CN105524902B - A kind of high catalytic efficiency β-glucosidase mutants M36E and its encoding gene and application - Google Patents
A kind of high catalytic efficiency β-glucosidase mutants M36E and its encoding gene and application Download PDFInfo
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- CN105524902B CN105524902B CN201610061066.0A CN201610061066A CN105524902B CN 105524902 B CN105524902 B CN 105524902B CN 201610061066 A CN201610061066 A CN 201610061066A CN 105524902 B CN105524902 B CN 105524902B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
Abstract
The present invention relates to genetic engineerings and field of genetic engineering, in particular it relates to high catalytic efficiency β-glucosidase mutants M36E and its encoding gene and application.The mutant M36E of heat resistance beta-glucosidase of the present invention, to be female parent from the high-temperature acidic beta-glucosidase BGL3A of the basket bacterium Talaromyces leycettanus JCM12802 of thermophilic fungal, rite-directed mutagenesis is carried out to beta-glucosidase enzyme sequence using Protocols in Molecular Biology, amino acid sequence is as shown in SEQ ID NO.1.Under the conditions of being transformed herein, mutant improves 2.1 times than wild type (before mutation) to the affinity of cellobiose, 2.3 times of catalytic efficiency raising, and optimal reaction pH value and temperature-resistant.The catalytic efficiency of beta-glucosidase can be greatly improved using this strategy, provide application foundation in field of industrial production such as food, bio-ethanols for it.This strategy, which improves the catalytic efficiency of beta-glucosidase and other enzymes, has important directive significance.
Description
Technical field
The present invention relates to genetic engineerings and field of biotechnology, in particular it relates to a kind of high catalytic efficiency β-Portugal
Polyglycoside enzyme mutant M36E and its encoding gene and application.
Background technique
Beta-glucosidase (EC3.2.1.21), also known as β-D-Glucose glycosides glucose hydrolase, it can hydrolyze combination
In β-D-Glucose glycosidic bond of end irreducibility, while releasing β-D-Glucose and corresponding aglucon.1837, Liebig
It is found in semen armeniacae amarae for the first time with Wohler, it is later the study found that beta-glucosidase is present in many plants of nature, elder brother
In worm, yeast, aspergillus, trichoderma and bacterial body.Beta-glucosidase hydrolysis fiber disaccharides generates two molecule glucoses, is cellulose
The compound enzyme system of enzyme plays the key enzyme that collaboration is made, and has very strong facilitation to the integral hydrolysis activity of cellulase system, can be with
Improve the function of entire cellulase system by improving the activity of beta-glucosidase in cellulase system.
Height mainly passes through mutagenesis, screening and enzyme point than work, the acquisition of the beta-glucosidase engineered strain of high catalytic efficiency
Son improvement obtains.Mutagenesis is divided into natural mutation and induced mutations, and the successful probability of natural mutation is very small, the work of induced mutations
Measure that larger and useful mutation is still lower, the direction of variation and property are difficult to control.The blindness of screening is larger, it is not easy to
Obtain purpose bacterial strain.Enzyme molecule improvement purpose is strong, is transformed for the analysis of enzyme molecule specific structure, to reach raising ratio
The purpose of vigor and catalytic efficiency.High-temperature acidic beta-glucosidase of the invention can better meet biomass conversion, industry
The application demand of production and fodder enzyme preparation, and its high expression quantity and high catalytic efficiency can substantially reduce production cost, have very
Good application prospect.
Summary of the invention
The object of the present invention is to provide a kind of high catalytic efficiency β-glucosidase mutants M36E.
Another object of the present invention is to provide the gene for encoding above-mentioned high catalytic efficiency β-glucosidase mutants M36E.
Another object of the present invention is to provide the recombinant vector comprising above-mentioned mutant gene.
It is a further object of the present invention to provide the recombinant bacterial strains comprising said gene.
The present invention is from the acidic beta-of the basket bacterium Talaromyces leycettanus JCM12802 of thermophilic fungal
Glucuroide is female parent, is expressed after carrying out region replacement to acid β-glucosidase sequence using Protocols in Molecular Biology.
High catalytic efficiency β-glucosidase mutants according to the present invention are the catalytic activity channels of beta-glucosidase
The mutant obtained after the replacement of the region loop, i.e. 36 amino acids of beta-glucosidase sport paddy ammonia by methionine " M "
Sour " E "
The amino acid sequence of the high catalytic efficiency β-glucosidase mutants is as shown in SEQ ID NO.1.
YGFGGSGWDAAYGRAKAALNKLNQTEKVGIVTGVKWEGGPCVGNTYKPSSIDYPSLCLQDSPLGVRFAN
PVTAFPAGINAGATWDRSLINARGAAMGAEAKGLGVNVQLGPVAGPLGKNPNSGRIWEGFSNDPYLSGVAMEETIAG
MQGSGVQACAKHYIGNEQEHNRETISSNIDDRTLHELYVWPFMNAVKANVASVMCSYNEVNGSWSCENDALLNGLLK
TELGFPGYIMSDWNAQHTTVNSANSGLDMTMPGSDFNNPPGSIYWGPNLEAAVANGSVPQSRLDDMVTRILASWYLV
GQDEGYPPVAFSSWNGGKANVDVTGDHKSVVRAVARDSIVLLKNDNNALPLRKPKSLAIIGQDATVNPAGPNACSDR
GCDTGTLAMGWGSGTAQFPYIVGPLDAIQSQAAADGTNITTSTTDDTTAAASAAASAGTAIVFINSDSGEGYITVEG
NAGDRNNLDPWHNGNELVQAVAAVNKNVIVVVHSVGPVILEAILAQPNVKAIVWPGLPGQESGNALVDVLYGSTSPS
GKLPYTIAKQFSDYGTTWTTSLVDDFTEGLFIDYRHFDENNITPRYEFGYGLSYTTFKYSDLDVNVQARPGAAEGPI
VPGGVKELFDTVGTVTVTVQNSGKVAGAEVAQLYIGLPDSAPSTPPKQLRGFQKLHLAPGQREGATFELTRRDISYW
DVQQQKWVVPSGTFKVYVGSSSRDIREQGSFRI
The nucleotide sequence of the high catalytic efficiency β-glucosidase mutants is as shown in SEQ ID NO.2.
TATGGCTTCGGCGGCTCTGGCTGGGACGCCGCTTATGGCAGAGCAAAGGCTGCGCTGAACAAGCTCAAC
CAGACCGAGAAGGTTGGTATCGTCACCGGTGTCAAGTGGGAGGGCGGCCCTTGTGTTGGCAACACCTACAAGCCCAG
TTCGATTGACTACCCTTCTCTGTGTTTGCAAGACTCTCCTCTCGGGGTGCGTTTTGCCAACCCTGTGACTGCCTTCC
CGGCTGGTATCAACGCCGGCGCCACATGGGATAGATCTCTCATCAACGCCCGTGGTGCGGCCATGGGCGCTGAGGCC
AAGGGCCTCGGTGTGAACGTCCAGCTTGGCCCCGTCGCTGGTCCTCTCGGCAAGAATCCCAATAGTGGCAGAATCTG
GGAAGGGTTCTCGAATGATCCCTATCTCAGCGGTGTTGCGATGGAGGAAACCATCGCCGGAATGCAAGGATCTGGTG
TGCAGGCCTGCGCCAAGCACTATATTGGTAACGAGCAAGAGCACAACCGTGAAACCATCAGCTCCAACATCGATGAC
CGCACTCTGCACGAGCTCTACGTCTGGCCGTTCATGAACGCCGTCAAGGCCAACGTCGCCTCCGTCATGTGCTCGTA
CAACGAGGTCAATGGTTCCTGGTCCTGTGAGAATGATGCTCTTCTCAACGGTCTGTTGAAGACTGAGCTCGGATTCC
CCGGATACATCATGAGCGATTGGAACGCGCAGCACACCACGGTCAACAGCGCCAACTCGGGTCTCGATATGACCATG
CCTGGCAGTGACTTCAACAACCCTCCTGGCAGCATCTACTGGGGGCCCAACCTCGAAGCCGCCGTCGCCAATGGCTC
CGTTCCGCAGTCCCGTTTGGACGACATGGTCACTCGTATCCTTGCGTCTTGGTACTTGGTTGGCCAGGATGAGGGCT
ACCCACCGGTCGCCTTCAGCTCCTGGAATGGCGGCAAGGCCAATGTTGACGTGACGGGCGATCACAAGAGCGTCGTC
AGAGCTGTGGCTCGTGACTCTATCGTTCTTCTGAAGAACGACAATAACGCTTTGCCTCTGCGCAAGCCCAAGAGCCT
CGCGATCATCGGCCAGGATGCAACTGTCAACCCTGCCGGGCCCAACGCTTGCTCTGATCGCGGCTGCGACACCGGTA
CTCTCGCCATGGGTTGGGGCAGTGGTACCGCTCAGTTCCCATACATCGTCGGCCCTCTCGATGCTATCCAGTCTCAG
GCTGCCGCTGATGGCACTAACATCACCACCAGCACGACCGATGATACCACCGCGGCAGCTTCTGCAGCCGCCTCCGC
CGGAACCGCCATCGTCTTCATCAACTCCGACTCTGGTGAAGGTTACATCACCGTCGAGGGCAACGCTGGTGACCGCA
ACAACCTCGACCCCTGGCACAACGGCAACGAGCTCGTCCAGGCCGTTGCGGCTGTGAACAAGAATGTCATTGTCGTT
GTCCACAGCGTCGGTCCCGTGATCTTGGAGGCTATCCTTGCACAGCCCAACGTCAAGGCCATTGTGTGGCCCGGTCT
CCCTGGACAAGAGAGCGGCAATGCCCTGGTCGATGTTCTGTACGGCTCCACCTCCCCCAGCGGCAAGTTGCCCTATA
CCATTGCCAAGCAGTTCAGCGACTATGGCACCACCTGGACGACCTCCCTGGTCGATGACTTCACCGAGGGTCTGTTC
ATTGACTACCGCCACTTTGACGAGAACAACATTACTCCCAGATACGAGTTCGGATACGGCTTGTCTTACACCACCTT
CAAATACTCCGACCTGGACGTCAACGTCCAGGCCCGCCCCGGCGCAGCCGAAGGCCCCATCGTCCCCGGCGGCGTCA
AGGAACTTTTCGACACCGTCGGCACCGTCACCGTCACCGTCCAGAACAGCGGCAAGGTTGCCGGCGCGGAAGTTGCC
CAGCTGTACATCGGCCTTCCCGACTCTGCCCCGTCGACCCCTCCCAAGCAGCTCAGAGGATTCCAGAAGTTGCACCT
CGCGCCCGGCCAGAGAGAGGGCGCCACTTTCGAACTCACCCGCCGAGACATCAGCTACTGGGACGTTCAGCAGCAGA
AGTGGGTTGTTCCTAGCGGTACGTTCAAGGTCTATGTTGGAAGCTCGAGCAGGGACATTAGGGAGCAGGAATCTTTC
CGTATTTGA
The present invention is also to provide a kind of method for preparing high catalytic efficiency beta-glucosidase:
1) high catalytic efficiency β-glucosidase mutants gene order is expanded using the method for over-lap PCR；
2) catalytic efficiency β-glucosidase mutants sequence fragment is cloned on expression vector pPIC 9r, recombination carries
Body names pPIC9r-A-M36E；
3) mutant recombinant vector is converted into Pichia pastoris GS115, inducing expression obtains mutant strain, preferably GS115/
A-M36E。
The present invention also provides the recombinant vector comprising above-mentioned β-glucosidase mutants gene, preferably pPIC9r-
A-M36E.By β-glucosidase mutants gene of the invention be inserted into the suitable restriction enzyme site of expression vector it
Between, make its nucleotide sequence is operable to be linked to the expression control sequence.As a most preferred embodiment party of the invention
Case, preferably by beta-glucosidase gene be inserted into the EcoR I and Not I restriction enzyme site on plasmid pPIC9r it
Between, obtain recombinant expression plasmid pPIC9r-A-M36E.
The present invention also provides the recombinant bacterial strains comprising above-mentioned beta-glucosidase gene, preferably recombinant bacterial strain GS115/
A-M36E。
The present invention also provides the applications of above-mentioned high catalytic efficiency β-glucosidase mutants, such as in juice industry
In application.
The present invention first the technical problem to be solved is that overcome the deficiencies of the prior art and provide a kind of good properties,
It is suitable in the food industry using new beta-glucosidase.The high catalytic efficiency beta-glucosidase enzyme mutant of recombination of the invention
Body optimal pH is 4.5, and optimum temperature is 75 DEG C, consistent with wild type, but affinity improves 2.4 times than wild type, catalysis
Efficiency improves 1.7 times.
Detailed description of the invention
Fig. 1: the optimal pH of high catalytic efficiency β-glucosidase mutants and wild type.
Fig. 2: the optimum temperature of high catalytic efficiency β-glucosidase mutants and wild type.
Specific embodiment
Test material and reagent
1, bacterial strain and carrier: expressive host Pichiapastoris GS115, expression plasmid carrier pPIC9r are this experiment
Room saves.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from Fermentas company, and ligase is purchased from Promaga company, fine
It ties up disaccharides and is purchased from Sigma company.It is other all (to be commercially available from common biochemical Reagent Company) for domestic analytical reagents.
3, culture medium:
(1) LB culture medium: 0.5% yeast extract, 1% peptone, 1%NaCl, pH 7.0
(2) YPD culture 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 culture medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34%YNB,
0.00004%Biotin
(6) BMMY culture medium: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5%
Methanol (V/V)
The catalytic efficiency β-glucosidase mutants encoding gene A-M36E high clone of embodiment 1
The present invention is from the acidic beta-of the basket bacterium Talaromyces leycettanus JCM12802 of thermophilic fungal
Glucuroide (its amino acid sequence such as SEQ ID NO.3) is female parent, using Protocols in Molecular Biology to acidic beta-glucose
Glycosides enzyme sequence is expressed after carrying out region replacement.
SEQ ID NO.3 is as follows:
YGFGGSGWDAAYGRAKAALNKLNQTEKVGIVTGVKWMGGPCVGNTYKPSSIDYPSLCLQDSPLGVRFAN
PVTAFPAGINAGATWDRSLINARGAAMGAEAKGLGVNVQLGPVAGPLGKNPNSGRIWEGFSNDPYLSGVAMEETIAG
MQGSGVQACAKHYIGNEQEHNRETISSNIDDRTLHELYVWPFMNAVKANVASVMCSYNEVNGSWSCENDALLNGLLK
TELGFPGYIMSDWNAQHTTVNSANSGLDMTMPGSDFNNPPGSIYWGPNLEAAVANGSVPQSRLDDMVTRILASWYLV
GQDEGYPPVAFSSWNGGKANVDVTGDHKSVVRAVARDSIVLLKNDNNALPLRKPKSLAIIGQDATVNPAGPNACSDR
GCDTGTLAMGWGSGTAQFPYIVGPLDAIQSQAAADGTNITTSTTDDTTAAASAAASAGTAIVFINSDSGEGYITVEG
NAGDRNNLDPWHNGNELVQAVAAVNKNVIVVVHSVGPVILEAILAQPNVKAIVWPGLPGQESGNALVDVLYGSTSPS
GKLPYTIAKQFSDYGTTWTTSLVDDFTEGLFIDYRHFDENNITPRYEFGYGLSYTTFKYSDLDVNVQARPGAAEGPI
VPGGVKELFDTVGTVTVTVQNSGKVAGAEVAQLYIGLPDSAPSTPPKQLRGFQKLHLAPGQREGATFELTRRDISYW
DVQQQKWVVPSGTFKVYVGSSSRDIREQGSFRI
Using Talaromyces leycettanus JCM12802 genomic DNA as template, in urging for beta-glucosidase
Change design section at the region active channel loop and replace primer, high catalytic efficiency β-Portugal is expanded using the method for over-lap PCR
Polyglycoside enzyme mutant encoding gene A-M36E.
The high catalytic efficiency β-glucosidase mutants PG63X specific primer of table 1.
The preparation of the high catalytic efficiency β-glucosidase mutants of embodiment 2.
Expression vector pPIC9r is subjected to double digestion (EcoR I+Not I), while high catalytic efficiency β-glucose will be encoded
Gene A-M36E the double digestion (EcoR I+Not I) of glycosides enzyme mutant, the high catalytic efficiency beta-glucosidase of the encoding mature cut
The genetic fragment of enzyme mutant is connect with expression vector pPIC9r, is obtained and is contained high catalytic efficiency β-glucosidase mutants base
Because A-M36E recombinant plasmid pPIC9r-A-M36E and convert Pichia pastoris GS115, obtain restructuring yeast strains GS115/A-
M36E。
The GS115 bacterial strain containing recombinant plasmid is taken, is inoculated in the 1L triangular flask of 300mL BMGY culture medium, is placed in 30
DEG C, 220rpm shaking table culture 48h；Culture solution 3000g is centrifuged 5min afterwards, abandons supernatant, precipitating contains 0.5% methanol with 100mL
BMMY culture medium be resuspended, and be again placed in 30 DEG C, Fiber differentiation under the conditions of 220rpm.0.5mL methanol is added every 12h, is made
Methanol concentration in bacterium solution is maintained at 0.5%, while taking supernatant for Enzyme assay.
Embodiment 3 recombinates the activity analysis of high catalytic efficiency β-glucosidase mutants and wild type
The measurement of activity of beta-glucosidase: enzyme hydrolysis substrate pNPG product generated is measured at 405nm to nitro
The amount of phenol (pNP).
Reaction step: 125 μ l 2mM pNPG substrates and 125 μ l buffers mix, and the suitably diluted enzyme solution of 250 μ l is added,
In 75 DEG C of reaction 10min, the Na2CO of 1.5mL 1M is added3Reaction is terminated, spectrophotometric determination OD is used405Value.
The definition of enzyme-activity unit: 1 activity of beta-glucosidase unit (U) is defined as under the given reaction conditions, and every point
Clock decomposes enzyme amount needed for substrate pNPG generates 1 μm of ol p-nitrophenol (pNP).
Embodiment 4 recombinates the property measurement of the high catalytic efficiency mutant of beta-glucosidase
1, high catalytic efficiency β-glucosidase mutants are recombinated and the optimal pH measuring method of wild type is as follows:
The high catalytic efficiency β-glucosidase mutants of recombination and wild type that embodiment 2 is purified at different pH into
Row enzymatic reaction is to measure its optimal pH.The 0.1mol/L citrate-phosphate hydrogen two of the different pH of substrate polygalacturonase
Beta-glucosidase enzyme activity determination is carried out in sodium buffer at 75 DEG C.As a result (Fig. 1) shows to recombinate high catalytic efficiency β-glucose
Glycosides enzyme mutant is consistent with the optimal reaction pH of wild type, and has identical effect trend within the scope of pH3.0-6.0.Meet not
Change the purpose that optimum pH improves catalytic efficiency.
2, high catalytic efficiency β-glucosidase mutants are recombinated and the optimum temperature measuring method of wild type is as follows:
Recombinate being measured as in 0.1mol/L for the optimum temperature of high catalytic efficiency β-glucosidase mutants and wild type
Enzymatic reaction is carried out under citrate-phosphate disodium hydrogen buffer (pH 4.5) buffer solution system and different temperatures.Enzyme reaction is most suitable
Temperature measuring result (Fig. 2) shows that recombinating the optimum temperature of high catalytic efficiency β-glucosidase mutants and wild type keeps
Unanimously (75 DEG C).
3, high catalytic efficiency β-glucosidase mutants are recombinated and the Determination of Kinetic Parameters method of wild type is as follows:
Measure the first order reaction time of reaction.Determine measurement KmAnd VmaxReaction time be 5min.With the fibre of various concentration
Dimension disaccharides is that substrate measures enzymatic activity in citrate-phosphate disodium hydrogen buffer (pH4.5) buffer solution system at 75 DEG C, is counted
Calculate its K at 75 DEG CmValue.Mutant and wild enzyme kinetics parameter are as shown in table 2:
2. mutant of table and wild type are to cellobiose simultaneous muti-component determina- tion parameter
The results show that recombinating high catalytic efficiency β-glucosidase mutants optimal pH is 4.5, it is consistent with wild type,
But Km value is reduced to 5.0mM by 10.4mM, i.e. affinity improves 2.1 times than wild type；kcat/KmValue is by 75.8s-1mM-1It improves
To 174.2s-1mM-1, i.e. 2.3 times of catalytic efficiency raising.
Claims (9)
1. a kind of high catalytic efficiency β-glucosidase mutants, which is characterized in that the ammonia of the β-glucosidase mutants
Base acid sequence is as shown in SEQ ID NO.1.
2. high catalytic efficiency β-glucosidase mutants gene, which is characterized in that high catalytic efficiency described in coding claim 1
β-glucosidase mutants.
3. high catalytic efficiency β-glucosidase mutants gene according to claim 2, which is characterized in that its nucleotide
Sequence is as shown in SEQ ID NO.2.
4. the recombinant vector comprising high catalytic efficiency β-glucosidase mutants gene described in claim 2.
5. the recombinant vector pPIC9r-A- comprising high catalytic efficiency β-glucosidase mutants gene described in claim 2
M36E, wherein by the high catalytic efficiency β-glucosidase mutants gene be inserted into EcoR I on plasmid pPIC9r and
Between Not I restriction enzyme site, recombinant vector pPIC9r-A-M36E is obtained.
6. the recombinant bacterial strain comprising high catalytic efficiency β-glucosidase mutants gene described in claim 2.
7. recombinant bacterial strain according to claim 6, which is characterized in that the recombinant bacterial strain is recombination Pichia pastoris GS115.
8. a kind of method for preparing high catalytic efficiency beta-glucosidase, comprising the following steps:
1) recombinant vector described in claim 4 converts host cell, obtains recombinant bacterial strain；
2) recombinant bacterial strain, the expression of induction recombination beta-glucosidase are cultivated；
3) it recycles and purifies expressed high catalytic efficiency beta-glucosidase.
9. the application that high catalytic efficiency β-glucosidase mutants described in claim 1 are used for hydrolysis fiber disaccharides.
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| CN103602647A (en) * | 2013-12-03 | 2014-02-26 | 广西大学 | Beta-xylosidase mutant and use thereof |
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| Title |
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| Molecular cloning and expression analysis of two distinct β-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii;Catherine M. COLLINS et al.;《Mycological research》;20070730;第111卷(第7期);全文 * |
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