CN118147115A - Beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof - Google Patents
Beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof Download PDFInfo
- Publication number
- CN118147115A CN118147115A CN202410573800.6A CN202410573800A CN118147115A CN 118147115 A CN118147115 A CN 118147115A CN 202410573800 A CN202410573800 A CN 202410573800A CN 118147115 A CN118147115 A CN 118147115A
- Authority
- CN
- China
- Prior art keywords
- beta
- penicillium oxalicum
- mutant
- glucosidase
- glucosidase mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010047754 beta-Glucosidase Proteins 0.000 title claims abstract description 46
- 102000006995 beta-Glucosidase Human genes 0.000 title claims abstract description 43
- 241000985513 Penicillium oxalicum Species 0.000 title claims abstract description 40
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 66
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 33
- 235000019253 formic acid Nutrition 0.000 claims abstract description 33
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 23
- 239000008103 glucose Substances 0.000 claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000011534 incubation Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 241000499912 Trichoderma reesei Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 241000793189 Saccharomyces cerevisiae BY4741 Species 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 21
- 150000007524 organic acids Chemical class 0.000 abstract description 8
- 238000010306 acid treatment Methods 0.000 abstract description 6
- 239000003054 catalyst Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 239000002253 acid Substances 0.000 description 8
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 7
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 7
- 229940120668 salicin Drugs 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000000329 molecular dynamics simulation Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 150000008495 β-glucosides Chemical class 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013124 brewing process Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 150000002240 furans Chemical class 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000006959 non-competitive inhibition Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 150000008498 β-D-glucosides Chemical class 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to a beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof. The amino acid sequence of the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 is shown in SEQ ID NO: 2. The mutant V3-4-H6 has higher organic acid tolerance at a lower temperature, still maintains 72.7% relative activity after 10 mg/mL formic acid treatment, and has the glucose content which is 2.01 times of that of the original 16BGL generated by bran residues after the enzymolysis pretreatment of the mutant V3-4-H6 under the condition of 10 mg/mL formic acid treatment, thus showing that the mutant V3-4-H6 has higher organic acid tolerance, can be applied to various fields such as feed, medicine, food, chemical industry, energy source and the like, and provides a more reliable and efficient enzyme catalyst for related application fields.
Description
Technical Field
The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to a beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof.
Background
Beta-glucosidase is an enzyme that hydrolyzes the beta-1, 4-glycosidic bond of the non-reducing terminal residue in beta-D-glucoside while releasing glucose. However, the original beta-glucoside has poor enzymatic properties, such as low catalytic activity, poor organic acid tolerance and high catalytic temperature, so that the beta-glucoside is difficult to apply to various fields of feed, medicine, food, chemical industry, energy sources and the like.
The beta-glucosidase of penicillium oxalicum 16 has uninhibited activity on furan derivatives and phenolic compounds and excellent tolerance to high concentrations of KCl, naCl and ethanol, however, the wild type 16BGL has poor activity and very poor tolerance to formic acid, so that improving the enzyme activity of the beta-glucosidase of penicillium oxalicum 16 under formic acid conditions is of great significance in the field of bioenergy.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof, wherein the following technical scheme is adopted:
In a first aspect of the invention, a beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 is provided, wherein the amino acid sequence of the beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 is shown in SEQ ID NO: 2.
SEQ ID NO：2：
KDLAYSPPFYPSPWATGEGEWAEAYKKAVDFVSGLTLAEKVNITTGAGWEQERCVGETGGVPRLGMWGMCMQDSPLGVRNADYSSAFPAGVNVAATWDRRLAYQRGTAMGEEHRDKGVDVQLGPVAGPLGKNPDGGRGWEGFSPDPVLTGVMMAETIKGIQDAGVIACAKHFIMNEQEHFRQAGEAQGYGFNISQSLSSNVDDKTMHELYLWPFVDSVRAGVGSVMCSYNQINNSYGCSNSYTLNKLLKGELGFQGFVMSDWGAHHSGVGDALAGLDMSMPGDVILGSPYSFWGTNLTVSVLNSTIPEWRLDDMAVRIMAAYYKVGRDRHRTPPNFSSWTRDKYGYEHFIVQENYVKLNERVNVQRDHANVIRKIGSDSIVMLKNNGGLPLTHQERLVAILGEDAGSNAYGANGCSDRGCDNGTLAMGWGSGTANFPYLITPEQAIQNEVLNYGNGDTNVFAVTDNGALSQMAALASTASVALVFVNADSGEGYISVDGNEGDRKNMTLWKNGEELIKTATANCNNTIVIMHTPNAVLVDSWYDNENITAILWAGMPGQESGRSLVDVLYGRTNPGGKTPFTWGKERKDWGSPLLTKPNNGHGAPQDDFTDVLIDYRRFDKDNVEPIFEFGFGLSYTKFEFSDIQVKALNHGEYNATVGKTKPAPSLGKPGNASDSLFPSNINRVRQYLYPYLNSTDLKASANDPDYGMNASAYIPPHATDSDPQDLLPASGPSGGNPGLFEDLIEVTATVTNTGSVTGDEVPQLYVSLGGADDPVKVLRAFDRVTIAPGQKLRWTATLNRRDLSNWDVPSQNWIISDAPKKVWVGNSSRKLPLSADLPKVQ.
The amino acid sequence of the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 is SEQ ID NO:2 is represented by SEQ ID NO:1 into serine.
SEQ ID NO：1：
KDLAYSPPFYPSPWATGEGEWAEAYKKAVDFVSGLTLAEKVNITTGAGWEQERCVGETGGVPRLGMWGMCMQDSPLGVRNADYSSAFPAGVNVAATWDRRLAYQRGTAMGEEHRDKGVDVQLGPVAGPLGKNPDGGRGWEGFSPDPVLTGVMMAETIKGIQDAGVIACAKHFIMNEQEHFRQAGEAQGYGFNISQSLSSNVDDKTMHELYLWPFVDSVRAGVGSVMCSYNQINNSYGCSNSYTLNKLLKGELGFQGFVMSDWGAHHSGVGDALAGLDMSMPGDVILGSPYSFWGTNLTVSVLNSTIPEWRLDDMAVRIMAAYYKVGRDRHRTPPNFSSWTRDKYGYEHFIVQENYVKLNERVNVQRDHANVIRKIGSDSIVMLKNNGGLPLTHQERLVAILGEDAGSNAYGANGCSDRGCDNGTLAMGWGSGTANFPYLITPEQAIQNEVLNYGNGDTNVFAVTDNGALSQMAALASTASVALVFVNADSGEGYISVDGNEGDRKNMTLWKNGEELIKTATANCNNTIVIMHTPNAVLVDSWYDNENITAILWAGMPGQESGRSLVDVLYGRTNPGGKTPFTWGKERKDWGSPLLTKPNNGHGAPQDDFTDVLIDYRRFDKDNVEPIFEFGFGLSYTKFEFSDIQVKALNHGEYNATVGKTKPAPSLGKPGNASDHLFPSNINRVRQYLYPYLNSTDLKASANDPDYGMNASAYIPPHATDSDPQDLLPASGPSGGNPGLFEDLIEVTATVTNTGSVTGDEVPQLYVSLGGADDPVKVLRAFDRVTIAPGQKLRWTATLNRRDLSNWDVPSQNWIISDAPKKVWVGNSSRKLPLSADLPKVQ.
According to the invention, the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 is obtained through a molecular transformation technology, the reaction temperature of the mutant V3-4-H6 is 65 ℃, the specific activity of the mutant V3-4-H6 is 0.0165 IU/mg when acid is not added, the specific activity of the mutant V3-4-H6 after treatment by adding 10 mg/mL formic acid is 0.012 IU/mg, the catalytic activity of the mutant V3-4-H6 is only 0.43 times of the original 16BGL (beta-glucosidase of penicillium oxalicum 16), but the formic acid tolerance of the V3-4-H6 is obviously enhanced, the 1.64 times of the original 16BGL is achieved, the K m/KI value of 10 mg/mL formic acid inhibition intensity is 1.17x10 -2, and particularly, the K m/KI value of the mutant V3-4-H6 is 1.9 times of the mutant, the performance of the mutant V3-4-H6 in formic acid treatment is more excellent, and a more reliable and efficient enzyme catalyst is provided for related application fields.
In a second aspect of the invention, there is provided a gene of the beta-glucosidase mutant V3-4-H6 of Penicillium oxalicum 16, the gene sequence of which is as shown in SEQ ID NO: 3.
SEQ ID NO：3：
AAGGATCTTGCCTACTCTCCCCCCTTCTATCCTTCTCCATGGGCAACCGGTGAAGGTGAATGGGCCGAGGCCTACAAGAAGGCTGTGGACTTTGTTTCTGGTCTGACTCTTGCCGAGAAGGTCAACATCACGACCGGTGCTGGATGGGAACAGGAGCGTTGTGTGGGTGAGACCGGCGGTGTCCCTCGACTTGGAATGTGGGGAATGTGCATGCAAGATTCTCCTCTCGGCGTTCGTAATGCCGACTACAGCTCTGCCTTCCCCGCCGGTGTGAATGTGGCTGCCACCTGGGACCGACGACTCGCGTACCAGCGTGGTACGGCCATGGGCGAGGAGCATCGCGACAAGGGTGTCGACGTGCAGCTTGGCCCCGTCGCTGGTCCATTGGGCAAGAACCCCGACGGTGGTCGTGGCTGGGAAGGCTTTTCTCCCGATCCGGTTCTGACCGGTGTTATGATGGCCGAGACAATCAAGGGTATCCAAGATGCTGGTGTCATTGCTTGCGCCAAGCACTTCATCATGAATGAGCAGGAGCACTTCCGCCAGGCGGGTGAAGCCCAGGGATACGGATTCAATATTTCTCAGAGTTTGAGCTCCAACGTCGATGACAAGACCATGCACGAGCTGTACTTGTGGCCGTTTGTCGATTCGGTTCGGGCCGGTGTGGGTTCCGTCATGTGCTCTTACAACCAGATCAACAACAGCTACGGGTGCTCCAACAGCTACACGCTCAACAAATTGCTCAAGGGCGAGCTCGGCTTTCAGGGCTTCGTCATGAGCGACTGGGGTGCGCACCACAGCGGTGTCGGTGACGCCCTTGCCGGTCTCGACATGTCTATGCCCGGTGATGTGATTCTTGGTAGCCCCTACTCCTTCTGGGGAACTAACTTGACCGTCTCTGTGCTGAACAGCACCATCCCCGAATGGCGTCTGGATGACATGGCCGTTCGTATCATGGCTGCCTACTACAAGGTCGGCAGAGATCGTCATCGCACTCCTCCCAACTTCAGCTCCTGGACCCGCGATAAGTACGGCTACGAGCACTTTATTGTCCAGGAGAACTATGTCAAGCTCAACGAGCGTGTCAATGTTCAACGTGATCATGCCAACGTCATCCGCAAGATTGGCTCCGACAGTATCGTGATGCTCAAGAACAACGGGGGTCTGCCTTTGACTCATCAAGAGCGTCTGGTGGCTATCTTGGGCGAGGATGCTGGTTCCAACGCCTACGGCGCCAACGGCTGCAGTGACCGAGGCTGTGACAACGGTACCTTGGCCATGGGCTGGGGCAGTGGAACGGCCAACTTCCCCTACCTGATCACTCCCGAGCAAGCCATTCAGAATGAGGTTCTCAACTACGGCAACGGTGACACCAACGTCTTTGCTGTCACAGACAACGGTGCCCTCAGCCAAATGGCTGCCCTTGCTTCAACCGCAAGTGTTGCATTGGTGTTCGTCAACGCTGATTCGGGCGAGGGCTACATCAGTGTGGACGGCAACGAGGGCGATCGCAAGAACATGACCCTGTGGAAGAACGGCGAGGAGCTGATCAAGACCGCCACTGCCAACTGCAACAACACCATCGTCATCATGCACACCCCCAACGCCGTCCTGGTCGATTCATGGTACGACAATGAGAACATCACTGCCATTCTGTGGGCTGGTATGCCCGGCCAAGAGAGTGGTCGTAGCTTGGTTGATGTTCTCTACGGCCGCACGAACCCTGGTGGCAAGACCCCCTTCACCTGGGGTAAGGAGCGCAAGGATTGGGGATCTCCTCTTCTGACTAAACCCAACAACGGCCACGGTGCTCCTCAGGATGACTTCACCGATGTTCTGATTGACTATCGCCGTTTCGACAAGGACAACGTGGAGCCCATCTTCGAGTTCGGCTTCGGTCTGAGCTACACCAAATTTGAGTTCTCTGACATCCAGGTCAAGGCGCTGAATCACGGCGAGTACAACGCCACCGTGGGCAAGACCAAGCCTGCCCCTTCGTTGGGCAAGCCTGGTAATGCCTCCGATAGTCTGTTCCCCAGCAACATCAACCGTGTGCGCCAGTACCTTTACCCTTACCTGAACTCGACCGATCTGAAGGCGTCTGCCAACGACCCTGACTATGGCATGAATGCATCGGCGTACATTCCTCCCCATGCCACCGACAGCGACCCACAGGACCTTCTCCCCGCCAGCGGACCTTCCGGTGGCAACCCTGGTTTGTTTGAGGATCTTATTGAGGTGACTGCTACTGTCACCAACACCGGCTCAGTTACTGGTGACGAGGTTCCCCAGCTGTATGTTTCGCTTGGCGGTGCCGATGACCCCGTTAAGGTCCTCCGTGCCTTCGACCGTGTCACGATCGCCCCTGGTCAGAAGCTCCGGTGGACAGCAACCCTCAACCGTCGTGATCTGTCCAACTGGGATGTCCCATCACAGAACTGGATCATCTCAGACGCCCCCAAGAAGGTGTGGGTGGGCAACTCGTCGCGCAAGCTGCCTCTTTCAGCCGATCTGCCCAAGGTGCAG.
In a third aspect of the present invention, there is also provided a method for preparing the above β -glucosidase mutant V3-4-H6 of penicillium oxalicum 16, comprising the steps of:
error-prone PCR is carried out on the beta-glucosidase gene of the penicillium oxalicum 16, then a PCR product is connected with a vector pYAT BY using a seamless cloning kit, and then the PCR product is converted into an expression host saccharomyces cerevisiae BY4741 BY using a yeast conversion kit, so that a recombinant strain containing the beta-glucosidase mutant gene of the penicillium oxalicum 16 is obtained; and finally, placing the recombinant strain in a culture medium for culture, expression and screening to finally obtain the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16.
In a fourth aspect of the invention, the application of the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 in the production of glucose by the enzymolysis of bran is provided, and the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 is subjected to formic acid treatment and then the bran is subjected to enzymolysis to obtain glucose.
In a fifth aspect of the present invention, there is also provided a method for enzymatic hydrolysis of bran to glucose, comprising the steps of:
And (3) placing formic acid into beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16, incubating at 37 ℃, adding NaOH solution after incubation to adjust the pH to 5.0, adding Trichoderma reesei residue enzyme solution and bran residue, and performing water bath reaction to obtain glucose.
The beneficial effects of the invention are as follows:
according to the invention, the beta-glucosidase mutant V3-4-H6 gene of the penicillium oxalicum 16 is obtained BY a molecular transformation technology through a directed evolution technology, the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 is obtained BY recombination and expression in saccharomyces cerevisiae BY4741 through a genetic engineering technology, the mutant V3-4-H6 has higher organic acid tolerance at a lower temperature, the relative activity of 72.7% is still maintained after 10 mg/mL formic acid treatment, and the glucose content generated BY bran residues after enzymolysis pretreatment of the mutant V3-4-H6 reaches 2.01 times of the original 16BGL under the condition of 10 mg/mL formic acid treatment, so that the beta-glucosidase mutant V3-4-H6 has higher organic acid tolerance, can be applied to a plurality of fields such as feed, medicine, food, chemical industry, energy sources and the like, and provides a more reliable and efficient enzyme catalyst for related application fields.
Drawings
FIG. 1 shows the relative specific activities of original 16BGL and mutant V3-4-H6 at different temperatures;
FIG. 2 shows the relative specific activities of the original 16BGL and mutant V3-4-H6 after the addition of 10 mg/mL of formic acid and the incubation without the addition of acid;
FIG. 3 shows the glucose content of the bran residue after enzymatic pretreatment of the original 16BGL and mutant V3-4-H6 after incubation with 10 mg/mL formic acid without acid addition.
Detailed Description
The conception, specific structure, and technical effects produced by the present application will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects, and effects of the present application. It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other.
Example 1
Preparation of beta-glucosidase mutant V3-4-H6 of Penicillium oxalicum 16
1. Obtaining the beta-glucosidase gene of Penicillium oxalicum 16
Inoculating penicillium oxalicum 16 serving as a material, performing induction culture and collecting mycelium; trizol reagent of Tarkara is firstly used for extracting total RNA by a Trizol method, then reverse transcription is carried out by using reverse transcription kit of Tarkara to obtain cDNA, and gene of beta-glucosidase (the sequence of which is shown as SEQ ID NO: 8) is amplified by using cDNA as a template through two specific references (the sequences of which are shown as SEQ ID NO:4 and SEQ ID NO: 5) by using a conventional PCR method.
SEQ ID NO：4：5’-TCTCTCGAGAAAAGAGAGGCTGAAGCTAAGGATCTTGCCTACTCTCCCCCCTTCTATCC-3’。
SEQ ID NO：5：5’-CATTTAAATTAGTGATGGTGATGGTGATGCTGCACCTTGGGCAGATCGGCTGAAAGAGG-3’。
SEQ ID NO：8：
AAGGATCTTGCCTACTCTCCCCCCTTCTATCCTTCTCCATGGGCGACCGGTGAAGGTGAATGGGCCGAGGCCTACAAGAAGGCTGTGGACTTTGTTTCTGGTCTGACTCTTGCCGAGAAGGTCAACATCACGACCGGTGCTGGATGGGAACAGGAGCGTTGTGTGGGTGAGACCGGCGGTGTCCCTCGACTTGGAATGTGGGGAATGTGCATGCAAGATTCTCCTCTCGGCGTTCGTAATGCCGACTACAGCTCTGCCTTCCCCGCCGGTGTGAATGTGGCTGCCACCTGGGACCGACGACTCGCGTACCAGCGTGGTACGGCCATGGGCGAGGAGCATCGCGACAAGGGTGTCGACGTGCAGCTTGGCCCCGTCGCTGGTCCATTGGGCAAGAACCCCGACGGTGGTCGTGGCTGGGAAGGCTTTTCTCCCGATCCGGTTCTGACCGGTGTTATGATGGCCGAGACAATCAAGGGTATCCAAGATGCTGGTGTCATTGCTTGCGCCAAGCACTTCATCATGAATGAGCAGGAGCACTTCCGCCAGGCGGGTGAAGCCCAGGGATACGGATTCAATATTTCTCAGAGTTTGAGCTCCAACGTCGATGACAAGACCATGCACGAGCTGTACTTGTGGCCGTTTGTCGATTCGGTTCGGGCCGGTGTGGGTTCCGTCATGTGCTCTTACAACCAGATCAACAACAGCTACGGGTGCTCCAACAGCTACACGCTCAACAAATTGCTCAAGGGCGAGCTCGGCTTTCAGGGCTTCGTCATGAGCGACTGGGGTGCGCACCACAGCGGTGTCGGTGACGCCCTTGCCGGTCTCGACATGTCTATGCCCGGTGATGTGATTCTTGGTAGCCCCTACTCCTTCTGGGGAACTAACTTGACCGTCTCTGTGCTGAACAGCACCATCCCCGAATGGCGTCTGGATGACATGGCCGTTCGTATCATGGCTGCCTACTACAAGGTCGGCAGAGATCGTCATCGCACTCCTCCCAACTTCAGCTCCTGGACCCGCGATAAGTACGGCTACGAGCACTTTATTGTCCAGGAGAACTATGTCAAGCTCAACGAGCGTGTCAATGTTCAACGTGATCATGCCAACGTCATCCGCAAGATTGGCTCCGACAGTATCGTGATGCTCAAGAACAACGGGGGTCTGCCTTTGACTCATCAAGAGCGTCTGGTGGCTATCTTGGGCGAGGATGCTGGTTCCAACGCCTACGGCGCCAACGGCTGCAGTGACCGAGGCTGTGACAACGGTACCTTGGCCATGGGCTGGGGCAGTGGAACGGCCAACTTCCCCTACCTGATCACTCCCGAGCAAGCCATTCAGAATGAGGTTCTCAACTACGGCAACGGTGACACCAACGTCTTTGCTGTCACAGACAACGGTGCCCTCAGCCAAATGGCTGCCCTTGCTTCAACCGCAAGTGTTGCATTGGTGTTCGTCAACGCTGATTCGGGCGAGGGCTACATCAGTGTGGACGGCAACGAGGGCGATCGCAAGAACATGACCCTGTGGAAGAACGGCGAGGAGCTGATCAAGACCGCCACTGCCAACTGCAACAACACCATCGTCATCATGCACACCCCCAACGCCGTCCTGGTCGATTCATGGTACGACAATGAGAACATCACTGCCATTCTGTGGGCTGGTATGCCCGGCCAAGAGAGTGGTCGTAGCTTGGTTGATGTTCTCTACGGCCGCACGAACCCTGGTGGCAAGACCCCCTTCACCTGGGGTAAGGAGCGCAAGGATTGGGGATCTCCTCTTCTGACTAAACCCAACAACGGCCACGGTGCTCCTCAGGATGACTTCACCGATGTTCTGATTGACTATCGCCGTTTCGACAAGGACAACGTGGAGCCCATCTTCGAGTTCGGCTTCGGTCTGAGCTACACCAAATTTGAGTTCTCTGACATCCAGGTCAAGGCGCTGAATCACGGCGAGTACAACGCCACCGTGGGCAAGACCAAGCCTGCCCCTTCGTTGGGCAAGCCTGGTAATGCCTCCGATCATCTGTTCCCCAGCAACATCAACCGTGTGCGCCAGTACCTTTACCCTTACCTGAACTCGACCGATCTGAAGGCGTCTGCCAACGACCCTGACTATGGCATGAATGCATCGGCGTACATTCCTCCCCATGCCACCGACAGCGACCCACAGGACCTTCTCCCCGCCAGCGGACCTTCCGGTGGCAACCCTGGTTTGTTTGAGGATCTTATTGAGGTGACTGCTACTGTCACCAACACCGGCTCAGTTACTGGTGACGAGGTTCCCCAGCTGTATGTTTCGCTTGGCGGTGCCGATGACCCCGTTAAGGTCCTCCGTGCCTTCGACCGTGTCACGATCGCCCCTGGTCAGAAGCTCCGGTGGACAGCAACCCTCAACCGTCGTGATCTGTCCAACTGGGATGTCCCATCACAGAACTGGATCATCTCAGACGCCCCCAAGAAGGTGTGGGTGGGCAACTCGTCGCGCAAGCTGCCTCTTTCAGCCGATCTGCCCAAGGTGCAG.
2. Preparation of beta-glucosidase mutant V3-4-H6 of Penicillium oxalicum 16
The 16BGL gene sequence (SEQ ID NO: 8) obtained above was subjected to error-prone PCR (10 XPCR Buffer at 3. Mu.L, dNTPs at 3. Mu.L, mnCl 2 at 0.5 mmol/L, upstream primer SP at 0.3. Mu.M (its sequence is shown as SEQ ID NO: 4), downstream primer XM at 0.3. Mu.M (its sequence is shown as SEQ ID NO:5 respectively), taq DNA polymerase at 5U, beta-glucosidase DNA template at 5 ng, adding sterilized deionized water to 30. Mu.L, pre-denaturing at 94℃for 4 min, denaturation at 94℃for 30 sec, annealing at 60℃for 30 sec, 72℃for 40 min, 40 cycles, and final extension for 10 min for random mutagenesis, then ligated with constitutive expression vector pYAT22 (amplified BY two specific primers, its sequences are shown as SEQ ID NO:6 and SEQ ID NO: 7), transformed into host cell-expressing yeast DNA template at 94℃for 4 min, and further performing a colony selection of yeast containing NO glucose at 40% and 2 mg/30% of glucose BY using a transformation kit, and culturing yeast colony containing NO 2mg of 2-15% glucose in the yeast and 4 mg/or 50mg of glucose.
SEQ ID NO：6：5’-CCTCTTTCAGCCGATCTGCCCAAGGTGCAGCATCACCATCACCATCACTAATTTAAATG-3’。
SEQ ID NO：7：5’-AGAAGGGGGGAGAGTAGGCAAGATCCTTAGCTTCAGCCTCTCTTTTCTCGAGAGATACC-3’。
Positive mutants selected from the above first round of screening were used for the second round of screening; inoculating the selected positive mutants into a 96-well plate containing 200 mu L of uracil-free liquid basal medium, and culturing at 30 ℃ to obtain a culture solution of 60 h; centrifuging the bacterial liquid 10000 rpm for 5 minutes, and respectively preserving supernatant and precipitate; taking 50 mu L of diluted supernatant, adding 5.5 mu L of 100 mg/mL formic acid (the final concentration of the formic acid is 10 mg/mL), incubating in a 37 ℃ water bath for 1 h, adding 5 mu L of 2 mol/L NaOH, and mixing with 39.5 mu L of 0.1% salicin (dissolved in 50mM citrate buffer, pH 5) in a 96-well plate at 50 ℃ for 30 minutes; then, 100 μl DNS was added to the mixture to terminate the reaction; the reaction mixture was boiled for another 10 minutes, then cooled at room temperature, and finally, the OD value of the mixture was measured at 540 nm using a microplate reader.
Positive mutants with higher OD values were selected from the second round of screening for further screening and inoculated into liquid basal medium containing 25 mL uracil-free medium and incubated at 30 ℃ for 2 days. Then, the bacterial liquid is centrifuged for 15 minutes at 10000g to obtain supernatant, and mutants V3-4-H6 with higher activity and high organic acid tolerance are screened according to a DNS method, wherein the amino acid sequence of the mutants V3-4-H6 is shown as SEQ ID NO:2, the DNA sequence of which is shown as SEQ ID NO: 3.
Example 2
Enzymatic property detection of beta-glucosidase mutant V3-4-H6 of Penicillium oxalicum 16
Measurement of enzyme activity: taking 50 mu L of diluted enzyme solution (original 16BGL and mutant V3-4-H6), adding 450 mu L of 0.1% salicin (salicin is dissolved in pH 5.0 and 50 mM of citric acid buffer solution in advance), uniformly mixing, reacting at 50 ℃ for 30 min, adding 500 mu L of DNS reagent to terminate the reaction, boiling the reactant in boiling water for 10 min immediately, and detecting an OD value at 540 nm by using a spectrophotometer after cooling to room temperature, wherein the result is shown in Table 1.
TABLE 1 OD of primordial enzyme and mutant V3-4-H6 540
Determination of the optimum temperature: the beta-glucosidase and its mutant V3-4-H6 are respectively reacted with 50mM citric acid buffer solution containing salicin, pH 5 is reacted at different temperatures (35-80 ℃ for 30 minutes), and the activity is measured by DNS method, the result is shown in figure 1, and as can be seen from figure 1, the reaction temperature of the beta-glucosidase mutant V3-4-H6 is 65 ℃; from FIGS. 2 and Table 2, it is seen that the specific activity without adding acid is 0.0165 IU/mg, the specific activity after incubation with 10 mg/mL of formic acid is 0.012 IU/mg, and the formic acid tolerance of V3-4-H6 is significantly enhanced to 1.64 times of the original 16BGL, although the catalytic activity is only 0.43 times of the original 16 BGL.
TABLE 2 relative specific Activity of the original enzyme and mutant V3-4-H6 without acid and relative specific Activity after incubation with 10 mg/mL formic acid
| Enzymes | Specific activity (IU/mg) | Specific activity after formic acid incubation (IU/mg) |
| 16BGL | 0.0386 | 0.0073 |
| V3-4-H6 | 0.0165 | 0.012 |
Determination of organic acid tolerance: 50. Mu.L of diluted enzyme solution is taken, 5.5 mu.L of 100 mg/mL formic acid (final concentration of formic acid is 10 mg/mL) is added, the mixture is incubated in a water bath at 37 ℃ for 1h, 5 mu.L of 2 mol/L NaOH is added, and 439.5 mu.L of salicin with different concentrations (the salicin is dissolved in a citrate buffer solution with pH of 5.0 and 50mM in advance) is added. After reaction at 50 ℃ for 30min, 500 μl DNS reagent was added to the mixture to terminate the reaction, and the OD of the mixture was obtained at 540 nm, with final concentrations of salicin of 0.2 mmol/L, 0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L, 1.0 mmol/L, and 1.5 mmol/L, respectively. V max,Km and K I (formic acid inhibition constants) were calculated from the Lineweaver-Burk reciprocal plot using (1) the Michaelis-Menten equation and (2) the non-competitive inhibition equation.
（1）
（2）
As a result, the results are shown in Table 3, and the formic acid inhibition strength K m/KI was 1.17X10- -2 (final formic acid concentration 10 mg/mL), specifically, the K m/KI value of 16BGL was 1.9 times that of V3-4-H6, and this data reflects that the tolerance of V3-4-H6 in formic acid environment was superior to 16BGL. It is described that in a high organic acid concentration production environment, it can maintain good stability and activity, thereby improving production efficiency and product quality.
TABLE 3 molecular dynamics constants and formic acid inhibiting molecular dynamics constants
Example 3
Determination of beta-glucosidase mutant V3-4-H6 of Penicillium oxalicum 16 for glucose production
And under the condition of no acid addition, measuring the glucose content of the bran residue after enzymolysis pretreatment: taking 3.8 mg protease liquid, adding 250 mu L of trichoderma reesei residue enzyme liquid and 0.2 g of pretreated bran residues, carrying out water bath 2h at 50 ℃, and finally adding 500 mu L of DNS, and carrying out boiling water bath 10min to terminate the reaction. After cooling to room temperature, the OD was measured at 540 nm using a spectrophotometer.
10 After incubation with mg/mL formic acid, glucose content of the bran residue after enzymolysis pretreatment is determined: taking 3.8 mg enzyme liquid, adding 27.5 mu L of 100 mg/mL formic acid, incubating at 37 ℃ for 1h, and adding 25 mu L of 2mol/L NaOH to adjust the pH to about 5.0. Subsequently 250 μl of trichoderma reesei residue enzyme solution and 0.2g of pretreated bran residue were added, the mixture was water-bath at 50 ℃ 2h, and finally 500 μl DNS was added, and the reaction was stopped in a boiling water bath 10 min. After cooling to room temperature, the OD was measured at 540 nm using a spectrophotometer.
The detection result is shown in fig. 3, and as can be seen from fig. 3, the content of glucose generated when the beta-glucosidase mutant V3-4-H6 is not added with acid is 687.3 mu g, the content of glucose generated when 10 mg/mL formic acid is added for treatment is 511.2 mu g, and the content of glucose generated by bran residues after enzymolysis pretreatment is only 0.69 times of the original 16BGL, but the content of glucose generated when the mutant V3-4-H6 is added with formic acid is 2.01 times of the original 16 BGL. The method has potential application value in the fields of acid environment for industrial production of alcohol or glucose, fruit juice preparation, brewing process, feed additives and the like.
While the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any such detail or embodiments or any particular embodiment, but is to be construed as providing broad interpretation of such claims by reference to the appended claims in view of the prior art so as to effectively encompass the intended scope of the invention. Furthermore, the foregoing description of the invention has been presented in its embodiments contemplated by the inventors for the purpose of providing a useful description, and for the purposes of providing a non-essential modification of the invention that may not be presently contemplated, may represent an equivalent modification of the invention.
Claims (5)
1. The beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 is characterized in that the amino acid sequence of the beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 is shown in SEQ ID NO: 2.
2. A gene of the beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 according to claim 1, wherein the gene sequence is as set forth in SEQ ID NO: 3.
3. A method for preparing the β -glucosidase mutant V3-4-H6 of penicillium oxalicum 16 according to claim 1, comprising the steps of:
error-prone PCR is carried out on the beta-glucosidase gene of the penicillium oxalicum 16, then a PCR product is connected with a vector pYAT BY using a seamless cloning kit, and then the PCR product is converted into an expression host saccharomyces cerevisiae BY4741 BY using a yeast conversion kit, so that a recombinant strain containing the beta-glucosidase mutant gene of the penicillium oxalicum 16 is obtained; and finally, placing the recombinant strain in a culture medium for culture, expression and screening to finally obtain the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16.
4. The use of the beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 according to claim 1 for enzymatic hydrolysis of bran to glucose, wherein the beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 is treated with formic acid and the bran is enzymatically hydrolyzed to glucose.
5. A method for producing glucose by enzymatic hydrolysis of bran, comprising the steps of:
Placing formic acid into beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16, incubating at 37 ℃, adding NaOH solution after incubation to adjust pH to 5.0, adding Trichoderma reesei residue enzyme solution and bran residue, and performing water bath reaction to obtain glucose; the amino acid sequence of the beta-glucosidase mutant V3-4-H6 of the penicillium oxalicum 16 is shown in SEQ ID NO: 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410573800.6A CN118147115A (en) | 2024-05-10 | 2024-05-10 | Beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410573800.6A CN118147115A (en) | 2024-05-10 | 2024-05-10 | Beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118147115A true CN118147115A (en) | 2024-06-07 |
Family
ID=91297021
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410573800.6A Pending CN118147115A (en) | 2024-05-10 | 2024-05-10 | Beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118147115A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120077216A1 (en) * | 2009-06-16 | 2012-03-29 | Codexis, Inc. | Beta-Glucosidase Variants |
| US20150218593A1 (en) * | 2012-09-05 | 2015-08-06 | Proteus | Polypetide with Reinforced Beta-Glucosidase Activity at Low Temperature |
| CN111518792A (en) * | 2020-05-26 | 2020-08-11 | 江西师范大学 | Beta-glucosidase mutant TLE of penicillium oxalicum 16 and application thereof |
| CN111621490A (en) * | 2020-05-26 | 2020-09-04 | 江西师范大学 | Beta-glucosidase mutant SVS of penicillium oxalicum 16 and application thereof |
-
2024
- 2024-05-10 CN CN202410573800.6A patent/CN118147115A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120077216A1 (en) * | 2009-06-16 | 2012-03-29 | Codexis, Inc. | Beta-Glucosidase Variants |
| US20150218593A1 (en) * | 2012-09-05 | 2015-08-06 | Proteus | Polypetide with Reinforced Beta-Glucosidase Activity at Low Temperature |
| CN111518792A (en) * | 2020-05-26 | 2020-08-11 | 江西师范大学 | Beta-glucosidase mutant TLE of penicillium oxalicum 16 and application thereof |
| CN111621490A (en) * | 2020-05-26 | 2020-09-04 | 江西师范大学 | Beta-glucosidase mutant SVS of penicillium oxalicum 16 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张念等: "草酸青霉16 β⁃葡萄糖苷酶的异源表达", 江西师范大学学报（自然科学版）, vol. 47, no. 3, 31 May 2023 (2023-05-31), pages 237 - 241 * |
| 黄秋霞: "β-葡萄糖苷酶活性和有机酸耐受性的定向进化", 中国优秀硕士学位论文全文数据库基础科学辑, no. 03, 15 March 2022 (2022-03-15), pages 006 - 296 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| da Silva Delabona et al. | Using Amazon forest fungi and agricultural residues as a strategy to produce cellulolytic enzymes | |
| KR20110119386A (en) | Gene coding for cellulase from bacillus velezensis a-68 and production method of cellulase by transformed escherichia coli a-68 thereof | |
| WO2020073866A1 (en) | Glucoamylase tlga15 and gene and application thereof | |
| JP2012235729A (en) | Alcohol fermentative yeast and ethanol production method using the same | |
| WO2023236638A1 (en) | Thermal stability-improved glucose oxidase goxm10 mutant e361p, derivative mutant thereof, and use thereof | |
| CN111621490B (en) | Beta-glucosidase mutant SVS of penicillium oxalicum 16 and application thereof | |
| CN111518792A (en) | Beta-glucosidase mutant TLE of penicillium oxalicum 16 and application thereof | |
| CN107129976B (en) | Xylanase, coding gene thereof and application thereof | |
| CN110373403B (en) | High-temperature-resistant neutral pullulanase and application thereof | |
| CN117402858B (en) | Beta-glucosidase mutant with improved heat resistance | |
| Singh et al. | Evaluation and characterization of new α-L-rhamnosidase-producing yeast strains | |
| Jmel et al. | Efficient enzymatic saccharification of macroalgal biomass using a specific thermostable GH 12 endoglucanase from Aspergillus terreus JL1 | |
| Zhao et al. | Influence of cellulase gene expression and cellulolytic activity on cellulose utilization in different Volvariella volvacea strains | |
| Thomas et al. | Identification and characterization of a highly alkaline and thermotolerant novel xylanase from Streptomyces sp. | |
| CN107201351B (en) | Preparation method and application of amylase mutant | |
| US8227220B2 (en) | Process for the preparation of ethanol from starch | |
| CN114045293B (en) | Gene Aokap1 for improving kojic acid yield of aspergillus oryzae, method and application | |
| CN118147115A (en) | Beta-glucosidase mutant V3-4-H6 of penicillium oxalicum 16 and application thereof | |
| Finkelman | Yeast strain development for extracellular enzyme production | |
| CN114736881B (en) | Glucose oxidase GoxM10 mutant A4D with improved acid stability and derivative mutant and application thereof | |
| Parmar et al. | Isolation, purification and characterization of carboxymethyl cellulase (CMCase) from psychrotolerant yeast Rhodotorula mucilaginosa BPT1 | |
| Cristóbal et al. | Isolation and molecular characterization of Shewanella sp. G5, a producer of cold‐active β‐D‐glucosidases | |
| CN101280290B (en) | Genetic engineering bacteria for producing high-thermal stability recombinant beta-glucanase and construction thereof | |
| KR100767383B1 (en) | New starch utilizing yeast and use thereof | |
| Kham et al. | A Thermotolerant yeast Cyberlindnera rhodanensis DK isolated from Laphet-so capable of extracellular thermostable β-glucosidase production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |