CN112481240A - GH16 family heat-resistant glucanase mutant and construction method and application thereof - Google Patents
GH16 family heat-resistant glucanase mutant and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a GH16 family heat-resistant glucanase mutant and a construction method and application thereof, relating to the field of genetic engineering and genetic engineering. The invention takes GH16 family high specific activity dextranase BisGlu16B _ delta C derived from Bispora sp MEY-1 as a female parent, and the heat stability of the dextranase mutant obtained by adopting a molecular biology technology is obviously improved compared with that of the wild type of the female parent. Wherein, the amino acid sequence of the mutant is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3. The method greatly improves the temperature tolerance and the catalytic efficiency of the glucanase, and creates conditions for the application of the glucanase in the industrial production field.
Description
Technical Field
The invention relates to the field of genetic engineering and genetic engineering, in particular to a GH16 family heat-resistant glucanase mutant and a construction method and application thereof.
Background
Cellulose, hemicellulose, lignin and the like are the main components of plant cell walls. Cellulose accounts for about 40-45% of the dry weight of the cell, and is a linear structure molecule formed by connecting glucose through beta-1, 4-and beta-1, 3-glycosidic bonds. Lignin is a complex phenolic polymer, and accounts for about 15-25% of the dry cell weight. Hemicellulose constitutes about 30-35% of the dry cell weight, the most abundant renewable biological resource behind cellulose, and consists of heteropolysaccharides, named as glucans, mannans, and glucomannans, among others, by the majority of the backbone constituents (Schulze E1891. Ber Dtsch Chem Ges 24, 2277-. Wherein the beta-glucan is a structural non-starch polysaccharide in the cell wall of monocotyledonous gramineous plants, and is mainly present in aleurone layer and endosperm cells, such as barley and oat endosperm cell walls containing about 70-75% of glucan (Philippe S et al 2006.plant 224(2), 449) 461.).
Beta-glucanases are a generic term for a class of enzymes that break down the glucose polymers formed by beta-glycosidic linkages. The different modes of action can be divided into an inner cutting type and an outer cutting type. Wherein the endo beta-1, 3-1, 4-glucanase (E.C.3.2.1.73) can specifically act on beta-1, 4 glycosidic bonds connected with the beta-1, 3 bonds, so that the endo beta-1, 3-glucanase is degraded into low molecular weight fragments, loses hydrophilicity and viscosity, reduces the viscosity of contents in intestinal tracts of monogastric animals, improves the activity of endogenous digestive enzymes, improves the environment of intestinal microorganisms, and improves the growth performance and the feed conversion rate (Mathlouthi N et al 2002.Amin Res 51, 395-406.). The method is widely applied to food brewing, particularly in the beer brewing process, glucan in malt causes difficulty in beer filtration and blockage of a filtration membrane, the production cost and the quality of beer are increased, and the problems can be solved by adopting the synergistic effect of acidic glucanase and glucanase. Therefore, the structural basis of the production, purification, thermostability, catalytic efficiency and acidity profile of acidic dextranase and its application in the fields of feed processing, brewing industry, juice processing, and energy are going to be deepened.
Because of the different demands of different industries on the properties of glucanases, the improvement of glucanase research with potential applications is still of great significance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a GH16 family heat-resistant glucanase mutant and a construction method and application thereof, the mutant is an acidic glucanase mutant obtained by carrying out point mutation on wild amino acid of glucanase, the mutant can tolerate high temperature treatment of more than 65 ℃, has very high hemicellulase activity under acidic and neutral pH values, and particularly has very good application potential in the industrial fields of feed, beer brewing, food and the like.
A GH16 family heat-resistant glucanase mutant is prepared by carrying out point mutation on a glucanase wild type sequence SEQ ID NO.4 to obtain a glucanase mutant E36R, D127K or E36R/D127K; when the glucanase mutant is E36R, the amino acid sequence is shown as SEQ ID NO. 1; when the glucanase mutant is D127K, the amino acid sequence is shown as SEQ ID NO. 2; when the glucanase mutant is E36R/D127K, the amino acid sequence is shown as SEQ ID NO. 3.
SEQ ID NO.1:
QYTLQQDYMADGNFFSQFSFWDTADPTDGFVAYKNRTYCTDNDLISSSSTNVQIRVDSSNVTPNGRPSVRITSNQSYNPGTLVILDLEHMPGGICGTWPAFWMVGPNWPDDGEIDIIEGVNQQTTNDMTLHTSEGCTISSSGDFSGSIVSTDCWVDDPNQSDNEGCQITTSNTETYGSGFNANNGGVYATDFQDAAISIYFFPRGSIPSDITDGSPDPSGWGTPIAQFTDSSCDIQSYFTDLQIVFDTTFCGQWAGNVWSSGSCASVASTCDDYVENNPAAFVDAYWSINSLQVYSGTSNGPMQNDTSSSSWGPSASANVAV
SEQ ID NO.2:
QYTLQQDYMADGNFFSQFSFWDTADPTDGFVAYKNETYCTDNDLISSSSTNVQIRVDSSNVTPNGRPSVRITSNQSYNPGTLVILDLEHMPGGICGTWPAFWMVGPNWPDDGEIDIIEGVNQQTTNKMTLHTSEGCTISSSGDFSGSIVSTDCWVDDPNQSDNEGCQITTSNTETYGSGFNANNGGVYATDFQDAAISIYFFPRGSIPSDITDGSPDPSGWGTPIAQFTDSSCDIQSYFTDLQIVFDTTFCGQWAGNVWSSGSCASVASTCDDYVENNPAAFVDAYWSINSLQVYSGTSNGPMQNDTSSSSWGPSASANVAV
SEQ ID NO.3:
QYTLQQDYMADGNFFSQFSFWDTADPTDGFVAYKNRTYCTDNDLISSSSTNVQIRVDSSNVTPNGRPSVRITSNQSYNPGTLVILDLEHMPGGICGTWPAFWMVGPNWPDDGEIDIIEGVNQQTTNKMTLHTSEGCTISSSGDFSGSIVSTDCWVDDPNQSDNEGCQITTSNTETYGSGFNANNGGVYATDFQDAAISIYFFPRGSIPSDITDGSPDPSGWGTPIAQFTDSSCDIQSYFTDLQIVFDTTFCGQWAGNVWSSGSCASVASTCDDYVENNPAAFVDAYWSINSLQVYSGTSNGPMQNDTSSSSWGPSASANVAV
The nucleotide sequence of the encoding dextranase mutant E36R is shown in SEQ ID NO. 5; the nucleotide sequence of the encoding dextranase mutant D127K is shown in SEQ ID NO. 6; the nucleotide sequence of the encoding dextranase mutant E36R/D127K is shown in SEQ ID NO. 7.
SEQ ID NO.5：
CAATATACCCTTCAGCAGGATTACATGGCAGACGGCAACTTTTTTAGCCAATTTTCATTTTGGGATACCGCCGACCCTACAGATGGCTTTGTGGCTTATAAAAATCGCACTTATTGCACCGACAACGATCTCATCAGCAGTTCCAGCACGAACGTGCAGATTCGGGTGGACAGCTCCAATGTTACACCGAATGGACGGCCTAGTGTTCGCATTACCAGCAACCAGTCGTACAATCCAGGCACACTTGTAATCCTGGACCTTGAACACATGCCAGGTGGCATCTGCGGTACCTGGCCAGCATTTTGGATGGTTGGGCCGAATTGGCCCGCCGATGGGGAAATCGACATCATTGAGGGTGTCAACCAGCAAACTACCAATGACATGACCCTCCACACTAGTGAAGGCTGCACAATATCCAGCAGTGGCGATTTCTCGGGCTCGATAGTTAGCACCGACTGCTGGGTCGATGACCCCAACCAATCCGACAATGAAGGCTGTCAGATCACTACGAGCAATACCGAAACTTACGGTTCCGGTTTTAATGCTAACAATGGCGGCGTCTATGCGACGGACTTCCAAGACGCCGCTATCAGCATCTATTTCTTCCCCCGTGGTTCCATACCTTCGGACATTACAGACGGCTCTCCAGACCCGTCCGGCTGGGGTACGCCAATTGCGCAGTTCACGGATAGCAGCTGTGACATTCAAAGCTATTTCACCGATTTACAGATCGTTTTCGATACGACGTTCTGTGGACAATGGGCTGGCAACGTCTGGTCAAGTGGCTCTTGTGCCTCTGTGGCAAGTACCTGCGACGACTACGTGGAAAACAACCCGGCTGCCTTCGTCGATGCATACTGGTCGATCAACAGTCTTCAGGTTTATTCGGGAACCTCCAATGGTCCCATGCAGAATGATACTTCGAGCAGCAGCTGGGGTCCATCTGCTTCTGCAAATGTGGCAGTGCCGTCATCGGTACGTGCCATTGTCGGTGGCTCTGGATCAGCAGCCAGCTCCACTACATTTGCGATCTCCACTAAATCTGCTCCATTCCCCGTCGGGAACTCAACTTCCGTCGTTGGAACTACTGGCGCCAGTTCGAATGGCGCATGGGCTGCTATAGTCACGGGAACGGGACCTATTGGAGTTGCTCAAGAAACTAGCGTTTCCGCTGCTTCAGCAGCTTGA
SEQ ID NO.6：
CAATATACCCTTCAGCAGGATTACATGGCAGACGGCAACTTTTTTAGCCAATTTTCATTTTGGGATACCGCCGACCCTACAGATGGCTTTGTGGCTTATAAAAATGACACTTATTGCACCGACAACGATCTCATCAGCAGTTCCAGCACGAACGTGCAGATTCGGGTGGACAGCTCCAATGTTACACCGAATGGACGGCCTAGTGTTCGCATTACCAGCAACCAGTCGTACAATCCAGGCACACTTGTAATCCTGGACCTTGAACACATGCCAGGTGGCATCTGCGGTACCTGGCCAGCATTTTGGATGGTTGGGCCGAATTGGCCCGCCGATGGGGAAATCGACATCATTGAGGGTGTCAACCAGCAAACTACCAATAAGATGACCCTCCACACTAGTGAAGGCTGCACAATATCCAGCAGTGGCGATTTCTCGGGCTCGATAGTTAGCACCGACTGCTGGGTCGATGACCCCAACCAATCCGACAATGAAGGCTGTCAGATCACTACGAGCAATACCGAAACTTACGGTTCCGGTTTTAATGCTAACAATGGCGGCGTCTATGCGACGGACTTCCAAGACGCCGCTATCAGCATCTATTTCTTCCCCCGTGGTTCCATACCTTCGGACATTACAGACGGCTCTCCAGACCCGTCCGGCTGGGGTACGCCAATTGCGCAGTTCACGGATAGCAGCTGTGACATTCAAAGCTATTTCACCGATTTACAGATCGTTTTCGATACGACGTTCTGTGGACAATGGGCTGGCAACGTCTGGTCAAGTGGCTCTTGTGCCTCTGTGGCAAGTACCTGCGACGACTACGTGGAAAACAACCCGGCTGCCTTCGTCGATGCATACTGGTCGATCAACAGTCTTCAGGTTTATTCGGGAACCTCCAATGGTCCCATGCAGAATGATACTTCGAGCAGCAGCTGGGGTCCATCTGCTTCTGCAAATGTGGCAGTGCCGTCATCGGTACGTGCCATTGTCGGTGGCTCTGGATCAGCAGCCAGCTCCACTACATTTGCGATCTCCACTAAATCTGCTCCATTCCCCGTCGGGAACTCAACTTCCGTCGTTGGAACTACTGGCGCCAGTTCGAATGGCGCATGGGCTGCTATAGTCACGGGAACGGGACCTATTGGAGTTGCTCAAGAAACTAGCGTTTCCGCTGCTTCAGCAGCTTGA
SEQ ID NO.7：
CAATATACCCTTCAGCAGGATTACATGGCAGACGGCAACTTTTTTAGCCAATTTTCATTTTGGGATACCGCCGACCCTACAGATGGCTTTGTGGCTTATAAAAATCGCACTTATTGCACCGACAACGATCTCATCAGCAGTTCCAGCACGAACGTGCAGATTCGGGTGGACAGCTCCAATGTTACACCGAATGGACGGCCTAGTGTTCGCATTACCAGCAACCAGTCGTACAATCCAGGCACACTTGTAATCCTGGACCTTGAACACATGCCAGGTGGCATCTGCGGTACCTGGCCAGCATTTTGGATGGTTGGGCCGAATTGGCCCGCCGATGGGGAAATCGACATCATTGAGGGTGTCAACCAGCAAACTACCAATAAGATGACCCTCCACACTAGTGAAGGCTGCACAATATCCAGCAGTGGCGATTTCTCGGGCTCGATAGTTAGCACCGACTGCTGGGTCGATGACCCCAACCAATCCGACAATGAAGGCTGTCAGATCACTACGAGCAATACCGAAACTTACGGTTCCGGTTTTAATGCTAACAATGGCGGCGTCTATGCGACGGACTTCCAAGACGCCGCTATCAGCATCTATTTCTTCCCCCGTGGTTCCATACCTTCGGACATTACAGACGGCTCTCCAGACCCGTCCGGCTGGGGTACGCCAATTGCGCAGTTCACGGATAGCAGCTGTGACATTCAAAGCTATTTCACCGATTTACAGATCGTTTTCGATACGACGTTCTGTGGACAATGGGCTGGCAACGTCTGGTCAAGTGGCTCTTGTGCCTCTGTGGCAAGTACCTGCGACGACTACGTGGAAAACAACCCGGCTGCCTTCGTCGATGCATACTGGTCGATCAACAGTCTTCAGGTTTATTCGGGAACCTCCAATGGTCCCATGCAGAATGATACTTCGAGCAGCAGCTGGGGTCCATCTGCTTCTGCAAATGTGGCAGTGCCGTCATCGGTACGTGCCATTGTCGGTGGCTCTGGATCAGCAGCCAGCTCCACTACATTTGCGATCTCCACTAAATCTGCTCCATTCCCCGTCGGGAACTCAACTTCCGTCGTTGGAACTACTGGCGCCAGTTCGAATGGCGCATGGGCTGCTATAGTCACGGGAACGGGACCTATTGGAGTTGCTCAAGAAACTAGCGTTTCCGCTGCTTCAGCAGCTTGA
A recombinant vector comprising the above-mentioned nucleotide sequence encoding a mutant thermotolerant dextranase of the GH16 family;
when the sequence is SEQ ID NO.5, the recombinant vector is pPIC9 r-E36R;
when the sequence is SEQ ID NO.6, the recombinant vector is pPIC9 r-D127K;
when the sequence is SEQ ID NO.7, the recombinant vector is pPIC9 r-E36R/D127K.
A recombinant strain expressing the recombinant vector;
when the recombinant vector is pPIC9r-E36R, the recombinant strain is GS 115/E36R;
when the recombinant vector is pPIC9r-D127K, the recombinant strain is GS 115/D127K;
when the recombinant vector is pPIC9r-E36R/D127K, the recombinant strain is GS 115/E36R/D127K.
The method for constructing the GH16 family thermotolerant dextranase mutant comprises the following steps:
1) amplifying sequence segments of the high-temperature dextranase mutants by adopting an over-lap PCR method;
2) cloning the sequence fragment of the dextranase mutant between EcoR I and Not I restriction sites of an expression vector pPIC9r to obtain a recombinant vector;
3) transforming the mutant recombinant vector into pichia pastoris GS115, and carrying out induced expression to obtain a mutant strain;
4) culturing the recombinant strain, and inducing the expression of the recombinant glucanase;
5) recovering and purifying the expressed heat-resistant dextranase mutant.
The glucanase mutant provided by the invention has excellent thermal stability, and the optimal temperatures of the mutants E36R, D127K and E36R/D127K are respectively increased by 5 ℃, 10 ℃ and 5 ℃ compared with the wild type; half life (t) at 65 ℃1/2) Respectively extending 5.7 times, 1.5 times and 1.1 times compared with wild type; t ismThe values are respectively improved by 9.6 ℃, 7.1 ℃ and 3.3 ℃ compared with the wild type. The catalytic efficiency is respectively improved by 55%, 31% and 1.2% compared with the wild type; the optimum pH for the enzymatic reaction was shifted by 0.5 units towards an alkaline environment.
The GH16 family thermostable glucanase mutant is applied to degradation of hemicellulose.
Has the advantages that:
compared with the prior art, the GH16 family heat-resistant glucanase mutant and the preparation method and application thereof have the following advantages:
the invention provides a glucanase mutant with excellent property and suitable for being applied to hemicellulose degradation, and the optimum temperature of the glucanase mutant is increased by 5-10 ℃ compared with the wild type; half life (t) at 65 ℃1/2) The elongation is 1.1 to 5.7 times that of the wild type; t ismThe value is improved by 3.3-9.6 ℃ compared with the wild type. The catalytic efficiency is not lost but improved by 1.2-55% while the thermal stability is improved.
Compared with the means such as blind-mesh bacteria or artificial (natural) mutagenesis and the like, the enzyme molecule improvement shortens the modification time of the enzymology property, so that the glucanase mutant which is stable in the acid pH environment and in the medium and low temperature range and has high enzyme activity is shown by a bran degradation experiment, has obvious hydrolysis effect on bran, meets various industrial requirements, and has wide application prospect in producing reducing sugar by degrading hemicellulose.
Drawings
FIG. 1 shows the optimum pH requirement of the mutant of the thermostable dextranase in comparison with the wild type;
FIG. 2 shows the pH stability of the thermostable dextranase mutants compared to wild-type;
FIG. 3 shows the optimal temperature requirements of the mutant and wild type of the thermostable dextranase;
FIG. 4 shows the thermostability of the thermotolerant dextranase mutant at 65 ℃ with wild type;
FIG. 5 shows the thermostability of the thermotolerant dextranase mutant at 70 ℃ with wild type.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
1. Bacterial strain and carrier: the expression host Pichia pastoris GS115, expression plasmid vector pPIC9r from Invitrogen;
2. enzymes and other biochemical reagents: endonuclease was purchased from Fermentas, ligase from Promaga, and barley glucan from Sigma; other reagents are domestic analytical pure reagents (all purchased from the national pharmaceutical group);
3. culture medium:
(1) LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;
(2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;
(3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;
(4) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;
(5) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin;
(6) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004%
Biotin, 0.5% methanol (V/V)
EXAMPLE 1 cloning of Gene encoding Heat-resistant dextranase mutant
GH16 family BisGlu16_ delta C derived from Bispora sp.MEY-1 is taken as a female parent, and the encoding genes of the heat-resistant glucanase mutant, namely SEQ ID NO.5(E36R, 1188bp), SEQ ID NO.6(D127K, 1188bp) and SEQ ID NO.7(E36R/D127K, 1188bp), a mutation method and a cloning method reference are respectively amplified by adopting an over-lap PCR method (You, et al., 2018).
The primer sequences used are shown in table 1:
TABLE 1 primer Synthesis List
Example 2 preparation of thermostable dextranase mutants
Carrying out double enzyme digestion (EcoR I + Not I) on the expression vector pPIC9r, simultaneously carrying out double enzyme digestion (EcoR I + Not I) on the gene coding the heat-resistant glucanase mutant, connecting the cut gene segment (removing signal peptide segment) coding the mature heat-resistant glucanase mutant with the expression vector pPIC9r to obtain a recombinant plasmid containing the heat-resistant glucanase mutant gene, and transforming Pichia pastoris GS115 to obtain recombinant yeast strains GS115/E36R, GS115/D127K and GS 115/E36R/D127K.
Taking three GS115 strains containing recombinant plasmids, respectively inoculating the three GS115 strains into a 1L triangular flask of 300mL BMGY medium, and placing the three GS115 strains at 30 ℃ for shake cultivation at 220rpm for 48 hours; after this time, the culture broth was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 100mL BMMY medium containing 0.5% methanol and again placed at 30 ℃ for induction culture at 220 rpm. 0.5mL of methanol is added every 12h, so that the concentration of the methanol in the bacterial liquid is kept at 0.5%, and meanwhile, the supernatant is taken for enzyme activity detection.
The optimal pH value of the recombinant heat-resistant glucanase mutant is 4.0, the heat stability is greatly improved, and the catalytic efficiency is improved by 1.2-55 percent compared with the wild type. The recombinant dextranase is expressed in Pichia pastoris. After the expressed glucanase is purified, the protein content of the glucanase reaches more than 98 percent of the total protein.
Example 3 Activity analysis of recombinant thermostable dextranase mutants and wild type
Firstly, a DNS method: the specific method comprises the following steps: under the conditions of pH 3.5 and 55 ℃, 1mL of reaction system comprises 100 μ L of 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 mixture 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 glucan to produce 1. mu. mol reducing sugar per minute under the given conditions.
II, determining the properties of the mutant and the wild type of the recombinant heat-resistant glucanase
1. The method for measuring the optimal pH of the recombinant heat-resistant glucanase mutant and the wild type comprises the following steps:
the recombinant thermostable glucanase mutant purified in example 2 and the wild type were subjected to enzymatic reactions at different pH to determine the optimum pH. Substrate dextrans dextranase activity assays were performed at 55 ℃ in 0.1mol/L citrate-disodium phosphate buffer at various pH's. The results are shown in FIG. 1, and the optimal reaction pH of the recombinant heat-resistant dextranase mutant is shifted to alkaline environment by 0.5 unit and is pH 4.0 compared with the wild type.
2. The method for measuring the optimal temperature of the recombinant heat-resistant dextranase mutant and the wild type comprises the following steps:
the optimal temperature of the recombinant heat-resistant dextranase mutant and the wild type is determined by performing the enzymatic reaction in a 0.1mol/L citrate-disodium hydrogen phosphate buffer (pH 4.5) buffer system at different temperatures. The results of the enzyme reaction optimum temperature determination are shown in FIG. 2, the optimum temperature of the recombinant heat-resistant dextranase mutant is improved by 5-10 ℃ compared with that of the wild type (55 ℃), and the relative enzyme activity of the mutant at high temperature is obviously improved compared with that of the wild enzyme.
3. The thermostability of the recombinant thermostable dextranase mutants and the wild type at 65 ℃ and 70 ℃ was determined as follows:
in the detection method reference (You, et al.,2019), the thermal stability of all mutants is better than that of the wild type, and after treatment at 65 ℃ for 30min, the residual enzyme activities of the three mutants are respectively 4.2 times, 3.1 times and 2.6 times of that of the wild type (as shown in figure 4); after 10min treatment at 70 ℃, the residual enzyme activities of the three mutants are 28.5 times, 25 times and 21 times of the wild type respectively (as shown in figure 5), and the detailed data are shown in table 1.
TABLE 1 comparison of thermostability parameters of Heat-resistant dextranase mutants with wild type
4. The kinetic parameters of the mutant and the wild type of the recombinant heat-resistant glucanase are determined as follows:
the detection method the first order reaction time of the reaction was determined with reference to literature (You, et al., 2019). Determination of assay KmAnd VmaxThe reaction time of (3) was 5 min. Measuring enzyme activity under optimum conditions (temperature, pH) with dextran (1.25, 1.0, 0.8, 0.4, 0.2, 0.15 and 0.1%) of different concentrations as substrate, calculating corresponding reaction rate, and calculating K with GraFit7 softwaremValue and Vmax。
When glucan is taken as a substrate, K of wild type and mutant of recombinant heat-resistant glucanase mutant under the optimal conditionmValues of 5.4mg/mL, 4.5mg/mL, 4.6mg/mL and 3.8mg/mL, respectively, and catalytic efficiencies (kcat/Km) of 21000 mL/s.mg, 32600 mL/s.mg, 27600 mL/s.mg and 23500 mL/s.mg, respectively (Table 2).
TABLE 2 comparison of specific activity and kinetic parameters of the thermotolerant dextranase mutants with wild type
The mutant is an acidic glucanase mutant obtained by carrying out point mutation on wild amino acid of glucanase, can tolerate high temperature treatment of more than 65 ℃, has very high hemicellulase activity under acidic and neutral pH values, and particularly has good application potential in the industrial fields of feeds, beer brewing, foods and the like.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Sequence listing
<110> university of Jiangsu science and technology
<120> GH16 family heat-resistant glucanase mutant and construction method and application thereof
<141> 2020-12-09
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 322
<212> PRT
<213> Amino acid sequence (Amino acid sequence)
<400> 1
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Arg Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Asp Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Asp Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Asp Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val
<210> 2
<211> 322
<212> PRT
<213> Amino acid sequence (Amino acid sequence)
<400> 2
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Glu Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Lys Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Asp Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Asp Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val
<210> 3
<211> 322
<212> PRT
<213> Amino acid sequence (Amino acid sequence)
<400> 3
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Arg Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Lys Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Asp Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Asp Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val
<210> 4
<211> 1188
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatcgcac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgcc gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagcttga 1188
<210> 5
<211> 1188
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgacac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgcc gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaataa gatgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagcttga 1188
<210> 6
<211> 1188
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatcgcac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgcc gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaataa gatgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgccgt catcggtacg tgccattgtc ggtggctctg gatcagcagc cagctccact 1020
acatttgcga tctccactaa atctgctcca ttccccgtcg ggaactcaac ttccgtcgtt 1080
ggaactactg gcgccagttc gaatggcgca tgggctgcta tagtcacggg aacgggacct 1140
attggagttg ctcaagaaac tagcgtttcc gctgcttcag cagcttga 1188
<210> 7
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gtagaattcc aatataccct tcagcagg 28
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
attcgcggcc gctcaagctg ctgaagcagc 30
<210> 9
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cagatggctt tgtggcttat aaaaatcgca c 31
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cggtgcaata agtgcgattt ttataag 27
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caaccagcaa actaccaata agatgacc 28
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctagtgtgga gggtcatctt attggtag 28
<210> 13
<211> 644
<212> PRT
<213> Amino acid sequence (Amino acid sequence)
<400> 13
Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe Phe Ser
1 5 10 15
Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe Val Ala
20 25 30
Tyr Lys Asn Arg Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser Ser Ser
35 40 45
Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr Pro Asn
50 55 60
Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn Pro Gly
65 70 75 80
Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp Asp Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn Asp Met
115 120 125
Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly Asp Phe
130 135 140
Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro Asn Gln
145 150 155 160
Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu Thr Tyr
165 170 175
Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr Asp Phe
180 185 190
Gln Asp Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser Ile Pro
195 200 205
Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly Thr Pro
210 215 220
Ile Ala Gln Phe Thr Asp Ser Ser Cys Asp Ile Gln Ser Tyr Phe Thr
225 230 235 240
Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp Ala Gly
245 250 255
Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr Cys Asp
260 265 270
Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr Trp Ser
275 280 285
Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro Met Gln
290 295 300
Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala Asn Val
305 310 315 320
Ala Val Gln Tyr Thr Leu Gln Gln Asp Tyr Met Ala Asp Gly Asn Phe
325 330 335
Phe Ser Gln Phe Ser Phe Trp Asp Thr Ala Asp Pro Thr Asp Gly Phe
340 345 350
Val Ala Tyr Lys Asn Glu Thr Tyr Cys Thr Asp Asn Asp Leu Ile Ser
355 360 365
Ser Ser Ser Thr Asn Val Gln Ile Arg Val Asp Ser Ser Asn Val Thr
370 375 380
Pro Asn Gly Arg Pro Ser Val Arg Ile Thr Ser Asn Gln Ser Tyr Asn
385 390 395 400
Pro Gly Thr Leu Val Ile Leu Asp Leu Glu His Met Pro Gly Gly Ile
405 410 415
Cys Gly Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asp
420 425 430
Asp Gly Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Thr Thr Asn
435 440 445
Asp Met Thr Leu His Thr Ser Glu Gly Cys Thr Ile Ser Ser Ser Gly
450 455 460
Asp Phe Ser Gly Ser Ile Val Ser Thr Asp Cys Trp Val Asp Asp Pro
465 470 475 480
Asn Gln Ser Asp Asn Glu Gly Cys Gln Ile Thr Thr Ser Asn Thr Glu
485 490 495
Thr Tyr Gly Ser Gly Phe Asn Ala Asn Asn Gly Gly Val Tyr Ala Thr
500 505 510
Asp Phe Gln Asp Ala Ala Ile Ser Ile Tyr Phe Phe Pro Arg Gly Ser
515 520 525
Ile Pro Ser Asp Ile Thr Asp Gly Ser Pro Asp Pro Ser Gly Trp Gly
530 535 540
Thr Pro Ile Ala Gln Phe Thr Asp Ser Ser Cys Asp Ile Gln Ser Tyr
545 550 555 560
Phe Thr Asp Leu Gln Ile Val Phe Asp Thr Thr Phe Cys Gly Gln Trp
565 570 575
Ala Gly Asn Val Trp Ser Ser Gly Ser Cys Ala Ser Val Ala Ser Thr
580 585 590
Cys Asp Asp Tyr Val Glu Asn Asn Pro Ala Ala Phe Val Asp Ala Tyr
595 600 605
Trp Ser Ile Asn Ser Leu Gln Val Tyr Ser Gly Thr Ser Asn Gly Pro
610 615 620
Met Gln Asn Asp Thr Ser Ser Ser Ser Trp Gly Pro Ser Ala Ser Ala
625 630 635 640
Asn Val Ala Val
Claims (6)
1. A GH16 family heat-resistant glucanase mutant is characterized in that a glucanase mutant E36R, D127K or E36R/D127K is obtained by point mutation based on a glucanase wild type sequence SEQ ID NO. 4; when the glucanase mutant is E36R, the amino acid sequence is shown as SEQ ID NO. 1; when the glucanase mutant is D127K, the amino acid sequence is shown as SEQ ID NO. 2; when the glucanase mutant is E36R/D127K, the amino acid sequence is shown as SEQ ID NO. 3.
2.A nucleic acid molecule encoding the glucanase mutant of claim 1, wherein the glucanase mutant is E36R, wherein the glucanase mutant has the nucleotide sequence of SEQ ID No. 5; the nucleotide sequence of the encoding dextranase mutant D127K is shown in SEQ ID NO. 6; the nucleotide sequence of the encoding dextranase mutant E36R/D127K is shown in SEQ ID NO. 7.
3. A recombinant vector comprising the nucleotide sequence encoding the dextranase mutant of claim 2; when the sequence is SEQ ID NO.5, the recombinant vector is pPIC9 r-E36R; when the sequence is SEQ ID NO.6, the recombinant vector is pPIC9 r-D127K; when the sequence is SEQ ID NO.7, the recombinant vector is pPIC9 r-E36R/D127K.
4. A recombinant strain, characterized by a strain expressing the recombinant vector of claim 3; when the recombinant vector is pPIC9r-E36R, the recombinant strain is GS 115/E36R; when the recombinant vector is pPIC9r-D127K, the recombinant strain is GS 115/D127K; when the recombinant vector is pPIC9r-E36R/D127K, the recombinant strain is GS 115/E36R/D127K.
5. The method for constructing the GH16 family thermotolerant dextranase mutant based on claim 1, which comprises the following steps:
1) amplifying sequence segments of the high-temperature dextranase mutants by adopting an over-lap PCR method;
2) cloning the sequence fragment of the dextranase mutant between EcoR I and Not I restriction sites of an expression vector pPIC9r to obtain a recombinant vector;
3) transforming the mutant recombinant vector into pichia pastoris GS115, and carrying out induced expression to obtain a mutant strain;
4) culturing the recombinant strain, and inducing the expression of the recombinant glucanase;
5) recovering and purifying the expressed heat-resistant dextranase mutant.
6. The use of a GH16 family thermotolerant glucanase mutant according to claim 1 for degrading hemicellulose.
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| CN113234705A (en) * | 2021-04-21 | 2021-08-10 | 江南大学 | Acid-tolerant 1,3-1, 4-beta-glucanase mutants |
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| CN113373131B (en) * | 2021-06-04 | 2022-07-22 | 江苏科技大学 | GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutant and application thereof |
| CN113699136A (en) * | 2021-06-04 | 2021-11-26 | 江苏科技大学 | Beta-1, 3-1, 4-glucanase mutant with high catalytic activity at animal body temperature and application thereof |
| CN113373131A (en) * | 2021-06-04 | 2021-09-10 | 江苏科技大学 | GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutant and application thereof |
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| CN114752583A (en) * | 2022-03-30 | 2022-07-15 | 齐鲁工业大学 | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof |
| CN114752583B (en) * | 2022-03-30 | 2023-07-21 | 齐鲁工业大学 | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof |
| CN114836402A (en) * | 2022-04-26 | 2022-08-02 | 江苏科技大学 | GH16 family heat stability enhanced glucanase mutant and construction method and application thereof |
| CN114836402B (en) * | 2022-04-26 | 2023-11-21 | 江苏科技大学 | Glucanase mutant with enhanced GH16 family thermal stability, construction method and application thereof |
| CN117511918A (en) * | 2023-11-16 | 2024-02-06 | 山东弥美生物科技股份有限公司 | Beta-1, 3-glucanase mutant and application thereof |
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