CN114703165B - Beta-glucosidase mutant and application thereof - Google Patents
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Abstract
The invention mainly utilizes a site-directed mutagenesis technology to construct a mutant strain of beta-glucosidase from humicola insolens, and obtains a beta-glucosidase mutant T331Q with improved glucose tolerance, and the amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID NO: 4. Under the same conditions, the mutant can reach the relative activity of 239.0% at the maximum under the activation of 400mmol/L glucose, and the relative activity can still reach 112.61% at the glucose concentration of 1.5mol/L, so that the mutant has good glucose tolerance. The mutant has higher application value in the fields of biofuel, food health care, biosynthesis and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to a beta-glucosidase mutant with improved glucose tolerance and application thereof.
Background
Beta-glucosidase (beta-Glucosidases EC 3.2.1.21), known as beta-D-glucosidase, belongs to the class of hydrolases, also known as cellobiase, gentiobinase or amygdalase. The enzyme can hydrolyze non-reducing beta-D-glycosidic bonds bound to the terminal end, releasing beta-D-glucose and the corresponding ligands. The beta-glucosidase has hydrolytic activity to various glycoside compounds, can act on various bioactive substances, and the ligand generated by hydrolysis of the beta-glucosidase is a functional aglycone or aromatic component; some beta-glucosidase also has transglycosylation activity and can be converted into functional oligosaccharides, so that the beta-glucosidase has wide application prospect in the fields of food health care, biological energy sources and the like.
Because the enzyme is always in a unfavorable environment in the actual production process, the beta-glucosidase is influenced by the product glucose, so that the production efficiency is low, and the substrate is not fully reflected. The inhibition of the products is usually relieved by adopting measures such as increasing enzyme amount, synchronous Saccharification and Fermentation (SSF), etc., but the methods have complex process, large pollution and higher cost. In order for the catalytic reaction to proceed efficiently, it is currently highly desirable to obtain beta-glucosidase with high glucose tolerance. At the same time, glucose tolerance is also becoming more and more important as a key property of beta-glucosidase in biofuel, food health care, biosynthesis and other production processes. Beta-glucosidase with high tolerance shows unique application potential because it can reduce the inhibition effect of glucose on enzyme activity. CN102220302a is based on beta-glucosidase from a marine uncultured microorganism source, and the mutant gene is obtained by PCR site-directed mutagenesis, the glucose tolerance concentration of the obtained mutant protein is improved by 13 times compared with wild type beta-glucosidase protein. CN103266096A is prepared by mutating tryptophan at 386 position of beta-glucosidase gene Pbgl into cysteine to obtain beta-glucosidase mutant Pbgl-W386C, and the tolerance of glucose is 11.88 times that of the mutant Pbgl-W386C before mutation, and is improved by 10.88 times. CN105754973a the beta-glucosidase mutant obtained by mutating tryptophan at position 233 on the beta-glucosidase S-bgl6 molecule of streptomyces into aspartic acid has a 208.9-fold improvement in glucose tolerance compared to before mutation. CN107142254A mutates the 315-site amino acid of beta-glucosidase from acidophilic heat-resistant alicyclic bacillus sp.A4 from histidine to arginine to obtain beta-glucosidase mutant with higher glucose tolerance, and under the same condition, the enzyme activity promotion effects of glucose on the mutant and wild type are 212% and 163%, respectively.
Disclosure of Invention
Aiming at the current industrial demand and the deficiency of the prior art, the invention mainly aims to construct a beta-glucosidase mutant with good glucose tolerance by a site-directed mutagenesis technology.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a beta-glucosidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 4.
In a second aspect, the invention provides a gene encoding said β -glucosidase mutant.
In a third aspect, the invention provides a vector comprising the gene of the β -glucosidase mutant.
In a fourth aspect, the present invention provides a recombinant strain comprising a gene or vector as described above.
In a fifth aspect, the present invention provides the use of a β -glucosidase mutant as described above for hydrolyzing β -D-glucosidic bonds at the non-reducing end of a glycoside or oligosaccharide compound, producing β -D-glucose and the corresponding ligand; and, the mutant has improved glucose tolerance compared to the wild type.
In a sixth aspect, the present invention provides a method for preparing the beta-glucosidase mutant as described above by gene recombination and expression using the gene encoding the beta-glucosidase mutant of the invention or the vector of the invention.
In a seventh aspect, the present invention provides a method for hydrolysing a glycoside compound, in particular cellobiose or cellooligosaccharide, comprising the step of contacting the glycoside compound with a β -glucosidase mutant according to the invention or with a recombinant strain according to the invention under conditions whereby β -D-glucosidic bonds of the compound can be hydrolysed by enzymatic catalysis of the β -glucosidase mutant or the recombinant strain.
The beneficial effects are that:
the beta-glucosidase mutant obtained by the site-directed mutagenesis technology is recombined and expressed in a pichia pastoris system by a genetic engineering technology, under the same condition, the relative activity of the mutant can reach 239.0% at most under the activation of 400mmol/L glucose, and when the glucose concentration is 1.5mol/L, the relative activity can still reach 112.61%, and compared with a wild type, the mutant has obviously improved glucose tolerance. The mutant has higher application value in the fields of biofuel, food health care, biosynthesis and the like.
Drawings
Fig. 1: PCR amplification electrophoretogram of wild-type beta-glucosidase gene in examples; wherein: 1 is a negative control, 2 is a DNA Marker, and 3 is a wild bglHi gene.
Fig. 2: the relative enzyme activities of the wild type and mutant at different concentrations of glucose activation are shown in the examples.
Fig. 3: purified protein electrophoresis verification of mutants in examples; wherein: 1 is protein Marker,2, 3 is recombinant strain GS115/pPIC9K-bglHiM, and 4 is control strain GS115/pPIC9K.
Fig. 4: amino acid sequence of the beta-glucosidase mutant of the invention.
Detailed Description
The invention is further described below by means of specific embodiments. Unless otherwise indicated, the technical means, materials, etc. to which the following embodiments relate may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
In a first aspect, the invention provides a beta-glucosidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 4.
In a second aspect, the invention provides a gene encoding said β -glucosidase mutant.
According to a preferred embodiment of the invention, the nucleotide sequence of the gene is as set forth in SEQ ID NO: 3.
In a third aspect, the invention provides a vector comprising the gene of the β -glucosidase mutant. The vector may be one of vectors known to those skilled in the art for producing proteins by gene recombination, such as an expression vector.
According to a preferred embodiment of the invention, the vector is a pPIC9K plasmid.
In a fourth aspect, the present invention provides a recombinant strain comprising a gene or vector as described above. The recombinant strain may be any host suitable for producing the β -glucosidase mutant of the invention from the gene or vector of the invention, e.g., pichia pastoris.
According to a preferred embodiment of the invention, the host is pichia pastoris GS115.
According to another embodiment of the present invention, the host is E.coli JM109.
In a fifth aspect, the present invention provides the use of a β -glucosidase mutant as described above for hydrolyzing β -D-glucosidic bonds at the non-reducing end of a glycoside or oligosaccharide compound, producing β -D-glucose and the corresponding ligand; and, the mutant has improved glucose tolerance compared to the wild type.
In a sixth aspect, the present invention provides a method for preparing the beta-glucosidase mutant as described above by gene recombination and expression using the gene encoding the beta-glucosidase mutant of the invention or the vector of the invention. Gene recombination methods and expression hosts known to those skilled in the art can be used, and the medium and culture conditions suitable for host expression are selected. The method may further comprise a step of recovering the beta-glucosidase mutant, which may involve a step of isolating or purifying the beta-glucosidase mutant from the culture or expression product of the host, using any method known to those skilled in the art.
According to a preferred embodiment of the invention, the mutant is prepared as follows: the mutant gene and plasmid pPIC9K are subjected to the same double digestion and connection by EcoR I and Avr II to obtain a recombinant vector, the recombinant vector is transformed into an escherichia coli host JM109, the plasmid is linearized by Sac I and finally electrically transferred into pichia pastoris GS115 to be expressed, and the protein is purified by an anion exchange column and the like and the enzyme activity is verified, so that the beta-glucosidase mutant with improved glucose tolerance compared with the wild type is obtained.
In a seventh aspect, the present invention provides a method for hydrolysing a glycoside compound, in particular cellobiose or cellooligosaccharide, comprising the step of contacting the glycoside compound with a β -glucosidase mutant according to the invention or with a recombinant strain according to the invention under conditions whereby β -D-glucosidic bonds of the compound can be hydrolysed by enzymatic catalysis of the β -glucosidase mutant or the recombinant strain.
The invention is defined as follows:
1. nomenclature of amino acids and DNA nucleic acid sequences
Amino acid residues are in the form of three letter abbreviations or single letter symbols using accepted IUPAC nomenclature. The DNA nucleic acid sequence uses accepted IUPAC nomenclature.
2. Identification of beta-glucosidase mutants
"amino acid substituted at the original amino acid position" is used to denote the mutated amino acid in the β -glucosidase mutant. The mutant is shown as SEQ ID NO:2, and the threonine (T) at position 331 in the amino acid sequence of the beta-glucosidase is replaced by glutamine (Q), which is represented by T331Q or Thr331 Gln. The gene mutation sites of the mutants were as follows:
the present invention will be described in more detail with reference to specific examples. In the following examples, unless otherwise specified:
the culture medium and the enzyme activity determination method used in the invention are as follows:
LB medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 18g/L of agar and other components are consistent.
MD solid medium: glucose 20g/L and agar 18g/L.
YPD medium: 20g/L of peptone, 10g/L of yeast powder, 20g/L of glucose, 18g/L of agar and other components are consistent.
YPG medium: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glycerol.
BMGY medium: 10g of yeast powder, 20g of peptone are dissolved in 700mL of water and sterilized at 121 ℃ for 20min, and 100mL pH 6.0,1mol/L of potassium phosphate buffer solution, 100mL of 10 XYNB, 2mL of 500 Xbiotin and 100mL of 10 Xglycerol are added at 60 ℃ during use.
BMMY medium: 10g of yeast powder, 20g of peptone are dissolved in 700mL of water and sterilized at 121 ℃ for 20min, 100mL of 1mol/L potassium phosphate buffer pH 6.0, 100mL of 10 XYNB, 2mL of 500 Xbiotin and 2.5mL of methanol are added at 60 ℃ during use.
MES buffer: 3.91g MES was weighed and dissolved in water, the pH was adjusted to 6.5 with Tris base solution, and water was added to a volume of 1L.
buffer a: the final concentration was 20mmol/L, pH 6.5.5 MES buffer and sterilized membrane was used.
buffer B: the final concentration of 20mmol/L, pH 6.5.5 MES buffer and the final concentration of 1mol/L NaCl solution are passed through a 0.22 μm sterilizing film for standby.
G418 screening media: melting YPD culture according to concentration (0.5 mg/mL,1mg/mL,2 mg/mL), adding at about 55deg.C, and mixing; other resistance selection media were added to the corresponding concentrations of resistance.
Competent preparation of Escherichia coli culture medium
Coli resuscitates, LB medium.
Wash medium: caCl is added into each liter 2 11.1g；
Resuspension medium: 1.5mL glycerol, 8.5mL 11.1g/L CaCl 2 A solution;
pichia pastoris competent preparation medium:
YPD medium: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
sterile water: sterilizing the film-coated water at 121 ℃ for 20 min;
1mol/L sorbitol: 18.21g of sorbitol is dissolved in every 100mL of deionized water, sterilized at 121 ℃ for 20min and stored at 4 ℃ for later use.
The enzyme activity detection of the beta-glucosidase used in the invention is mainly based on the ligand released by enzymolysis reaction and glucose, takes the artificial synthetic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPG) as a substrate, and hydrolysis products of the beta-glucosidase are p-nitrobenzene (pNP) and glucose, wherein the p-nitrobenzene has a specific light absorption value at 400 nm.
The specific detection method comprises the following steps: mixing 100. Mu.L of the properly diluted enzyme solution with 2.9mL of a substrate solution (pNPG is dissolved in disodium hydrogen phosphate-citric acid buffer solution, the final concentration is 3mmol/L, pH 5.0), reacting at 50℃for 4min, adding 1mL of 1mol/L Na 2 CO 3 The solution terminated the reaction. The control was an inactivated fermentation broth supernatant boiled at 100℃for 10min. Determination of the reaction solution OD 400 nm, and the content of pNP was calculated with reference to a standard curve. The amount of enzyme required to produce 1. Mu. Mol of p-nitrobenzene (pNP) per minute in the reaction system was defined as 1 enzyme activity unit (U).
The p-nitrobenzene (pNP) standard curve is as follows; 0.1mg/mL of paranitrophenol mother solution is prepared, diluted to the concentration of 0.002mg/mL, 0.004mg/mL, 0.006mg/mL, 0.008mg/mL and 0.010mg/mL respectively, sterile water is used as a blank, and the absorbance is measured at 400 nm. Taking an OD value as an ordinate and a pNP concentration (mg/mL) as an abscissa, making a pNP standard curve, wherein a linear regression equation Y=40.8X-0.026; r is R 2 ＝0.9988。
Wherein, before the enzyme solution and the substrate solution are mixed, the substrate solution needs to be preheated for more than 2 minutes in a water bath at 50 ℃.
The calculation formula of the enzyme activity of the beta-glucosidase comprises the following steps: enzyme activity s=x×v 1 ×1000×n/139×t×V 2
Wherein X is the amount of p-NP (mg/mL); v (V) 1 Is the volume (mL) of the reaction solution; v (V) 2 Is the volume (mL) of enzyme solution; n is the dilution factor of the enzyme solution; t is the reaction time (min).
Definition of enzyme activity: the amount of enzyme required to produce 1. Mu. Mol of p-nitrobenzene (pNP) per minute in the reaction system was defined as 1 enzyme activity unit (U) at 50 ℃.
Example 1: construction of wild-type beta-glucosidase bglHi recombinant strain
1.1 Synthesis and amplification of wild-type beta-glucosidase Gene bglHi
According to GenBank: KF588650.1, a wild type beta-glucosidase bglHi gene sequence derived from specific humicola (Humicola insolens) is obtained, and in order to be suitable for pichia pastoris expression, codon optimization is performed, and the optimized nucleotide sequence is shown as SEQ ID NO:1, the sequence was synthesized by the company entrusted with biosystems and amplified by PCR, and the primer sequences were as follows:
primer P1: F5'-ATGTCTTTGCCTCCAGACTTC-3';
primer P2: R5'-CGACTCTTTGATTAGAAAGGAGTAA-3';
amplifying by taking P1 and P2 as an upstream primer and a downstream primer and taking a wild type gene of bglHi as a template;
the reaction system for amplification is as follows:
| upstream primer P1 | 1.5μL |
| Downstream primer P2 | 1.5μL |
| DNA template | 2μL |
| PrimeSTAR enzyme | 25μL |
| ddH 2 O | 20μL |
The amplification procedure was set up as follows: pre-denaturation: 95 ℃ for 5min; denaturation: 98 ℃ for 10s; annealing: 20s at 50 ℃; extension: 72 ℃ for 10s; reacting for 32 cycles; extension: and at 72℃for 10min.
The PCR product is subjected to agarose gel electrophoresis, the band of the wild type beta-glucosidase gene can be seen, which is 1431bp (shown in figure 1), and then the PCR product is recovered by a small amount of DNA recovery kit, so that the wild type beta-glucosidase gene, namely bglHi, is obtained.
1.2 Linearization of expression vectors
The pPIC9K plasmid was extracted, and the extraction process was performed by referring to the manual of the kit. And (3) carrying out agarose gel electrophoresis after double digestion of EcoR I and Avr II, and recovering the product by a DNA gel recovery kit to obtain the linearized vector sequence.
1.3 Construction of recombinant strains
The target fragment (bglHi) and the vector fragment which are subjected to double digestion by EcoR I and Avr II are connected to form a recombinant plasmid pPIC9K-bglHi, the recombinant plasmid is transformed into escherichia coli JM109, and the sequence of the recombinant plasmid is verified to be shown as SEQ ID NO:1.
example 2: obtaining beta-glucosidase mutant by utilizing site-directed mutagenesis method
2.1, carrying out directional mutation based on a site-directed mutagenesis technology, constructing a novel beta-glucosidase mutant, and designing primers as follows:
primer P3:5'-CAGTTGTTCTACAACAAGTACGGTGATTGTATCGGTCCA G-3';
primer P4: R5'-CTCCAAGTTTCCCAAAAAGTCGTCCTC-3';
in a site-directed mutagenesis PCR reaction system, P3 and P4 are used as upstream and downstream primers, and a recombinant plasmid pPIC9K-bglHi is used as a template to carry out the site-directed mutagenesis PCR.
The reaction system for amplification is as follows:
| upstream primer P3 | 1.5μL |
| Downstream primer P4 | 1.5μL |
| DNA template | 2μL |
| PrimerSTAR enzyme | 25μL |
| ddH 2 O | 20μL |
The amplification conditions were: pre-denaturation: 95 ℃ for 5min; denaturation: 98 ℃ for 10s; annealing: 20s at 62 ℃; extension: 72 ℃ for 1min; reacting for 32 cycles; extension: and at 72℃for 10min.
2.2 the obtained linear plasmid with the beta-glucosidase mutant gene is self-connected into a ring to obtain a recombinant plasmid pPIC9K-bgl HiM, and the recombinant plasmid pPIC9K-bgl Hi and the recombinant plasmid pPIC9K-bgl HiM are transformed into pichia pastoris to obtain a recombinant strain GS115/pPIC9K-bgl HiM capable of expressing the beta-glucosidase mutant.
Example 3: verification of beta-glucosidase mutant
3.1 Shake flask verification
The recombinant strain obtained in example 2 was referred to "Pichia pastoris expression handbook", single colonies were streaked out from the recombinant yeasts with high copy number selected, and they were inoculated into 30mL YPG medium (containing kanamycin at a final concentration of 50. Mu.g/mL) with the single colonies of the control strain P.pastoris GS115/pPIC9K, respectively, and cultured at 30℃for 16-18h at 180 r/min; respectively inoculating the seed solution into 50mL BMGY liquid culture medium according to 2% inoculum size, culturing at 30deg.C and 180r/min to OD 600 Up to 5-6 (about 16-20 h); collecting bacterial liquid in sterilized 50mL centrifuge tube, centrifuging at 8000r/min for 5min, pouring out supernatant, re-suspending bacterial cells with 20mL BMMY culture medium, washing repeatedly for 3 times, and re-culturing with BMMY culture mediumSuspension cell (final cell concentration OD) 600 =1); and (3) after starving for 1h, adding an inducer methanol, adding 0.5% methanol every 24h to induce expression, and carrying out timed sampling analysis to obtain the beta-glucosidase enzyme solution.
And respectively measuring the glucose tolerance of the beta-glucosidase of the enzyme liquid, and comparing the tolerance of the mutant with the tolerance of the wild type beta-glucosidase under different glucose concentrations to obtain a strain with the 1 strain beta-glucosidase with the glucose tolerance obviously stronger than that of the wild type.
3.2 shake flask duplicate experiments, purification and enzyme Activity studies
The single colony of the recombinant strain and the single colony of the control strain P.pastoris GS115/pPIC9K are respectively inoculated into 30mL YPG culture medium (containing kanamycin with the final concentration of 50 mu g/mL) and cultured for 16-18h at the temperature of 30 ℃ and 180 r/min; respectively inoculating the seed solution into 50mL BMGY liquid culture medium according to 2% inoculum size, culturing at 30deg.C and 180r/min to OD 600 Up to 5-6 (about 16-20 h); collecting bacterial solution in sterilized 50mL centrifuge tube, centrifuging at 8000r/min for 5min, pouring out supernatant, cell washing with 20mL BMMY culture medium, repeating washing for 3 times, and finally re-suspending bacterial cells with BMMY culture medium (final bacterial cell concentration OD 600 =1); after starvation for 1h, the inducer methanol is added, 0.5% methanol is added every 24h to induce expression, and the culture is carried out for 96h. Placing the fermentation broth into a 50mL sterile centrifuge tube, centrifuging at 4deg.C and 12000r/min for 20min, and removing thalli; the supernatant of the fermentation broth is put into a 30kD ultrafiltration tube for ultrafiltration concentration after passing through a 0.22 mu m sterilization membrane, and is centrifuged at 4 ℃ for 60min at 5000r/min to prepare about 1mL of concentrated solution. Transferring the concentrated fermentation liquor into a dialysis bag for dialysis, putting the dialysis bag into a beaker filled with deionized water, and dialyzing at 4 ℃ under magnetic stirring to remove salt ions in the fermentation liquor. Placing the dialyzed enzyme solution into a 10mL centrifuge tube, centrifuging at 8000r/min and 4 ℃ for 10min, removing insoluble bottom impurities, and removing the insoluble impurities through a water-based film of 0.22 mu m; balanced anion exchange column (sources): 20mmol/L MES buffer as buffer A, washing the column with buffer A, washing 3-5 column volumes to equilibrate the anion exchange column; loading: loading the whole sample into the equilibrated column at a flow rate of 0.5mL/min, and opening with buffer AThe washing is started for 3-5 column volumes, and the collection of protein peaks is started from the completion of loading. After the sample was equilibrated, elution was performed with buffer B and a different gradient was set until 100% nacl concentration was reached. The elution flow rate was set at 1mL/min, and each time an elution peak occurred, the collection tube was replaced, and all elution peaks were collected and numbered separately. The purified enzyme solution was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 5% concentrated gel and 12% separated gel, and the results are shown in FIG. 3. Lane 4 of fig. 3 is the fermentation supernatant of the control strain GS115/pPIC9K, which has substantially no protein secretion, since pichia pastoris GS115 has been specifically engineered to secrete little or no extracellular protein, and lanes 2 or 3 are purified enzyme solutions of recombinant strain GS115/pPIC9K-bglHiM, which have a distinct protein band with an apparent molecular weight of about 60kDa. And (3) measuring the activity and glucose tolerance of the purified enzyme solution to obtain a beta-glucosidase mutant T331Q, wherein the activity of the beta-glucosidase can reach 239.0% at most under the activation of 400mmol/L glucose, and the activity can still reach 112.61% at the concentration of 1.5mol/L glucose (the enzyme activity is shown in figure 2).
Example 4: sequencing of beta-glucosidase mutant
The recombinant strain is used for extracting the beta-glucosidase gene sequence and sequencing (Jin Weizhi biotechnology limited company), and the result shows that the nucleotide sequence of the beta-glucosidase mutant gene is amplified to be shown as SEQ ID NO:3, the coding gene was designated bglHiM.
The amino acid sequence of the beta-glucosidase bglHiM obtained above is respectively compared with the amino acid sequence of the wild beta-glucosidase bglHi of SEQ ID NO:1, performing comparative analysis, and displaying the result: in comparison with the wild-type β -glucosidase bglHi, the amino acid at position 331 of the β -glucosidase bglHiM is mutated from Thr to Gln (as shown in fig. 4).
Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.
Sequence listing
<120> a beta-glucosidase mutant and use thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1431
<212> DNA
<213> Humicola insolens (Humicola insolens)
<400> 1
atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60
tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120
aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180
gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240
agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300
gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360
tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420
gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480
tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540
gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600
tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660
gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720
ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780
atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840
ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900
gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960
ccagaggacg actttttggg aaacttggag actttgttct acaacaagta cggtgattgt 1020
atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080
ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140
tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200
aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260
gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320
accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380
tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431
<210> 2
<211> 476
<212> PRT
<213> Humicola insolens (Humicola insolens)
<400> 2
Met Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala Tyr
1 5 10 15
Gln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp
20 25 30
Asp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly
35 40 45
Ala Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala Leu
50 55 60
Leu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp Ser
65 70 75 80
Arg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys Gly
85 90 95
Ile Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly Ile
100 105 110
Thr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu Asp
115 120 125
Lys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp Phe
130 135 140
Glu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys His
145 150 155 160
Trp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr Asn
165 170 175
Thr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser Pro
180 185 190
Val Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile Leu
195 200 205
Ile Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys Pro
210 215 220
Thr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr Leu
225 230 235 240
Pro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg Lys
245 250 255
Ile Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly Lys
260 265 270
Tyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu Phe
275 280 285
Thr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr Gly
290 295 300
Met Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val Pro
305 310 315 320
Pro Glu Asp Asp Phe Leu Gly Asn Leu Glu Thr Leu Phe Tyr Asn Lys
325 330 335
Tyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg Pro
340 345 350
His Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg Tyr
355 360 365
Gly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys Gly
370 375 380
Glu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg Val
385 390 395 400
Lys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala Glu
405 410 415
Asp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp Asn
420 425 430
Phe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr Val
435 440 445
Asp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys Ser
450 455 460
Leu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys Glu
465 470 475
<210> 3
<211> 1431
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60
tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120
aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180
gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240
agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300
gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360
tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420
gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480
tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540
gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600
tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660
gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720
ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780
atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840
ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900
gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960
ccagaggacg actttttggg aaacttggag cagttgttct acaacaagta cggtgattgt 1020
atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080
ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140
tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200
aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260
gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320
accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380
tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431
<210> 4
<211> 476
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Met Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala Tyr
1 5 10 15
Gln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp
20 25 30
Asp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly
35 40 45
Ala Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala Leu
50 55 60
Leu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp Ser
65 70 75 80
Arg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys Gly
85 90 95
Ile Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly Ile
100 105 110
Thr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu Asp
115 120 125
Lys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp Phe
130 135 140
Glu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys His
145 150 155 160
Trp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr Asn
165 170 175
Thr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser Pro
180 185 190
Val Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile Leu
195 200 205
Ile Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys Pro
210 215 220
Thr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr Leu
225 230 235 240
Pro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg Lys
245 250 255
Ile Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly Lys
260 265 270
Tyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu Phe
275 280 285
Thr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr Gly
290 295 300
Met Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val Pro
305 310 315 320
Pro Glu Asp Asp Phe Leu Gly Asn Leu Glu Gln Leu Phe Tyr Asn Lys
325 330 335
Tyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg Pro
340 345 350
His Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg Tyr
355 360 365
Gly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys Gly
370 375 380
Glu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg Val
385 390 395 400
Lys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala Glu
405 410 415
Asp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp Asn
420 425 430
Phe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr Val
435 440 445
Asp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys Ser
450 455 460
Leu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys Glu
465 470 475
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atgtctttgc ctccagactt c 21
<210> 6
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgactctttg attagaaagg agtaa 25
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
cagttgttct acaacaagta cggtgattgt atcggtccag 40
<210> 8
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ctccaagttt cccaaaaagt cgtcctc 27
Claims (9)
1. A beta-glucosidase mutant, which is characterized in that the amino acid sequence of the beta-glucosidase mutant is shown in SEQ ID NO: 4.
2. A gene encoding the β -glucosidase mutant of claim 1.
3. A recombinant vector comprising the gene of claim 2.
4. A recombinant strain comprising the gene of claim 2 or the recombinant vector of claim 3.
5. The recombinant vector of claim 3, wherein the starting vector of the recombinant vector is a pPIC9K plasmid.
6. The recombinant strain of claim 4, wherein the starting strain of the recombinant strain is pichia pastoris GS115 or escherichia coli JM109.
7. Use of a β -glucosidase mutant according to claim 1 for hydrolyzing β -D-glucosidic bonds at the non-reducing end of a glycoside or oligosaccharide compound to produce β -D-glucose and the corresponding ligand.
8. A method of making the β -glucosidase mutant of claim 1, comprising the steps of:
(1) The nucleotide sequence is shown as SEQ ID NO:3, connecting the beta-glucosidase mutant gene shown in the step 3 with a linearization vector pPIC9K after double digestion of EcoR I and Avr II, so as to obtain a recombinant vector containing the beta-glucosidase mutant gene;
(2) The recombinant vector is transformed into pichia pastoris GS115 to obtain a recombinant strain containing a beta-glucosidase mutant gene;
(3) Culturing the recombinant strain under proper conditions, inducing expression, collecting and purifying the expression product to obtain the beta-glucosidase mutant.
9. A method of hydrolyzing a glycoside compound, the method comprising the step of contacting the glycoside compound with the β -glucosidase mutant of claim 1 under conditions capable of hydrolyzing the β -D-glucosidic bond of the compound by enzymatic catalysis of the β -glucosidase mutant.
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