the rotating speed is 200-350 rpm;
the fermentation medium comprises the following components: 3 to 5 percent of glycerin, 2 to 3 percent of monoammonium phosphate, 0.1 to 0.2 percent of monopotassium phosphate, 2 to 3 percent of magnesium sulfate, 0.8 to 1.2 percent of potassium sulfate, 0.1 to 0.2 percent of calcium sulfate, 0.2 to 0.4 percent of potassium hydroxide and the balance of water.
9. Use of the polygalacturonase mutant according to claim 1 for hydrolyzing polygalacturonic acid.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108315314A (en) * 2018-03-21 2018-07-24 中国农业科学院饲料研究所 A kind of polygalacturonase mutant TePG28b_ △ S88 and its gene and application
CN111926001A (en) * 2020-10-15 2020-11-13 中国农业科学院北京畜牧兽医研究所 Polygalacturonase mutant T316C/G344C with high thermal stability and gene and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2396403T3 (en) * 2009-02-10 2016-01-11 Coldzymes Aps COLD-ACTIVE beta-galactosidase, MANUFACTURING METHOD THEREOF AND USE OF SUCH ENZYME

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108315314A (en) * 2018-03-21 2018-07-24 中国农业科学院饲料研究所 A kind of polygalacturonase mutant TePG28b_ △ S88 and its gene and application
CN111926001A (en) * 2020-10-15 2020-11-13 中国农业科学院北京畜牧兽医研究所 Polygalacturonase mutant T316C/G344C with high thermal stability and gene and application thereof

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