CN113373131B - GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutant and application thereof - Google Patents
GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutant and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2448—Licheninase (3.2.1.73)
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
- A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C5/00—Other raw materials for the preparation of beer
- C12C5/004—Enzymes
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01073—Licheninase (3.2.1.73)
Abstract
The mutants are BisGlu16B-D47A, BisGlu16B-D175A or BisGlu16B-D47A/D175A, and the optimal temperature of the mutant is consistent with that of a wild type and is 60 ℃;T mthe value is improved by 2.3-4.3 ℃ compared with that of a wild type; half life at 65 ℃ ((t 1/2) The elongation is 1.1 to 1.4 times compared with the wild type;T 50the value is improved by 0.5-3.0 ℃ compared with the wild type. The thermal stability is improved without loss of catalytic efficiency. The heat-resistant high-catalytic acidic beta-1, 3-1, 4-glucanase mutant has great application potential in the fields of beer brewing, feed addition, juice clarification and the like.
Description
Technical Field
The invention relates to gene engineering and protein engineering, in particular to a group of GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutants and application thereof.
Background
Plant cell walls are mainly composed of cellulose, hemicellulose, lignin, pectin, and the like. The cellulose content is highest, accounting for about 40-50% of the dry weight of the cells, and is formed by connecting glucose through beta-1, 4 and beta-1, 3-glycosidic bonds. The content of hemicellulose is today higher than that of cellulose, and accounts for about 30-35% of the dry weight of cells, and the hemicellulose is the most abundant renewable resource after cellulose. Hemicellulose consists of heteropolysaccharides, named for the sugars with the predominant backbone composition, and is known as dextran, mannan, and glucomannan, et al (Schulze E1891. Ber Dtsch Chem Ges 24, 2277-2287.). 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 70-75% of glucan (Philippe S et al plant 224(2), 449-461).
Beta-glucan is a generic name for a class of enzymes that break down glucose polymers that are linked by beta-glycosidic bonds. The mode of action can be divided into an inner cutting type and an outer cutting type. Wherein the endo-beta-1, 3-1, 4-glucose (E.C.3.2.1.73) can specifically act on the beta-1, 4 glycosidic bond connected with the beta-1, 3 bond, thereby reducing the molecular weight, hydrophilicity and viscosity of the beta-1, 3-glycosidic bond. In the feed industry, glucanase can promote digestion of animals, reduce the risk of intestinal diseases, improve the microbial environment of the intestinal tract and promote the growth of the animals. In the food industry, the glucanase can be applied to brewing of beer and fruit juice to improve the product quality. Therefore, the improvement and application of the glucanase in thermal stability and catalytic efficiency are continuously advanced.
The glucanase is required to have stronger thermal stability and catalytic efficiency in different industrial applications, so that the research on the thermal stability and the catalytic efficiency of the glucanase is of great significance.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a group of GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutants and application thereof, wherein the mutants can tolerate high temperature treatment at the temperature of more than 65 ℃, have enzyme activity of more than 80% under acidic and neutral pH, and have better application potential in the fields of feed and beer brewing and the like.
The technical scheme is as follows: a group of GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutants, wherein the mutants are BisGlu16B-D47A, BisGlu16B-D175A or BisGlu16B-D47A/D175A, and the nucleotide sequence of the glucanase mutant BisGlu16B-D47A is shown as SEQ ID NO. 1; the nucleotide sequence of the glucanase mutant BisGlu16B-D175A is shown in SEQ ID NO. 2; the nucleotide sequence of the glucanase mutant BisGlu16B-D47A/D175A is shown as SEQ ID NO. 3; all the mutation sites are labeled with the sequence without signal peptide.
The amino acid sequence of the glucanase mutant BisGlu16B-D47A is shown in SEQ ID NO. 4; the amino acid sequence of the glucanase mutant BisGlu16B-D175A is shown as SEQ ID NO. 5; the amino acid sequence of the glucanase mutant BisGlu16B-D47A/D175A is shown as SEQ ID NO.6, wherein the sequence without the signal peptide is used for the reference of all mutation sites.
A recombinant vector comprising any one of the nucleotide sequences described above.
A recombinant strain comprising the above recombinant vector.
The GH16 family thermostable beta-1, 3-1, 4-glucanase mutant can be applied to beer brewing, feed addition and juice clarification.
Has the beneficial effects that: the invention provides a glucanase mutant with stronger thermal stability. The optimum temperature of the mutant is consistent with that of a wild type and is 60 ℃; t is a unit ofmThe value is improved by 2.3-4.3 ℃ compared with the wild type; half life at 65 ℃ (t)1/2) The elongation is 1.1 to 1.4 times compared with the wild type; t is50The value is improved by 0.5-3.0 ℃ compared with the wild type. The thermal stability is improved without loss of catalytic efficiency. Compared with the method of obtaining the glucanase suitable for industrial large-scale production by blind screening or natural mutagenesis and other means, the rational design of the enzyme shortens the modification time of the enzymology property. The heat-resistant high-catalytic acidic beta-1, 3-1, 4-glucanase mutant has great application potential in the fields of beer brewing, feed addition, juice clarification and the like.
Drawings
FIG. 1 shows protein purification of heat-resistant dextranase mutants and wild-type.
FIG. 2 is the optimum pH for the mutant thermostable glucanase from the wild type;
FIG. 3 shows the pH stability of the thermotolerant dextranase mutant versus wild type;
FIG. 4 shows the optimal temperature for the heat-resistant dextranase mutant versus wild type;
FIG. 5 shows heat-resistant dextranase at 65 ℃Half-life of mutant and wild type (t)1/2)；
FIG. 6 shows T of heat-resistant dextranase mutant and wild type50；
FIG. 7 shows the thermostable dextranase mutants and wild type Tm。
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, the expression plasmid vector pPIC9r for the laboratory preservation; the nucleotide sequence of BisGlu16B is shown in SEQ ID NO. 7.
2. Enzymes and other biochemical reagents: pfu enzyme was purchased from allkin, and oat glucan was purchased from Sigma; other reagents are domestic analytical pure reagents (all purchased from the national drug 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
Uses glucanase BisGlu16B from GH16 family as a starting material, and uses FastPfu polymerase to amplify the coding gene of the glucanase by adopting a site-directed mutagenesis PCR method. Mutation methods and cloning methods references (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
Transferring the PCR product into DMT competence, carrying out heat shock transformation and smearing on LB solid culture medium with Amp resistance, carrying out bacterium p verification and sequencing verification to obtain a recombinant plasmid containing the heat-resistant glucanase mutant gene and transforming Pichia pastoris GS115 to obtain recombinant yeast strains BisGlu16B-D47A, BisGlu16B-D175A and BisGlu 16B-D47A/D175A.
Taking a GS115 strain containing the recombinant plasmid, inoculating the strain into a 1L triangular flask of 300mL BMGY medium, and carrying out shake culture at 30 ℃ and 220rpm for 48 h; 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 methanol in the bacterial liquid is kept at 0.5%, and meanwhile, the supernatant is taken for enzyme activity detection.
Example 3 Activity analysis of recombinant thermostable dextranase mutants and wild type
Enzyme activity determination of beta-1, 3-1, 4-glucanase
The activity of the beta-1, 3-1, 4-glucanase was determined according to the method previously reported by Yang et al (Yang 10.1021/jf800303b) with 1% (w/v, g/mL) of oat beta-glucan as substrate. The specific method comprises the following steps: under the given conditions of pH and temperature, 1mL of reaction system comprises 100 μ L of 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. The beta-1, 3-1, 4-glucanase activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol glucose-equivalent reducing sugars per minute under the above conditions.
II, determining the properties of the mutant and the wild type of the recombinant heat-resistant glucanase
1. The optimum pH of the recombinant thermostable glucanase mutant and the wild type was determined as follows:
the purified recombinant thermostable glucanase mutants of example 2 and wild type were subjected to enzymatic reactions at different pH to determine their optimum pH. The substrate (oat glucan) was diluted with 0.1mol/L citrate-disodium phosphate buffer at different pH and the glucanase activity assay was performed at 60 ℃.
The results (FIG. 2) show that the optimal reaction pH value of the recombinant heat-resistant glucanase mutant and the wild type is between 2.5 and 4.5, and the recombinant heat-resistant glucanase mutant has higher enzyme catalytic activity in the pH range of 1.0 to 9.0 (FIG. 3).
2. The optimal temperature of the recombinant heat-resistant glucanase mutant and the wild type is determined as follows:
the optimal temperatures of the recombinant thermostable dextranase mutants and the wild type were determined by performing the enzymatic reactions in a 0.1mol/L citrate-disodium phosphate buffer (pH 3.5) buffer system at different temperatures. The results of the measurement of the optimum temperature for the enzymatic reaction (FIG. 4) show that the optimum temperature of the recombinant thermostable glucanase mutant and the wild type are both 60 ℃. The catalytic activity at 65 ℃ is higher than that of the wild type, particularly the mutant BisGlu 16B-D47A/D175A.
3. The thermostability at 65 ℃ of the recombinant thermostable glucanase mutants and the wild type was determined as follows:
the heat stability of the heat-resistant dextranase mutant and the wild type is gradually reduced after being treated at 65 ℃ for a certain time, but the heat stability of all mutants is better than that of the wild type. By fitting curves, it can be found that the half-life of the mutant BisGlu16B-D47A is prolonged by 5min compared with that of the wild-type enzyme, while the combined mutant BisGlu16B-D47A/D175A has the best effect, and the half-life is prolonged to 52min and is almost twice of that of the wild-type enzyme.
4. The kinetics of the recombinant thermotolerant dextranase mutant and the wild type are determined as follows:
under the optimal conditions, the kinetic parameters and specific activities of the thermostable glucanase and the wild type were determined by using oat glucan as a substrate, and the results are shown in table 2.
Wherein, the K of BisGlu16B-D47A is the optimum condition with oat glucan as a substratemAnd Vm0.79mg/mL and 3979u mol/min. mg. K of BisGlu16B-D175AmAnd VmRespectively 1.53mg/mL and 30300. mu. mol/min. mg. BisGlu16B-D47A/D1K of 75AmAnd VmRespectively 2.27mg/mL and 40700. mu. mol/min. mg. Wild type KmAnd VmRespectively 2.55mg/mL and 3970000. mu. mol/min. mg. The catalytic efficiency of the mutant was almost lossless compared to the wild type. In addition, mutant BisGlu16B-D47A/D175A catalyzed efficiency (k)cat/Km) And the specific activity is improved by 1.2 times and 1.1 times compared with the wild type. In conclusion, the thermal stability of the three mutants is enhanced compared with that of the wild type, and the loss of enzyme activity is not accompanied, wherein the mutant BisGlu16B-D47A/D175A is the most superior.
TABLE 2 kinetic parameters and specific activities of wild type BisGlu16B and its mutants under optimal conditions
Sequence listing
<110> university of Jiangsu science and technology
<120> group of GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutants and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
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caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agctggcttt gtggcttata aaaatgagac 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 ttggcccgac 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 cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
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caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac 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 ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgctga 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 cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
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<213> Artificial Sequence (Artificial Sequence)
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caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
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gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgctga 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 cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
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cacaagagac acgggccagg gaggctttga 1350
<210> 4
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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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 Ala 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 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 Pro Ser Ser Val Arg Ala Ile Val Gly Gly Ser Gly Ser Ala
325 330 335
Ala Ser Ser Thr Thr Phe Ala Ile Ser Thr Lys Ser Ala Pro Phe Pro
340 345 350
Val Gly Asn Ser Thr Ser Val Val Gly Thr Thr Gly Ala Ser Ser Asn
355 360 365
Gly Ala Trp Ala Ala Ile Val Thr Gly Thr Gly Pro Ile Gly Val Ala
370 375 380
Gln Glu Thr Ser Val Ser Ala Ala Ser Ala Ala Pro Ser Ser Pro Ala
385 390 395 400
Glu Ala Thr Pro Ala Ser Ser Val Ala Gly Ala Gln Ser Trp Asn Trp
405 410 415
Gln Ser His Ala Trp Gly Asn His Asn His His Glu Pro Ser Ala Ala
420 425 430
Ala Leu Lys Arg His Leu Arg His His Lys Arg His Gly Pro Gly Arg
435 440 445
Leu
<210> 5
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
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 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 Ala 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 Pro Ser Ser Val Arg Ala Ile Val Gly Gly Ser Gly Ser Ala
325 330 335
Ala Ser Ser Thr Thr Phe Ala Ile Ser Thr Lys Ser Ala Pro Phe Pro
340 345 350
Val Gly Asn Ser Thr Ser Val Val Gly Thr Thr Gly Ala Ser Ser Asn
355 360 365
Gly Ala Trp Ala Ala Ile Val Thr Gly Thr Gly Pro Ile Gly Val Ala
370 375 380
Gln Glu Thr Ser Val Ser Ala Ala Ser Ala Ala Pro Ser Ser Pro Ala
385 390 395 400
Glu Ala Thr Pro Ala Ser Ser Val Ala Gly Ala Gln Ser Trp Asn Trp
405 410 415
Gln Ser His Ala Trp Gly Asn His Asn His His Glu Pro Ser Ala Ala
420 425 430
Ala Leu Lys Arg His Leu Arg His His Lys Arg His Gly Pro Gly Arg
435 440 445
Leu
<210> 6
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
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 Ala 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 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 Ala 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 Pro Ser Ser Val Arg Ala Ile Val Gly Gly Ser Gly Ser Ala
325 330 335
Ala Ser Ser Thr Thr Phe Ala Ile Ser Thr Lys Ser Ala Pro Phe Pro
340 345 350
Val Gly Asn Ser Thr Ser Val Val Gly Thr Thr Gly Ala Ser Ser Asn
355 360 365
Gly Ala Trp Ala Ala Ile Val Thr Gly Thr Gly Pro Ile Gly Val Ala
370 375 380
Gln Glu Thr Ser Val Ser Ala Ala Ser Ala Ala Pro Ser Ser Pro Ala
385 390 395 400
Glu Ala Thr Pro Ala Ser Ser Val Ala Gly Ala Gln Ser Trp Asn Trp
405 410 415
Gln Ser His Ala Trp Gly Asn His Asn His His Glu Pro Ser Ala Ala
420 425 430
Ala Leu Lys Arg His Leu Arg His His Lys Arg His Gly Pro Gly Arg
435 440 445
Leu
<210> 7
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac 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 ttggcccgac 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 cagctcccag cagcccagcg 1200
gaggcaactc ctgcatctag cgtagctggg gcgcaatctt ggaactggca gtctcacgcg 1260
tggggcaatc ataatcatca cgaaccctcc gcagcagcct tgaaaaggca tctgagacat 1320
cacaagagac acgggccagg gaggctttga 1350
<210> 8
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
agctgtaggg tcggcggtat cccaaaatga aaat 34
<210> 9
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgaccctaca gctggctttg tggcttata 29
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
agcgacccag cagtcggtgc taactatcg 29
<210> 11
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgctgggtcg ctgaccccaa ccaatccgac a 31
Claims (5)
1. A group of GH16 family heat-resistant beta-1, 3-1, 4-glucanase mutants, wherein the mutants are BisGlu16B-D47A, BisGlu16B-D175A or BisGlu16B-D47A/D175A, and the nucleotide sequence of the glucanase mutant BisGlu16B-D47A is shown as SEQ ID NO. 1; the nucleotide sequence of the glucanase mutant BisGlu16B-D175A is shown as SEQ ID NO. 2; the nucleotide sequence of the glucanase mutant BisGlu16B-D47A/D175A is shown as SEQ ID NO. 3; all the mutation sites are labeled with the sequence without the signal peptide.
2. The GH16 family thermostable β -1,3-1, 4-glucanase mutant according to claim 1, wherein the glucanase mutant BisGlu16B-D47A has the amino acid sequence shown in SEQ ID No. 4; the amino acid sequence of the glucanase mutant BisGlu16B-D175A is shown as SEQ ID NO. 5; the amino acid sequence of the glucanase mutant BisGlu16B-D47A/D175A is shown as SEQ ID NO.6, wherein the sequence without the signal peptide is used for the reference of all mutation sites.
3. A recombinant vector comprising the nucleotide sequence of claim 1.
4. A recombinant strain comprising the recombinant vector according to claim 3.
5. The use of a mutant of the thermostable β -1,3-1, 4-glucanase of GH16 family of claim 1 for brewing beer, feed supplementation, and juice clarification.
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| CN114517191B (en) * | 2022-01-16 | 2024-03-15 | 江苏科技大学 | Acidic glucanase mutant with improved thermal stability and application thereof |
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