CN110438136A - The gene of beta-glucosidase and its mutant, amino acid sequence and application - Google Patents
The gene of beta-glucosidase and its mutant, amino acid sequence and application Download PDFInfo
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Abstract
The present invention relates to the gene of a kind of beta-glucosidase and its mutant, amino acid sequence and applications, belong to field of biotechnology.The albumen of the beta-glucosidase gene coding has the characteristic of glucose-tolerant, the mutant is that the phenylalanine of the 185th amino acids of N-terminal of the beta-glucosidase is replaced into tryptophan, glucose tolerance is improved, the beta-glucosidase and its mutant have broad application prospects in the exploitation of cellulolytic enzyme system.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of β-glucose of microbe-derived high glucose tolerance
The gene of glycosides enzyme and its mutant, amino acid sequence and application.
Background technique
Beta-glucosidase (EC 3.2.1.21) is also known as cellobiase, which can hydrolyze glucosides and oligosaccharides release is non-also
Former terminal saccharide, can also be with catalyzing hydrolysis back reaction or Transglycosylation, for a variety of glucosides such as synthesizing alkyl polyglycoside or oligosaccharides
Compound.Beta-glucosidase is the intracorporal functional endo enzyme of biology, is distributed widely in animal, in plant and microorganism,
Wherein microorganism also includes bacterium, archaeal, fungi and yeast etc..
The important application of one of beta-glucosidase is one of three enzymes in the compound enzyme system of cellulase, and cellulose is multiple
Synthase owner will be by Endo-β-glucanase, circumscribed 1,4 beta-glucanase (also known as cellobiohydrolase) and beta-glucosidase three
Kind enzyme composition.In hydrolyzation system, carried out first by Endo-β-glucanase in the intramolecular amorphous region of cellulose long-chain
Digestion generates new free-end, then by cellobiohydrolase as unit of cellobiose by newly generated free-end
It is hydrolyzed, cuts cellobiose molecule, it is finally by beta-glucosidase that the fiber of the cellobiose cut and short chain is few
Sugar is hydrolyzed to glucose.In hydrolytic process, a large amount of glucose is had accumulated, and the catalysis of existing many beta-glucosidases
Activity can be inhibited by reaction product glucose, lead to the decline of reaction rate, greatly limit their application.Therefore, in order to
Reduce Product inhibiton and improve cellulose percent hydrolysis, there is an urgent need to excellent catalysis characteristics and adapt to high glucose product
The beta-glucosidase of tired environment.
Summary of the invention
The technical problem to be solved in the present invention is that overcome the problems, such as that existing beta-glucosidase glucose tolerance is poor,
Provide the expression and application of a kind of high glucose tolerance beta-glucosidase and its mutant, the beta-glucosidase enzyme mutant
Body has higher glucose tolerance and thermal stability compared with wild type, and high glucose is resistant to beta-glucosidase and its dashes forward
Variant is hydrolyzed in compound enzyme system in cellulose and is had broad application prospects.
The present invention is achieved by the following technical solution:
The present invention provides a kind of nucleotide sequence of encoding beta-glucosidase, nucleotide sequence such as SEQ ID No:
Shown in 1.
A kind of above-mentioned nucleotide sequence coded beta-glucosidase, amino acid sequence is as shown in SEQ ID No:2.
A kind of recombinant expression carrier, it contains nucleotide sequence shown in SEQ ID No:1；Recombinant expression carrier is pET-
bgla。
A kind of recombinant bacterium, it contains nucleotide sequence shown in SEQ ID No:1, and the bacterial strain is e. coli bl21
(DE3)。
The present invention also provides the nucleotides sequences that a kind of coding improves the β-glucosidase mutants of glucose tolerance
Column, nucleotide sequence is as shown in SEQ ID No:3.
A kind of above-mentioned nucleotide sequence coded β-glucosidase mutants, amino acid sequence such as SEQ ID No:4
It is shown.
A kind of recombinant expression carrier, it contains nucleotide sequence shown in SEQ ID No:3；Recombinant expression carrier is pET-
f185w。
A kind of recombinant bacterium, it contains nucleotide sequence shown in SEQ ID No:3, and the bacterial strain is e. coli bl21
(DE3)。
β-glucosidase mutants have carried out a rite-directed mutagenesis compared with wild type, by β-shown in SEQ ID No:2
The phenylalanine (F) that the N-terminal of glucuroide plays the 185th amino acids is replaced into tryptophan (W), and glucose tolerance is aobvious
It writes and improves.
Using the method for above-mentioned beta-glucosidase and its nucleotide sequence preparation and reorganization beta-glucosidase of mutant,
Steps are as follows:
(1) the gene order SEQ ID No:1 and SEQ ID No:3 of beta-glucosidase and its mutant is cloned respectively
Into coli expression carrier pET-24a, recombinant expression carrier pET-bgla and pET-f185w are constructed, expression vector is turned
Change in e. coli bl21 (DE3), goes out the recombination containing beta-glucosidase or mutant gene with kanamycin resistance screening
Bacterial strain；
(2) strain inoculated for filtering out step (1) is in LB liquid medium, 37 DEG C culture 10-12 hours, planted
Sub- liquid；Seed liquor is inoculated in LB liquid medium with the amount of volume ratio 1%-2%, it is to be generated to grow to OD600When for 0.6-0.8,
The IPTG induction of final concentration 0.1-1mmol/L is added, inducing temperature is 16-37 DEG C, and induction time is 3-16 hours；
(3) thallus obtained after collection step (2) induction, after ultrasonication, using affinity column to recombinant beta-grape
Glycosidase and its mutant are purified, and electrophoretically pure recombination beta-glucosidase and its mutant are obtained.
The present invention also provides application of the above-mentioned recombination beta-glucosidase in cellobiose hydrolyzation system.
The present invention also provides application of the above-mentioned recombination β-glucosidase mutants in cellobiose hydrolyzation system.
Above-mentioned recombination beta-glucosidase and its mutant have following zymologic property:
(1) using chemical substrate PNPG as substrate when, the optimum temperature for recombinating beta-glucosidase is 40 DEG C, is in temperature
At 50 DEG C, enzyme activity only retains the 20% of highest enzyme activity；The optimum temperature for recombinating β-glucosidase mutants is 45 DEG C, In
Temperature still has the 60% of highest enzyme activity when being 50 DEG C.When reaction temperature is 60 DEG C, the vigor of two kinds of enzymes is lost.Recombinant beta-Portugal
Polyglycoside enzyme mutant has higher optimum temperature.
(2) when using PNPG as substrate, the optimal pH for recombinating beta-glucosidase is 7.5, and recombination beta-glucosidase is prominent
The optimal pH of variant is 8.
(3) beta-glucosidase and its mutant are recombinated at 4-30 DEG C, shows high stability；Recombinate beta-glucosidase
Thermal stability of the enzyme mutant at 40 DEG C is higher than wild type and recombinates beta-glucosidase；It is small more than 1 that temperature is higher than 45 DEG C of effects
When, the vigor for recombinating beta-glucosidase and its mutant is lost.
(4) it recombinates beta-glucosidase and its mutant is with good stability between pH range 7 to 9；Recombinant beta-
The optimal pH of glucuroide and mutant is respectively 7.5 and 8；It is stable under alkaline condition to recombinate β-glucosidase mutants
Property is higher.
(5) metal ion K+, Ca2+, Mn2+And work of the reducing agent DTT to the recombination beta-glucosidase and its mutant
Property has certain activation；Mg2+And Co2+It is smaller to the activity influence of recombination beta-glucosidase and its mutant；Zn2+,
Cu2+And surfactant SDS has high inhibition effect to recombination beta-glucosidase and its mutant；Metal chelating in reaction system
The addition of mixture EDTA has certain activation to recombination beta-glucosidase and its mutant, illustrates to recombinate beta-glucosidase
Enzyme and its mutant are not belonging to metal-binding protein.
(6) recombinating beta-glucosidase and its mutant has the characteristic of certain salt tolerant.It is added in the reaction system eventually
When concentration is the NaCl of 200mmol/L, recombinates beta-glucosidase and mutant keeps 56.1% He of highest vigor respectively
68%.The salt tolerance that beta-glucosidase is recombinated after mutation improves.
(7) it recombinates beta-glucosidase and its mutant is respectively to the half-inhibitory concentration IC50 of ethyl alcohol in system
16.3% and 14.7% (v/v), with the reported ethanol tolerance beta-glucosidase (IC50 from solution bacillus cellulosae
It is 15%) similar.The alcohol resistance of wild type recombination beta-glucosidase is slightly above mutant.
(8) recombinate beta-glucosidase and its mutant to the Km value rate of substrate cellobiose be respectively 16.4mmol/L and
193.9mmol/L, maximum reaction rate are respectively 22.9U/mg and 34.6U/mg.Wild type recombinates beta-glucosidase to substrate
Affinity be higher than mutant, and the maximum reaction rate of mutant is higher.
(9) recombination beta-glucosidase belongs to glucose-tolerant enzyme.In the presence of 100mmol/L glucose, have most to enzyme
Big activation；Activity keeps the 72.1% of highest vigor in the presence of 1000mmol/L glucose；Its inhibition constant Ki is
1292.3mmol/L.The glucose tolerance of recombination β-glucosidase mutants is greatly improved than wild type, In
Activity is barely affected in the presence of 1000mmol/L glucose, and inhibition constant Ki is 2573.1mmol/L, compares wild-type enzyme
1 times is improved, and glucokinase activator characteristic is kept.
The application of glucose-tolerant beta-glucosidase and its mutant of the present invention in cellobiose hydrolyzation system
Refer to:
Recombinate the cellobiose (100g/L) of beta-glucosidase and its mutant hydrolysis high concentration.It is small in 30 DEG C of reactions 8
Shi Hou, cellobiose hydrolysis reach balance, and the hydrolysis to cellobiose for recombinating beta-glucosidase and its mutant produces
Rate is respectively 90% and 70%.
The present invention compared with prior art the utility model has the advantages that
The present invention provides the gene order of a kind of beta-glucosidase and its mutant, utilizes the recombination of the gene expression
Beta-glucosidase and its mutant have stronger glucose tolerance compared with known enzyme, wherein mutant for glucose
Inhibition constant be doubled than wild type, hydrolyze in compound enzyme system and have broad application prospects in cellulose.
Detailed description of the invention
Fig. 1, the expression of recombination beta-glucosidase and its mutant and purifying electrophoretogram；(A) swimming lane M, albumen Marker；
Swimming lane 1, BL21 (DE3) cell pyrolysis liquid comprising pET-bgla；Swimming lane 2, the BL21 (DE3) comprising pET-bgla pass through IPTG
Cell pyrolysis liquid after induction；Swimming lane 3, recombination beta-glucosidase after purification；(B) swimming lane 1, the BL21 comprising pET-f185w
(DE3) cell pyrolysis liquid after IPTG is induced；Swimming lane 2, recombination β-glucosidase mutants after purification；Swimming lane M, albumen
Marker；
The temperature curve of Fig. 2, recombination beta-glucosidase and its mutant hydrolysis PNPG:: square block broken line is recombinant beta-
Glucuroide)；Dotted broken line recombinates β-glucosidase mutants)；
The pH curve of Fig. 3, recombination beta-glucosidase and its mutant hydrolysis PNPG: square block broken line is recombinant beta-grape
Glycosidase；Dotted broken line is recombination β-glucosidase mutants；
Fig. 4, the thermal stability curve for recombinating beta-glucosidase；
Fig. 5, the thermal stability curve for recombinating β-glucosidase mutants；
The pH stability curve of Fig. 6, recombination beta-glucosidase and its mutant: square block broken line is recombinant beta-glucose
Glycosides enzyme；Dotted broken line is recombination β-glucosidase mutants；
The influence of Fig. 7, salinity to recombination beta-glucosidase and its mutant: square block broken line is recombinant beta-glucose
Glycosides enzyme；Dotted broken line is recombination β-glucosidase mutants；
The influence of Fig. 8, ethyl alcohol to recombination beta-glucosidase and its mutant: square block broken line is recombination beta-glucosidase
Enzyme；Dotted broken line is recombination β-glucosidase mutants；
The tolerance of Fig. 9, recombination beta-glucosidase and its mutant for glucose: square block broken line is recombinant beta-grape
Glycosidase；Dotted broken line is recombination β-glucosidase mutants；
Figure 10, recombination beta-glucosidase and its mutant hydrolyze concentration cellulose disaccharides: square block broken line is recombinant beta-
Glucuroide；Dotted broken line is recombination β-glucosidase mutants.
Specific embodiment
Technical solution of the present invention is further explained below by embodiment, but protection scope of the present invention not by
The limitation of embodiment in any form.
The building of 1 beta-glucosidase gene expression vector of embodiment
Grown on picking 2216E plate marine bacteria L82 (J.Sun, W.Wang, C.Yao, F.Dai, X.Zhu,
J.Liu,J.Hao.Overexpression and characterization of a novel cold-adapted and
salt-tolerant GH1 β-glucosidase from the marine bacterium Alteromonas
Sp.L82.J.Microbiol.2018,56,656-664, the bacterial strain are deposited in China Aquatic Science Research Institute's Huanghai Sea aquatic products at present
Research institute, and freely provided to the public in 20 years from the applying date.) monoclonal is inoculated into 2216E fluid nutrient medium, 25 DEG C of trainings
It supports 2 days.Fresh bacterium solution is taken to extract genomic DNA, the DNA of extraction passes through 1 (5'-CG of primerGGATCCATGGATATATTGGCCG
) and primer 2 (5'-CCG AAGCAGGTTCT-3'CTCGAGAAGCATCTCTTTACGTTGCATGAGCAG-3') PCR is carried out (to draw
Line part is respectively restriction enzyme site BamH I and Xho I), obtain beta-glucosidase gene.
PCR system (LA Taq enzyme is purchased from TaKaRa)
PCR condition
1.94 DEG C, 5min
2.94 DEG C, 1min
55 DEG C, 30s
72 DEG C, 1min 30s
Circulation 25 times
3.72 DEG C, 5min
PCR product is connected to carrier T, carries out gene sequencing.Its open reading frame of post analysis is sequenced, finds β-glucose
Glycoside enzyme gene has 1347 bases, encodes 448 amino acid altogether.Theoretical relative molecular mass is 53KDa, and theoretical isoelectric point is
5.45.The beta-glucosidase similarity height that the sequence has zymologic property to report with oneself is derived from the resistance to Portugal of the macro genome in ocean
The beta-glucosidase of grape sugar, similarity 59.8%, the beta-glucosidase with the bacterial strain L82 another salt tolerant isolated
(J.Sun,W.Wang,C.Yao,F.Dai,X.Zhu,J.Liu,J.Hao.Overexpression and
characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase
From the marine bacterium Alteromonas sp.L82.J.Microbiol.2018,56,656-664) phase
It is 56.9% like degree.It is analyzed according to NCBI conserved amino acid sequences, the beta-glucosidase belongs to glycosyl hydrolase as the result is shown
1 family of enzyme.
After PCR product is carried out double digestion, it is connected on expression plasmid pET-24a, connexon is transformed into bacillus coli DH 5
In α, positive colony is gone out by antibiotic kanamycin screening, and carry out sequencing analysis, has beta-glucosidase to obtain
The recombinant expression carrier pET-bgla of new gene.
The building of 2 β-glucosidase mutants expression vector of embodiment
Using wild type beta-glucosidase gene as template, mutant gene is obtained by the method for bridging PCR, is mutated position
185 amino acids are set to, tryptophan are sported by phenylalanine, codon sports UGG by UUU.Bridging primer is primer P1
(5'-CGTTAAACGAACCTTGG) and P2 (5'-CGCTGC TGCAGCG-3'ACCAAGGTTCGTTTAACG-3') (dashed part
For mutational site).Firstly, by the primer 1 in primer P1 and embodiment 1, primer 2 in primer P2 and embodiment 1, PCR respectively
Two segments are obtained, PCR product recycles after carrying out agarose electrophoresis.Then, using the two segments as template, primer is carried out each other
After 10 recycle, primer 1 and primer 2 in embodiment 1 is added in PCR, then carries out 25 circulations, and the PCR product of acquisition is β-
Glucoside enzyme mutant gene.PCR system and PCR conditioned reference embodiment 1.
PCR product is connected to carrier T, DNA sequencing is carried out, verifies the sequence of mutant.PCR product is subjected to double digestion
Afterwards, it is connected on expression plasmid pET-24a, connexon is transformed into bacillus coli DH 5 alpha, passes through antibiotic kanamycin screening
Positive colony out, and sequencing analysis is carried out, to obtain the recombinant expression carrier for having β-glucosidase mutants gene
pET-f185w。
Embodiment 3 recombinates the preparation method of beta-glucosidase and its mutant
By recombinant plasmid transformed described in embodiment 1 and embodiment 2 in e. coli bl21 (DE3), β-glucose is obtained
The engineering strain of glycosides enzyme and its mutant.Bacterial strain is inoculated into the LB Liquid Culture containing 50 μ g/mL kanamycins respectively
In base, it is incubated overnight at 37 DEG C.1% culture solution is taken to be inoculated with, culture to OD600Value 0.6, be added IPTG to final concentration 0.2mM, 20
DEG C induction 16 hours.4 DEG C, 8500rpm is centrifuged 15min and collects thallus, and thallus is resuspended with 50mM phosphate buffer (pH 7).It is super
Sound smudge cells are crushed liquid at 4 DEG C, and 12000rpm is centrifuged 10min, collects supernatant, obtains crude enzyme liquid.Use equilibration buffer
Affinity column Ni-NTA Agarose is rinsed 5 column volumes by (50mM phosphate buffer, 500mM NaCl, pH 7), slightly
Enzyme solution enters pillar after 0.22 μm of membrane filtration, then uses 5 column volumes of Equilibration buffer wash, non-specific band logical
It crosses the equilibrium liquid containing 20mM imidazoles and rinses 5 column volumes to remove.Gradient is carried out with the equilibrium liquid of the imidazoles containing 20-250mM to wash
It is de-, obtain pure recombination beta-glucosidase and its mutant.SDS-PAGE detects purity of protein, and result is as shown in Figure 1.
BCA method measures protein concentration, using BSA as standard protein.
Embodiment 4 recombinates the zymologic property of beta-glucosidase and its mutant
The optimal reactive temperature for recombinating beta-glucosidase and its mutant is measured in 4-60 DEG C of temperature range, right
In chemical substrate 4- nitrophenols-beta-glucosidase (PNPG), the relationship of temperature and enzyme activity is as shown in Fig. 2, recombinant beta-glucose
Glycosides enzyme and mutant optimal reactive temperature are respectively 40 DEG C and 45 DEG C.Recombination beta-glucosidase and its optimal pH of mutant are
It is measured in the Britton-Robinson buffer of pH range 4-11.For substrate PNPG, the pass of pH and enzyme activity
System is as shown in figure 3, the optimal pH of recombination beta-glucosidase and mutant is respectively 7.5 and 8.Recombinate beta-glucosidase and its
The thermal stability of mutant is by acting on 3 hours pure enzyme in 4-50 DEG C of temperature range, and every 30min measures its remnant enzyme activity
Come what is realized, the thermal stability experimental result for recombinating beta-glucosidase and mutant is as shown in Figure 4 and Figure 5.Recombinant beta-grape
Glycosidase and its mutant show good stability at 0-30 DEG C.β-glucosidase mutants are recombinated at 40 DEG C
Thermal stability be higher than wild type recombinate beta-glucosidase；Temperature was higher than 45 DEG C of effects more than 1 hour, recombinated beta-glucosidase
The vigor of enzyme and its mutant is lost.It recombinates beta-glucosidase and its mutant pH stability experiment is by setting pure enzyme
After acting on 3 hours in the Britton-Robinson buffer of pH range 4-12, measure its remnant enzyme activity to realize.Experiment
As a result as shown in Figure 6.Beta-glucosidase and its mutant are recombinated in the buffer system of pH range 7-9, stability highest.
Different metal ions (the K of final concentration of 1mM is added in enzyme activity determination system+, Li+, Mg2+, Zn2+, Ca2+, Cu2 +, Co2+, Mn2+) and surfactant SDS, metal-chelator EDTA, reducing agent DTT, measure its remnant enzyme activity, experiment knot
Fruit is as shown in table 1.Metal ion K+, Ca2+, Mn2+And reducing agent DTT, metal-chelator EDTA are to recombination beta-glucosidase
And its activity of mutant has certain activation；Mg2+And Co2+To the activity of recombination beta-glucosidase and its mutant
It influences smaller；Li+There is certain inhibiting effect to recombination beta-glucosidase and its mutant；Zn2+, Cu2+And surface-active
Agent SDS has high inhibition effect to recombination beta-glucosidase and its mutant.
Influence of 1 chemical reagent of table to recombination beta-glucosidase and its mutant activity
It recombinates beta-glucosidase and its mutant is by the way that 0-2mol/L is added in live body system to the tolerance of salt
NaCl, its enzyme activity is measured to realize, result as shown in fig. 7, recombination beta-glucosidase and its mutant have it is certain
Salt tolerant characteristic.Beta-glucosidase and its mutant are recombinated to the tolerance of ethyl alcohol by the way that 0- is added in live body system
The ethyl alcohol of 30% (v/v), measures its enzyme activity to realize, result is as shown in Figure 8.Recombinate beta-glucosidase and mutant pair
The half-inhibitory concentration IC50 of ethyl alcohol is 16.3% and 14.7% (v/v) respectively in system.
Beta-glucosidase and its mutant is recombinated to join the dynamics of chemical substrate PNPG and natural substrate cellobiose
Number is as shown in table 2.The phosphate buffer (pH 7) that buffer is 50mM is measured, wherein PNPG concentration range is 0-16mM, fine
Dimension disaccharides concentration range is 0-200mM, recombinates beta-glucosidase and mutant measuring temperature is respectively 40 DEG C and 45 DEG C, pass through
Lineweaver-Burk method calculates Km, Vmax, kcatAnd kcat/KmEqual kinetic parameters.
Table 2 recombinates the kinetic parameter of beta-glucosidase and its mutant
Embodiment 5 recombinates the glucose tolerance of beta-glucosidase and its mutant
The glucose tolerance for recombinating beta-glucosidase and its mutant is by the way that 0-4mol/ is added in live body system
The glucose of L measures its enzyme activity to realize, result is as shown in Figure 9.The addition of 100mmol/L glucose is to recombinant beta-Portugal
Polyglycoside enzyme and its mutant have maximum activation effect.Recombination beta-glucosidase and the inhibition constant Ki of mutant are respectively
1292.3mmol/L and 2573.1mmol/L.Compared with existing beta-glucosidase, beta-glucosidase and its mutation are recombinated
Body belongs to glucose-tolerant enzyme, and wherein the glucose tolerance of mutant is apparently higher than wild type.
Embodiment 6 recombinates the application of beta-glucosidase and its mutant in cellobiose hydrolyzation system
Reaction system is the cellobiose of final concentration of 100g/L, 50mM phosphate buffer (pH 7) and 0.5mg/mL's
Beta-glucosidase or mutant are recombinated, reacts 8 hours, takes out per hour anti-in 30 DEG C, the constant-temperature table of revolving speed 180rpm
Liquid is answered, its percent hydrolysis is measured.Experimental result is as shown in Figure 10, and recombination beta-glucosidase is in first hour of reaction, hydrolysis
Rate has been more than 50%；After reaction carries out 8 hours, recombinates beta-glucosidase and mutant reaches the percent hydrolysis of cellobiose respectively
To 90% and 70%.Beta-glucosidase and its mutant are recombinated in cellobiose hydrolyzation system before application with higher
Scape.
Sequence table
<110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
<120>gene of beta-glucosidase and its mutant, amino acid sequence and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1413
<212> DNA
<213>beta-glucosidase (β-glucosidase)
<400> 2
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggatat attggccgaa 60
gcaggttctc cattcgcgca acgtgatttt acgattggcg tggccacgtc gtcgtttcag 120
attgaaggtg atgcagataa cagagagtta agcatctggg acactttctg taacaagccc 180
ggtaaaattg cagatgccag tcatggcctt gtggcctgtg accacgttaa tcgcatagaa 240
gaagacctct ctatcattaa cgccttaaat gttgatgcct atcgtttttc agtgagttgg 300
ccgcggataa taagacaaga tggtagcgtt aatcagcaag gcttagattt ttacggcaaa 360
attctagata cccttgcacg caacaatatt aaggcctacg tcactttgta tcactgggat 420
ttgcctcagt atttagaaga taccggcggg tggctgaata gaaagacagc cgacgcattt 480
gccaattatg tcgatgtcgt cacaaaagcc tttggtgaca gggttttttc ctacgcgacg 540
ttaaacgaac ctttttgcag cgcctattta ggttatgaaa ttggtgtgca cgcgccaggc 600
ttggtgggta aggaatatgg caaaaaagcc attcatcacc tattgttagc ccatggcaaa 660
gccatgccta ttatccgaaa aaatgcgcct aaggcagaag cggggatagt gctgaatttt 720
accccatttt attctaactc acccagcgaa gaggatatac gtgccacgaa actggctcac 780
gatcaccata acgactggta tataaagccg cttattcatg gtgagtatcc ctctcttatc 840
gagcaaatac cgccggcaca tcgtccggat attaaagacg gcgacatgga tattattgcg 900
gctcctttag actatttagg ggtgaattat tacacgcggg ctaaagtgaa agatgatggc 960
actagcgatc cgtgtcagct cccccctcct gaaggcagtg aaaccacggc aatggggtgg 1020
gaggtttatc cacaaggttt aacggattta cttgtacaac tgcatactga ttacacgtta 1080
ccgccgttgc tcattacaga aaatggcctc gcaagtgacg attttcttaa cgacgaaggt 1140
gaagttaacg atacgcagcg tattcgatat ttggccacgc acttacaggc ggtggccaac 1200
gcgatggaag ctggggtgaa tattacgggg tattttgtgt ggagcctgct agacaacttt 1260
gaatgggctt tagggtatga aaagcgattt ggcatagtct atgtggatta cagtacccaa 1320
aagcgcacgc taaaagcgag tgcaaagaaa ttacaaaacc tgctcatgca acgtaaagag 1380
atgcttctcg agcaccacca ccaccaccac tga 1413
<210> 2
<211> 470
<212> PRT
<213>beta-glucosidase (β-glucosidase)
<400> 2
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Asp
1 5 10 15
Ile Leu Ala Glu Ala Gly Ser Pro Phe Ala Gln Arg Asp Phe Thr Ile
20 25 30
Gly Val Ala Thr Ser Ser Phe Gln Ile Glu Gly Asp Ala Asp Asn Arg
35 40 45
Glu Leu Ser Ile Trp Asp Thr Phe Cys Asn Lys Pro Gly Lys Ile Ala
50 55 60
Asp Ala Ser His Gly Leu Val Ala Cys Asp His Val Asn Arg Ile Glu
65 70 75 80
Glu Asp Leu Ser Ile Ile Asn Ala Leu Asn Val Asp Ala Tyr Arg Phe
85 90 95
Ser Val Ser Trp Pro Arg Ile Ile Arg Gln Asp Gly Ser Val Asn Gln
100 105 110
Gln Gly Leu Asp Phe Tyr Gly Lys Ile Leu Asp Thr Leu Ala Arg Asn
115 120 125
Asn Ile Lys Ala Tyr Val Thr Leu Tyr His Trp Asp Leu Pro Gln Tyr
130 135 140
Leu Glu Asp Thr Gly Gly Trp Leu Asn Arg Lys Thr Ala Asp Ala Phe
145 150 155 160
Ala Asn Tyr Val Asp Val Val Thr Lys Ala Phe Gly Asp Arg Val Phe
165 170 175
Ser Tyr Ala Thr Leu Asn Glu Pro Phe Cys Ser Ala Tyr Leu Gly Tyr
180 185 190
Glu Ile Gly Val His Ala Pro Gly Leu Val Gly Lys Glu Tyr Gly Lys
195 200 205
Lys Ala Ile His His Leu Leu Leu Ala His Gly Lys Ala Met Pro Ile
210 215 220
Ile Arg Lys Asn Ala Pro Lys Ala Glu Ala Gly Ile Val Leu Asn Phe
225 230 235 240
Thr Pro Phe Tyr Ser Asn Ser Pro Ser Glu Glu Asp Ile Arg Ala Thr
245 250 255
Lys Leu Ala His Asp His His Asn Asp Trp Tyr Ile Lys Pro Leu Ile
260 265 270
His Gly Glu Tyr Pro Ser Leu Ile Glu Gln Ile Pro Pro Ala His Arg
275 280 285
Pro Asp Ile Lys Asp Gly Asp Met Asp Ile Ile Ala Ala Pro Leu Asp
290 295 300
Tyr Leu Gly Val Asn Tyr Tyr Thr Arg Ala Lys Val Lys Asp Asp Gly
305 310 315 320
Thr Ser Asp Pro Cys Gln Leu Pro Pro Pro Glu Gly Ser Glu Thr Thr
325 330 335
Ala Met Gly Trp Glu Val Tyr Pro Gln Gly Leu Thr Asp Leu Leu Val
340 345 350
Gln Leu His Thr Asp Tyr Thr Leu Pro Pro Leu Leu Ile Thr Glu Asn
355 360 365
Gly Leu Ala Ser Asp Asp Phe Leu Asn Asp Glu Gly Glu Val Asn Asp
370 375 380
Thr Gln Arg Ile Arg Tyr Leu Ala Thr His Leu Gln Ala Val Ala Asn
385 390 395 400
Ala Met Glu Ala Gly Val Asn Ile Thr Gly Tyr Phe Val Trp Ser Leu
405 410 415
Leu Asp Asn Phe Glu Trp Ala Leu Gly Tyr Glu Lys Arg Phe Gly Ile
420 425 430
Val Tyr Val Asp Tyr Ser Thr Gln Lys Arg Thr Leu Lys Ala Ser Ala
435 440 445
Lys Lys Leu Gln Asn Leu Leu Met Gln Arg Lys Glu Met Leu Leu Glu
450 455 460
His His His His His His
465 470
<210> 3
<211> 1413
<212> DNA
<213>β-glucosidase mutants (β-glucosidase)
<400> 3
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggatat attggccgaa 60
gcaggttctc cattcgcgca acgtgatttt acgattggcg tggccacgtc gtcgtttcag 120
attgaaggtg atgcagataa cagagagtta agcatctggg acactttctg taacaagccc 180
ggtaaaattg cagatgccag tcatggcctt gtggcctgtg accacgttaa tcgcatagaa 240
gaagacctct ctatcattaa cgccttaaat gttgatgcct atcgtttttc agtgagttgg 300
ccgcggataa taagacaaga tggtagcgtt aatcagcaag gcttagattt ttacggcaaa 360
attctagata cccttgcacg caacaatatt aaggcctacg tcactttgta tcactgggat 420
ttgcctcagt atttagaaga taccggcggg tggctgaata gaaagacagc cgacgcattt 480
gccaattatg tcgatgtcgt cacaaaagcc tttggtgaca gggttttttc ctacgcgacg 540
ttaaacgaac cttggtgcag cgcctattta ggttatgaaa ttggtgtgca cgcgccaggc 600
ttggtgggta aggaatatgg caaaaaagcc attcatcacc tattgttagc ccatggcaaa 660
gccatgccta ttatccgaaa aaatgcgcct aaggcagaag cggggatagt gctgaatttt 720
accccatttt attctaactc acccagcgaa gaggatatac gtgccacgaa actggctcac 780
gatcaccata acgactggta tataaagccg cttattcatg gtgagtatcc ctctcttatc 840
gagcaaatac cgccggcaca tcgtccggat attaaagacg gcgacatgga tattattgcg 900
gctcctttag actatttagg ggtgaattat tacacgcggg ctaaagtgaa agatgatggc 960
actagcgatc cgtgtcagct cccccctcct gaaggcagtg aaaccacggc aatggggtgg 1020
gaggtttatc cacaaggttt aacggattta cttgtacaac tgcatactga ttacacgtta 1080
ccgccgttgc tcattacaga aaatggcctc gcaagtgacg attttcttaa cgacgaaggt 1140
gaagttaacg atacgcagcg tattcgatat ttggccacgc acttacaggc ggtggccaac 1200
gcgatggaag ctggggtgaa tattacgggg tattttgtgt ggagcctgct agacaacttt 1260
gaatgggctt tagggtatga aaagcgattt ggcatagtct atgtggatta cagtacccaa 1320
aagcgcacgc taaaagcgag tgcaaagaaa ttacaaaacc tgctcatgca acgtaaagag 1380
atgcttctcg agcaccacca ccaccaccac tga 1413
<210> 4
<211> 470
<212> PRT
<213>β-glucosidase mutants (β-glucosidase)
<400> 4
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Asp
1 5 10 15
Ile Leu Ala Glu Ala Gly Ser Pro Phe Ala Gln Arg Asp Phe Thr Ile
20 25 30
Gly Val Ala Thr Ser Ser Phe Gln Ile Glu Gly Asp Ala Asp Asn Arg
35 40 45
Glu Leu Ser Ile Trp Asp Thr Phe Cys Asn Lys Pro Gly Lys Ile Ala
50 55 60
Asp Ala Ser His Gly Leu Val Ala Cys Asp His Val Asn Arg Ile Glu
65 70 75 80
Glu Asp Leu Ser Ile Ile Asn Ala Leu Asn Val Asp Ala Tyr Arg Phe
85 90 95
Ser Val Ser Trp Pro Arg Ile Ile Arg Gln Asp Gly Ser Val Asn Gln
100 105 110
Gln Gly Leu Asp Phe Tyr Gly Lys Ile Leu Asp Thr Leu Ala Arg Asn
115 120 125
Asn Ile Lys Ala Tyr Val Thr Leu Tyr His Trp Asp Leu Pro Gln Tyr
130 135 140
Leu Glu Asp Thr Gly Gly Trp Leu Asn Arg Lys Thr Ala Asp Ala Phe
145 150 155 160
Ala Asn Tyr Val Asp Val Val Thr Lys Ala Phe Gly Asp Arg Val Phe
165 170 175
Ser Tyr Ala Thr Leu Asn Glu Pro Trp Cys Ser Ala Tyr Leu Gly Tyr
180 185 190
Glu Ile Gly Val His Ala Pro Gly Leu Val Gly Lys Glu Tyr Gly Lys
195 200 205
Lys Ala Ile His His Leu Leu Leu Ala His Gly Lys Ala Met Pro Ile
210 215 220
Ile Arg Lys Asn Ala Pro Lys Ala Glu Ala Gly Ile Val Leu Asn Phe
225 230 235 240
Thr Pro Phe Tyr Ser Asn Ser Pro Ser Glu Glu Asp Ile Arg Ala Thr
245 250 255
Lys Leu Ala His Asp His His Asn Asp Trp Tyr Ile Lys Pro Leu Ile
260 265 270
His Gly Glu Tyr Pro Ser Leu Ile Glu Gln Ile Pro Pro Ala His Arg
275 280 285
Pro Asp Ile Lys Asp Gly Asp Met Asp Ile Ile Ala Ala Pro Leu Asp
290 295 300
Tyr Leu Gly Val Asn Tyr Tyr Thr Arg Ala Lys Val Lys Asp Asp Gly
305 310 315 320
Thr Ser Asp Pro Cys Gln Leu Pro Pro Pro Glu Gly Ser Glu Thr Thr
325 330 335
Ala Met Gly Trp Glu Val Tyr Pro Gln Gly Leu Thr Asp Leu Leu Val
340 345 350
Gln Leu His Thr Asp Tyr Thr Leu Pro Pro Leu Leu Ile Thr Glu Asn
355 360 365
Gly Leu Ala Ser Asp Asp Phe Leu Asn Asp Glu Gly Glu Val Asn Asp
370 375 380
Thr Gln Arg Ile Arg Tyr Leu Ala Thr His Leu Gln Ala Val Ala Asn
385 390 395 400
Ala Met Glu Ala Gly Val Asn Ile Thr Gly Tyr Phe Val Trp Ser Leu
405 410 415
Leu Asp Asn Phe Glu Trp Ala Leu Gly Tyr Glu Lys Arg Phe Gly Ile
420 425 430
Val Tyr Val Asp Tyr Ser Thr Gln Lys Arg Thr Leu Lys Ala Ser Ala
435 440 445
Lys Lys Leu Gln Asn Leu Leu Met Gln Arg Lys Glu Met Leu Leu Glu
450 455 460
His His His His His His
465 470
Claims (10)
1. a kind of nucleotide sequence of encoding beta-glucosidase, it is characterised in that the nucleotide sequence such as SEQ ID No:1
It is shown.
2. nucleotide sequence coded beta-glucosidase described in claim 1, it is characterised in that the beta-glucosidase
Amino acid sequence is as shown in SEQ ID No:2.
3. a kind of recombinant expression carrier, it is characterised in that the carrier contains SEQ ID No:1 nucleosides described in claim 1
Acid sequence；Recombinant expression carrier is pET-bgla.
4. a kind of recombinant bacterium, it contains recombinant expression carrier described in claim 3, and bacterial strain is e. coli bl21 (DE3).
5. a kind of β-glucosidase mutants, nucleotide sequence as shown in SEQ ID No:3, is dashed forward by SEQ ID No:1
Become.
6. nucleotide sequence coded β-glucosidase mutants described in claim 5, amino acid sequence such as SEQ ID
Shown in No:4.
7. a kind of recombinant expression carrier, it is characterised in that it contains SEQ ID No:3 nucleotides sequence described in claim 5
Column；Recombinant expression carrier is pET-f185w.
8. a kind of recombinant bacterium, it contains recombinant expression carrier as claimed in claim 7, and the bacterial strain is e. coli bl21
(DE3)。
9. application of the recombination beta-glucosidase in cellobiose hydrolysis described in claim 2.
10. application of the recombination β-glucosidase mutants in cellobiose hydrolysis described in claim 5.
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| CN111876399A (en) * | 2020-07-13 | 2020-11-03 | 中国水产科学研究院黄海水产研究所 | Arctic-pole-derived beta-glucosidase gene, and encoded protein and application thereof |
| CN113735942A (en) * | 2021-09-22 | 2021-12-03 | 山西大学 | Recombinant hypoglycemic polypeptide and preparation method and application thereof |
| CN114381448A (en) * | 2022-01-10 | 2022-04-22 | 鑫缘茧丝绸集团股份有限公司 | Glucanase mutant and application thereof |
| CN114703165A (en) * | 2022-04-02 | 2022-07-05 | 天津科技大学 | Beta-glucosidase mutant and application thereof |
| CN114752580A (en) * | 2022-05-25 | 2022-07-15 | 浙江工业大学 | Alpha-glycosidase mutant with improved transglycosylation activity, coding gene and application |
| CN114836482A (en) * | 2022-07-04 | 2022-08-02 | 云南英格生物技术有限公司 | Preparation method of oxyresveratrol |
| CN115710592A (en) * | 2023-01-09 | 2023-02-24 | 成都欧康医药股份有限公司 | Preparation process of 4',5, 7-trihydroxy flavanone |
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| CN111876399A (en) * | 2020-07-13 | 2020-11-03 | 中国水产科学研究院黄海水产研究所 | Arctic-pole-derived beta-glucosidase gene, and encoded protein and application thereof |
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| CN113735942A (en) * | 2021-09-22 | 2021-12-03 | 山西大学 | Recombinant hypoglycemic polypeptide and preparation method and application thereof |
| CN113735942B (en) * | 2021-09-22 | 2023-01-31 | 山西大学 | Recombinant hypoglycemic polypeptide and preparation method and application thereof |
| CN114381448A (en) * | 2022-01-10 | 2022-04-22 | 鑫缘茧丝绸集团股份有限公司 | Glucanase mutant and application thereof |
| CN114381448B (en) * | 2022-01-10 | 2024-02-20 | 鑫缘茧丝绸集团股份有限公司 | Glucanase mutant and application thereof |
| CN114703165A (en) * | 2022-04-02 | 2022-07-05 | 天津科技大学 | Beta-glucosidase mutant and application thereof |
| CN114752580A (en) * | 2022-05-25 | 2022-07-15 | 浙江工业大学 | Alpha-glycosidase mutant with improved transglycosylation activity, coding gene and application |
| CN114836482A (en) * | 2022-07-04 | 2022-08-02 | 云南英格生物技术有限公司 | Preparation method of oxyresveratrol |
| CN114836482B (en) * | 2022-07-04 | 2022-10-14 | 云南英格生物技术有限公司 | Preparation method of oxyresveratrol |
| CN115710592A (en) * | 2023-01-09 | 2023-02-24 | 成都欧康医药股份有限公司 | Preparation process of 4',5, 7-trihydroxy flavanone |
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