CN104560917A - Beta-glucosaccharase, beta-glucosaccharase mutant and application - Google Patents
Beta-glucosaccharase, beta-glucosaccharase mutant and application Download PDFInfo
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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 invention discloses a composition, containing an enzyme, wherein the enzyme has the activity of glucosaccharase and provides a degradation function in cellulose degradation, and the enzyme is the following protein (a) or (b): (a) protein with the amino acid sequence as shown in SEQ ID No: 1, (b) protein with one or more amino acids substituted, lost or added in the amino acid sequence in (a), having the activity of glucosaccharase and derived from (a). The invention also discloses a DNA molecule for encoding the enzyme, a recombinant vector containing the DNA molecule and a regulation sequence connected with the DNA molecule and used for expressing, and a host cell containing the DNA molecule or the recombinant vector. According to the beta-glucosaccharase mutant having the amino acid sequences as shown in SEQ ID No: 2, 3 and 4 provided by the invention, compared with wild beta-glucosaccharase, the thermostability is improved, and the integral hydrolysis rate of compound enzyme liquid in enzyme system compound can be improved.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of beta-glucosidase, the mutant of the mutant of beta-glucosidase and the beta-glucosidase of some thermotolerances raising is obtained with the method by site-directed point mutation, and the application in cellulose degradation of beta-glucosidase and beta-glucoside enzyme mutant.
Background technology
Because energy dilemma is day by day serious, the significant lignocellulose Mashing process of renewable and environment protecting more and more receives the concern of people.Utilize cellulose degraded lignocellulose to produce glucose, and then the biobased products that fermentative production comprises ethanol have important practical significance to socio-economic development.But because the efficiency of cellulose degraded natural cellulose substrate is low, cost is high, and the process being therefore biofuel by cellulose conversion still has very large challenge.Meanwhile, due to the specificity of substrate, and the imbalance of cellulase fermentations liquid enzyme system limits its application.In cellulose degradation process, a bottleneck of the hydrolysis efficiency of cellulase to be cellulose conversion be glucose.
Summary of the invention
The invention provides a kind of beta-glucosidase, beta-glucosidase of the present invention has the activity of glucuroide, and the activity of described glucuroide provides the function of degraded in cellulose degradation, can be used for degraded cellulose.
Meanwhile, in order to improve the thermotolerance of beta-glucosidase, the present invention suddenlys change to wild-type beta-glucosidase mutant, obtains the mutant of the beta-glucosidase that several thermotolerance significantly improves.
Technical scheme provided by the invention is:
A kind of composition, it contains a kind of enzyme, and described enzyme has the activity of glucuroide, and the activity of described glucuroide provides the function of degraded in cellulose degradation, and described enzyme is the protein of following (a) or (b):
(a) its aminoacid sequence as shown in SEQ ID NO:1,
B () aminoacid sequence in (a) passes through replacement, lacks or adds one or several amino acid and has the protein derivative by (a) of glucosidase activity.Be somebody's turn to do and be selected from by the amino acid whose sequence of (a) derivative protein: SEQ ID NO:2,3 and 4.
Chinese juniper shape mould (Penicillium piceum) H16 of a large amount of high-yield beta-glucosidase of energy having a strain to screen through selection by mutation at present, the preserving number of Chinese juniper shape mould (Penicillium piceum) H16 is CGMCC No.8339, the preservation time is: on October 15th, 2013, depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Applicant of the present invention is first by the genome sequencing of Chinese juniper shape mould (Penicillium piceum) H16, the beta-glucosidase protein sequence produced with all mould Pseudomonas by bioinformatics method builds storehouse, blastx is utilized to search in Chinese juniper shape mould full-length genome, and carry out sorting out and finally obtain the highest exocytosis beta-glucosidase of expression amount, its aminoacid sequence is as shown in SEQ ID NO:1.The expression amount of the beta-glucosidase as shown in SEQ ID NO:1 is the highest, and activity reaches more than 20IU/mL.The beta-glucosidase of high expression level amount, high vigor can be used for enzyme system composite in, balance lytic enzyme system, improves transformation efficiency.
Described enzyme provided by the invention and other enzyme actings in conjunction for degraded cellulose such as β-Isosorbide-5-Nitrae-glucolase, exoglucanase, for degraded cellulose.Enzyme provided by the invention also can separately for degradation of fibers disaccharides.
Preferably, in described composition, the Serine of the l-asparagine of the 335th that described enzyme is the aminoacid sequence shown in SEQ ID NO:1, the glutamine of the 341st and the 343rd is all substituted by tryptophane (N335W, Q341W and S343W), and its aminoacid sequence is as shown in SEQ ID NO:3.
Preferably, in described composition, the Serine that described enzyme is the aminoacid sequence shown in SEQ ID NO:1 the 336th is substituted by phenylalanine, the glutamine of the 341st is substituted by tryptophane and the Serine of the 343rd is substituted by tryptophane ((S336F, Q341W and S343W), its aminoacid sequence is as shown in SEQ ID NO:4.
Preferably, in described composition, the glutamine of the 341st that described enzyme is the aminoacid sequence shown in SEQ ID NO:1 and the Serine of the 343rd are all substituted by tryptophane, and its aminoacid sequence is as shown in SEQ ID NO:2.
Beta-glucoside enzyme mutant shown in aminoacid sequence SEQ ID NO:2 provided by the invention is compared with wild-type beta-glucosidase (aminoacid sequence SEQ ID NO:1), still there is the activity of glucuroide, can be used in degraded cellulose, and enzyme is lived roughly the same, and thermostability significantly improves, make it can improve compound enzyme liquid integral hydrolysis rate in enzyme system is composite.
The present invention also provides the shown beta-glucoside enzyme mutant of aminoacid sequence SEQ ID NO:3 and aminoacid sequence SEQ ID NO:4, compared with beta-glucosidase (aminoacid sequence SEQ ID NO:1), still there is the activity of glucuroide, can be used in degraded cellulose, and enzyme is lived roughly the same, and thermostability extremely significantly improves, make it can improve compound enzyme liquid integral hydrolysis rate in enzyme system is composite.Thermostability raising makes relative to wild-type beta-glucosidase (aminoacid sequence SEQ ID NO:1), and the shown beta-glucoside enzyme mutant of aminoacid sequence SEQ ID NO:3 and aminoacid sequence SEQ ID NO:4 is more being adapted to suitability for industrialized production.
Can be separately composite thus for degraded cellulose for enzyme system by wild-type beta-glucosidase (aminoacid sequence SEQ ID NO:1), also can by the one in beta-glucosidase and beta-glucoside enzyme mutant (aminoacid sequence SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4) this several enzyme or arbitrarily several simultaneously composite for enzyme system in thus for degraded cellulose.The present invention also provides a kind of DNA molecular, described DNA molecule encode described enzyme as above, and the sequence of this DNA molecular is selected from: SEQ ID NO:5,6,7 and 8.Because amino acid codes exists degeneracy, the phenomenon that same amino acid has two or more codons is called the degeneracy (degeneracy) of codon.Synonym (synonymous codon) is called corresponding to the amino acid whose different codon of same.Even if degeneracy makes base in those codons be changed, original acid of still encoding out.The degeneracy of password also makes based composition on DNA molecular have the variation in larger leeway.So DNA molecular of the present invention includes but not limited to above polynucleotide sequence, also there is the possible polynucleotide sequence of described enzyme of much can encoding, do not enumerating here.
The present invention also provides a kind of recombinant vectors, its described DNA molecular containing described enzyme of can encoding and the adjustment sequence for expressing be operably connected with described DNA molecular, the sequence of this DNA molecular is selected from: SEQ ID NO:5,6,7 and 8, what use in the present invention is pET serial carrier for the adjustment sequence expressed, certainly, also can use the expression vector of the nucleotide sequence of other described enzymes that are suitable for encoding, they can express in multiple eukaryotic host cell and prokaryotic host cell.
The present invention is also supplied to host cell, this host cell comprises the described DNA molecular of described enzyme of can encoding or described recombinant vectors, DNA molecular contained by described recombinant vectors can be encoded described enzyme, and the sequence of this DNA molecular is selected from: SEQ ID NO:5,6,7 and 8.In a preferred embodiment, this host cell is Bacillus coli cells.
The present invention also provides a kind of method producing the nucleotide sequence of codase mutant, comprising:
Step one, the nucleotide sequence that clone obtains as shown in SEQ ID NO:5 from Chinese juniper shape mould (Penicillium piceum) H16, and the adjustment sequence described polynucleotide sequence is connected to for expressing, build the connexon obtained with described nucleotide sequence, afterwards by being transformed in host cell by described connexon, obtain the recombinant vectors containing the nucleotide sequence shown in SEQ ID NO:5;
Step 2, utilize first pair of mutant primer, the recombinant vectors obtained with step one is template, carries out circulation extend amplification and obtain PCR primer by polymerase chain reaction method; And,
Step 3, utilize second pair of mutant primer and the 3rd pair of mutant primer respectively, with the PCR primer obtained in step 2 for template, again undertaken circulating by polymerase chain reaction method and extend the PCR primer that amplification obtains correspondence, afterwards by the PCR primer of the correspondence again obtained being transformed in different host cells separately, to obtain the object recombinant vectors containing mutational site.Preferably, in the method for the nucleotide sequence of described generation codase mutant, in described step 3, before the PCR primer again obtained is transformed in host cell, also comprises and utilize DpnI restriction endonuclease to process 3h at temperature 37 DEG C the PCR primer again obtained.
Preferably, in the method for the nucleotide sequence of described generation codase mutant, in described step 2, the base sequence of a wherein primer of described first pair of mutant primer is as shown in SEQ ID NO:11, the base sequence of another primer as shown in SEQ ID NO:12, the base sequence of SEQ ID NO:11 and the base sequence reverse complemental of SEQ ID NO:12; The primer sequence as shown in SEQ ID NO:11 and SEQ ID NO:12 is utilized again to obtain the nucleotide sequence as shown in SEQ ID NO:6 by pcr amplification;
In described step 3, the base sequence of a wherein primer of described second pair of mutant primer is as shown in SEQ ID NO:13, the base sequence of another primer as shown in SEQ ID NO:14, the base sequence of SEQ ID NO:13 and the base sequence reverse complemental of SEQ ID NO:14; The primer sequence as shown in SEQ ID NO:13 and SEQ ID NO:14 is utilized again to obtain the nucleotide sequence as shown in SEQ ID NO:7 by pcr amplification.
The base sequence of wherein one article of primer of described 3rd pair of mutant primer as shown in SEQ ID NO:15, the base sequence of another primer as shown in SEQ ID NO:16, the base sequence of SEQ ID NO:15 and the base sequence reverse complemental of SEQ ID NO:16; The primer sequence as shown in SEQ ID NO:15 and SEQ ID NO:16 is utilized again to obtain the nucleotide sequence as shown in SEQ ID NO:8 by pcr amplification.Primer sequence is not limited thereto the sequence listed, and also can be other sequences, as long as can obtain object nucleotide sequence.
Beneficial effect of the present invention is:
The expression amount of beta-glucosidase of the present invention is very high, and activity is very high.The beta-glucosidase of high expression level amount, high vigor can be used for enzyme system composite in, balance lytic enzyme system, improves transformation efficiency.
The such as beta-glucosidase shown in SEQ ID NO:1 is carried out as above two kinds of sudden changes by applicant of the present invention, the activity still with glucuroide of the two kinds of beta-glucoside enzyme mutants (aminoacid sequence SEQ ID NO:2 and 3) obtained, can be used in degraded cellulose.Wild-type beta-glucosidase can be used for separately enzyme system composite thus be used for degraded cellulose, also can by the one in this several enzyme or arbitrarily several be used for simultaneously enzyme system composite in thus be used for degraded cellulose.
And, the beta-glucoside enzyme mutant of aminoacid sequence SEQ ID NO:2 provided by the invention, SEQ ID NO:3 and SEQ ID NO:4, compared with beta-glucosidase (aminoacid sequence SEQ ID NO:1), thermotolerance improves, make it can improve compound enzyme liquid integral hydrolysis rate in enzyme system is composite, especially the beta-glucoside enzyme mutant of SEQ ID NO:3 and SEQ ID NO:4, thermotolerance improves extremely remarkable.Thermotolerance improves the hydrolysis efficiency that can improve beta-glucosidase in the unit time, reduces enzyme dosage, improves the effect improving integral hydrolysis rate in composite middle enzyme equilibrium system of enzyme system.In addition, flavor precursors can be hydrolyzed to by beta-glucosidase has strong natural flavour mountaineous aroma substance, and the raising of thermotolerance can make it in beer, tealeaves, play better effect, is with a wide range of applications.
Definition
For the ease of understanding the present invention, define a large amount of term and phrase
" reverse complemental " in the present invention, refers to the nucleotide sequence associated by basepairing rule.Such as, sequence " 5 '-A-T-G-3 ' " and sequence " 5 '-C-A-T-3 ' " reverse complemental.
Term " gene " refers to a kind of DNA molecular, and gene is the essential substance of heritable variation, and be the basic genetic unit controlling biological character, in gene, the base sequence of coding RNA or protein becomes structure gene, and gene alleged in the present invention is structure gene.
" wild type gene " refers to a kind of gene be separated from source, and " wild type gene " of the present invention obtains from Chinese juniper shape mould (Penicillium piceum) the H16 clone of mutagenesis.And " gene of sudden change " refers to a kind of gene, compared with " wild type gene ", it has the modification (feature namely changed) of sequence and/or functional property.
" wild-type enzyme " refers to a kind of protein, and " wild-type enzyme " of the present invention is separated from Chinese juniper shape mould (Penicillium piceum) H16 of mutagenesis to obtain, or the product of " wild type gene " allos or homology expression.And " enzyme mutant " refers to compared with " wild-type enzyme ", it has the modification (feature namely changed) of sequence and/or functional property.
" carrier " refers to the nucleic acid molecule can transporting another nucleic acid connected, and the carrier of a type is " plasmid ", and plasmid is that other DNA fragmentation can connected circular double stranded DNA ring.The carrier of another type is virus vector, and other DNA fragmentation can be connected to viral genome by it.Some vector integration in host cell gene group, and is able to copy together with host genome.Further, some carrier can instruct the expression of the gene be operatively connected with it, and the general such expression vector used is plasmid form.In the present invention, can use alternately " plasmid " and " carrier ".
" recombinant vectors " refers to the expression vector being connected to gene.In the present invention, can use alternately " recombinant plasmid " and " recombinant vectors ".
Primer, has another name called introduction.A bit of single stranded DNA or RNA, as the starting point of DNA replication dna, when nucleic acid building-up reactions, the starting point extended is carried out and the polynucleotide chain worked as each polynucleotide chain, on 3 '-OH of primer, Nucleotide synthesizes with diester chain form, therefore 3 '-OH of primer, must be free.Primer why is needed to be because archaeal dna polymerase only can be added to new Nucleotide on existing DNA chain in DNA synthesis.Primer is two sections of oligonucleotide sequences of synthetic, and a DNA profiling chain an of primer and area-of-interest one end is complementary, and another DNA profiling chain of another primer and the area-of-interest the other end is complementary.Chain DNA carrying the nucleotide sequence of coded protein amino acid information is called positive-sense strand, also known as coding strand.Another chain nucleotide sequence and positive-sense strand complementation, be called antisense strand.Generally a primer with positive-sense strand complementation is become upstream primer, be called downstream primer with a primer of antisense strand complementation.
Cellulase, cellulase refers to energy degraded cellulose β-Isosorbide-5-Nitrae-glucoside bond, and making Mierocrystalline cellulose become the general name of one group of enzyme of cellobiose and glucose, has been synergistic polycomponent enzyme system.The main ingredient of cellulase is inscribe β-Isosorbide-5-Nitrae-glucolase, exoglucanase and beta-glucosidase.First two enzyme mainly dissolves fiber, and cellobiose is converted into glucose by rear a kind of enzyme, suitably regulates the ratio of these three kinds of main component activity in composition (namely to component enzymes system), has realized cellulosic degraded.
Accompanying drawing explanation
The gel electrophoresis figure of the wherein PCR primer of two genes that Fig. 1 (a) is encoding beta-glucosidase;
The gel electrophoresis figure of the PCR primer of two other gene that Fig. 1 (b) is encoding beta-glucosidase; Wherein, b is depicted as G8221 gene, and a, c and d are depicted as other genes.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Embodiment 1:
First by the genome sequencing of Chinese juniper shape mould (Penicillium piceum) H16, the beta-glucosidase protein sequence produced with all mould Pseudomonas by bioinformatics method builds storehouse, blastx is utilized to search in Chinese juniper shape mould full-length genome, and carry out the sequence sorting out the beta-glucosidase obtaining multiple exocytosis, as shown in Fig. 1 (a) He 1 (b), adopt reverse transcription PCR (RT-PCR) methods analyst by the expression amount of these exocytosis albumen, find that the amount of one of them gene G8221 is the highest, thus to obtain aim sequence be the exocytosis beta-glucosidase that in Chinese juniper shape mould H16, expression amount is the highest, its aminoacid sequence is as shown in SEQ ID NO:1, DNA molecular sequence is as shown in SEQ ID NO:5.
Embodiment 2:
Utilize molecular simulation software Discovery studio that the beta-glucosidase of the beta-glucosidase of Chinese juniper shape mould (Penicillium piceum) H16 and fabulous aspergillus niger (Aspergillus niger) CGMCC 3.316 of thermostability is carried out modeling, build 3D model.Carrying out mutational site with following methods selects a. that the beta-glucosidase of Chinese juniper shape mould and aspergillus niger CGMCC3.316 is carried out 3D structure alignment, observes difference site; B. utilizing Discovery studio to carry out virtual sudden change, the feasibility of mutation scheme can be judged by calculating sudden change.According to above mutation scheme design mutant primer, be that template is suddenlyd change with the recombinant plasmid containing wild-type beta-glucosidase DNA sequence dna (SEQ ID NO:5).Utilize molecular simulation software to carry out design and rational to beta-glucosidase, selected mutational site, effectively can save the time of mutational site screening, improves mutation efficiency.
Described in above-mentioned construction process, expression vector refers to pET15, pET22 or pET28 etc.
1, pET28a (+)-bgl1 is built: the recombinant vectors of the wild type gene (nucleotide sequence SEQ ID NO:5) of encoding wild type beta-glucosidase (aminoacid sequence SEQ ID NO:1)
Utilize primer G8221F:5 '-ATA GCT AGC ATG CTC TCC AAA TTG CAA CAT C-3 ' (SEQ ID NO:9) and G8221R:5 '-ATA CTC GAG TCA AAC AGT GAA AGT GTC TGT T-3 ' (SEQ ID NO:10), with the genome of Chinese juniper shape mould (Penicillium piceum) H16 for template, wildtype gene sequence (SEQ ID NO:5) is obtained after carrying out pcr amplification, Nhe I and Xho I enzyme is used by a kind of expression vector pET28a of wildtype gene sequence (SEQ ID NO:5) and pET series to carry out respectively after enzyme cuts, purifying reclaim enzyme cut after wild type gene fragment and pET carrier, and wild type gene fragment is connected on pET28a carrier obtains connexon pET28a (+)-bgl1.This connexon pET28a (+)-bgl1 is transformed in E. coli DH5 α, whether be correct gene clone (identical with nucleotide sequence SEQ ID NO:5) to the transformant sequence verification obtained, after selecting corresponding bacterial strain E.coli BL21 (DE3)/pET28a (+)-bgl1 mass propgation containing correct sequence, extract recombinant vectors (the aminoacid sequence SEQ ID NO:1 that namely plasmid obtains pET28a (+)-bgl1 correct in a large number, nucleotide sequence SEQ ID NO:5), be the structure that template carries out follow-up mutator gene with the recombinant vectors of this pET28a (+)-bgl1.
Adopt directed mutagenesis method, first with the recombinant vectors of wild type gene fragment for template, suddenly change by designing first pair of mutant primer, obtain the PCR primer containing, for example the nucleotide sequence shown in SEQ ID NO:6, afterwards, with this PCR primer for template, then second pair of mutant primer and the 3rd pair of mutant primer is utilized to obtain the recombinant vectors respectively containing the nucleotide sequence shown in SEQ ID NO:7 and SEQ ID NO:8 by pcr amplification respectively.In the design of primer, upstream primer design in using mutational site as center, left and right each 12-30 base, downstream primer is the reverse complementary sequence of upstream primer.Mutant primer recombinant plasmid is utilized to carry out high-fidelity PCR amplification afterwards.The PCR primer obtained is the ring-type of a disappearance, namely be linear recombinant vectors, the phusion enzyme that the Thermo company that the present invention adopts produces has been increased recombinant plasmid total length by mutant primer, so obtaining PCR primer is ring-type (comprising gene order and the carrier of sudden change), after this PCR primer being transferred in escherichia coli DH5a cell, this linear recombinant vectors automatically can be repaired again and become the ring texture the same with recombinant vectors.
2, pET28a (+)-bgl1/Q341W is built
Utilize first pair of mutant primer 341343_F:5 '-TTC AAC TCG TGG AAT GGC GGC TGG ACC TGG GCG AAT GTG ACA GGA GAC CAT ' (SEQ ID NO:11) and 341343_R:5 '-ATG GTC TCC TGT CAC ATT CGC CCA GGT CCA GCC GCC ATT CCA CGA GTT GAA-3 ' (SEQ ID NO:12), with pET28a (+)-bgl1 for template, carry out high-fidelity PCR amplification, obtain pET28a (+)-bgl1/Q341W (aminoacid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:6).Now, pET28a (+)-bgl1/QQ341W is a linear recombinant vectors.
In some embodiments, can utilize as shown in following table 1 and table 2, adopt Thermo company phusion enzyme to carry out high-fidelity PCR amplification:
Table 1 PCR amplification system
The condition that table 2 PCR circulates
3, pET28a (+)-bgl1/N335W is built
Utilize second pair of mutant primer 335_F:5 '-GGC TAC CCA TCT GTA CAG TTC TGG TCG TGG AAT GGC GGC-3 ' (SEQ ID NO:13) and 335_R:5 '-GCC GCC ATT CCA CGA CCA GAA CTG TAC AGA TGG GTA GCC-3 ' (SEQ ID NO:14), with pET28a (+)-bgl1 for template, as above PCR amplification system shown in table 1 and table 2 and cycling condition is utilized to carry out high-fidelity PCR amplification, obtain pET28a (+)-bgl1/N335W (aminoacid sequence SEQ ID NO:3, nucleotide sequence SEQ ID NO:7).Now, pET28a (+)-bgl1/N335W is a linear recombinant vectors.
4, pET28a (+)-bgl1/S336F is built
Utilize the 3rd pair of mutant primer 336_F:5 '-AC CCA TCT GTA CAG TTC AAC TTC TGG AAT GGC GGC-3 ' (SEQ ID NO:15) and 336_R:5 '-GCC GCC ATT CCA GAA GTT GAA CTG TAC AGA TGG GT-3 ' (SEQ ID NO:16), with pET28a (+)-bgl1 for template, as above PCR amplification system shown in table 1 and table 2 and cycling condition is utilized to carry out high-fidelity PCR amplification, obtain pET28a (+)-bgl1/S336F (aminoacid sequence SEQ ID NO:4, nucleotide sequence SEQ ID NO:8).Now, pET28a (+)-bgl1/S336F is a linear recombinant vectors.
First the variants containing the nucleotide sequence shown in SEQ ID NO:7 and SEQ ID NO:8 PCR again obtained is according to after process of carrying out respectively shown in table 3 methylating, proceed to bacillus coli DH 5 alpha or escherichia coli jm109 competent cell, picking positive colony, and carry out determined dna sequence.And the bacterial plaque containing the correct DNA sequence dna that suddenlys change is cultivated and extracted plasmid, obtain the recombinant plasmid containing sudden change beta-glucosidase gene.Recombinant plasmid is proceeded to the expression strains such as e. coli bl21 (DE3) or E.coli Rosetta, screening positive clone, namely obtains the engineering strain containing mutator gene of the present invention.
It should be noted that, because the hosts such as E.coli DH5 α have the modification that methylates, and extract from the host cells such as E.coli DH5 α as pET28a (+)-bgl1 recombinant vectors of template, so its DNA sequence dna has methylation sites, DpnI can these sites of specific recognition, template DNA is cut into segment, thus the PCR primer after utilizing DpnI enzyme to cut is proceeded to after in bacillus coli DH 5 alpha competent cell, template DNA and pET28a (+)-bgl1 recombinant vectors can not grow containing on the solid plate of kantlex.And owing to there is no methylation sites in the PCR primer of successfully suddenling change, the enzyme that can not be subject to DpnI cuts impact, thus being transferred in E.coli DH5 α, the linear recombinant vectors of sudden change automatically can be repaired again and become the ring texture the same with recombinant vectors, and successfully copies.Therefore, when screening positive clone, the interference of residual template DNA is eliminated.
Table 3 utilizes the system of methylase DpnI process PCR primer
By above method, obtain engineering strain E.coli BL21 (DE3)/pET28a (+)-bgl1/N335W (the aminoacid sequence SEQ ID NO:3 containing mutator gene respectively, nucleotide sequence SEQ ID NO:7) and E.coli BL21 (DE3)/pET28a (+)-bgl1/S336F (aminoacid sequence SEQ ID NO:4, nucleotide sequence SEQ ID NO:8) etc.
Embodiment 3:
Containing expression and the protein purification of beta-glucosidase gene of the present invention and beta-glucoside enzyme mutant gene engineering bacteria
By engineering strain E.coli BL21 (DE3)/pET28a (+)-bgl1 (the aminoacid sequence SEQ ID NO:1 preserved, nucleotide sequence SEQ ID NO:5), E.coli BL21 (DE3)/pET28a (+)-bgl1/Q341W (aminoacid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:6), E.coli BL21 (DE3)/pET28a (+)-bgl1/N335W (aminoacid sequence SEQ ID NO:3, nucleotide sequence SEQ ID NO:7), with E.coli BL21 (DE3)/pET28a (+)-bgl1/S336F (aminoacid sequence SEQ ID NO:4, nucleotide sequence SEQ ID NO:8) etc. glycerol stock according to 2% volume ratio be inoculated in 20mL and contain in the LB liquid medium of kantlex, 28 DEG C of incubated overnight.Nutrient solution after activation is inoculated in the 100mL LB liquid nutrient medium containing kantlex, be positioned over 37 DEG C, be cultured to OD600=0.6 under 250rpm condition (with UNICO UV2102 ultraviolet-visible pectrophotometer, to cultivate with LB substratum as blank); Adding final concentration is afterwards that the IPTG of 0.8mM induces, and in 28 DEG C, continue cultivation 10 hours under 180rpm condition; 4 DEG C, 8000g collected by centrifugation thalline, add the binding buffer liquid (Binding buffer) that 0.1 times of bacteria liquid is long-pending, ultrasonic 40min smudge cells under 350W power, condition of ice bath, 30000g collected by centrifugation supernatant, obtains crude enzyme liquid.
Crude enzyme liquid carries out purifying through Ni-NTA column chromatography, and in elutriant, imidazole concentration is 500mM, wash-out 10 column volumes.The albumen obtained afterwards reaches the requirement of SDS-PAGE purity through detecting.Wild-type beta-glucosidase albumen (aminoacid sequence SEQ ID NO:1), beta-glucoside enzyme mutant Q341W (aminoacid sequence SEQ ID NO:2), beta-glucoside enzyme mutant N335W (aminoacid sequence SEQ ID NO:3) and beta-glucoside enzyme mutant S336F (aminoacid sequence SEQ ID NO:4) is obtained by above method.
Embodiment 4
The enzyme using cellobiose assay method to measure 4 kinds of albumen is lived, and actual is take cellobiose as the mensuration that substrate carries out, and concrete grammar and step are:
Sample: 1.5mL 0.5% cellobiose, at 50 DEG C of thermostat water bath preheating 5-10min, adds the certain density enzyme diluent of 0.5mL.Enzyme liquid needs at least two extent of dilution, and the glucose amount that dilution enzyme liquid discharges in 30min is slightly less than 1mg, and the glucose amount that dilution enzyme liquid discharges in 30min is slightly more than 1mg;
Water-bath: the control group of the sample mixed, substrate and enzyme is placed in simultaneously 50 DEG C of thermostat water baths, water-bath 30min.
Deactivation: at the end of reaction, boils 5min by sample, substrate and makes enzyme deactivation together with the control group of enzyme.
Measure: adopt biosensor to survey the glucose content produced.
Calculate: the beta-glucosidase enzyme amount being defined as the enzyme that per minute transforms required for product 1umol glucose alive is 1IU.The enzyme of various protein is lived and is asked for an interview following table 4.
The enzyme of the various protein of table 4 is lived
(note: in table, the beta-glucosidase of Chinese juniper shape mould secretion includes multiple beta-glucosidase, the work of its enzyme is the summation that multiple beta-glucosidase enzyme is lived.Wild-type beta-glucosidase and beta-glucoside enzyme mutant refer to the protein obtained by genetic engineering bacterium abduction deliverings such as E.coli in embodiment 3.)
Use cellobiose assay method to measure the thermotolerance of 4 kinds of albumen, concrete grammar is similar with the step that said determination enzyme is lived with step, just in water-bath step, is placed in 50 DEG C of thermostat water baths, after water-bath 48h, measures the remaining activity of various enzyme.
The calculating of remaining activity (very) is carried out: after such as wild-type beta-glucosidase places 48h at 50 degree, enzyme work also remains initial 60% by the numerical value of enzyme work, here to live as 100% for initial point defines enzyme during 0h, then its remaining activity is 60%.The remaining activity of the enzyme that each bacterial strain produces asks for an interview table 5.
The remaining activity of the enzyme that each bacterial strain of table 5 produces or mutant
It should be noted that, although the thermotolerance of the beta-glucosidase of aspergillus niger CGMCC3.316 production is higher, it is active and output is all lower, is not suitable for industrial production.
In Chinese juniper shape mould, the secretory volume of wild-type beta-glucosidase is maximum, it is the main body of beta-glucosidase, as can be seen from table 4 and table 5, the DNA molecular of these one or more series jumps can be transferred to Chinese juniper shape mould or other mould bacterial classifications, for generation of a large amount of beta-glucosidase, thus in composite for enzyme system, balance lytic enzyme system, improves transformation efficiency.
Beta-glucoside enzyme mutant Q341W (aminoacid sequence SEQ ID NO:2), beta-glucoside enzyme mutant N335W (aminoacid sequence SEQ ID NO:3) and beta-glucoside enzyme mutant S336F (aminoacid sequence SEQ ID NO:4) are compared with wild-type beta-glucosidase (aminoacid sequence SEQ ID NO:1), not only enzyme is lived roughly the same, thermotolerance improves in addition significantly, especially beta-glucoside enzyme mutant S336F (aminoacid sequence SEQ ID NO:4) and beta-glucoside enzyme mutant S336F (aminoacid sequence SEQ ID NO:4) thermostability improve extremely remarkable, this is for very important current production, can in the integral hydrolysis rate of the composite middle raising compound enzyme liquid of enzyme system, industrially there is using value greatly.
The 335/336th, 341 of wild-type beta-glucosidase and 343 amino acids are positioned at same structural domain, found by molecular simulation, 3 kinds of beta-glucoside enzyme mutants the increasing in a large number of hydrophobic interaction after sudden change, and hydrophobic interaction is conducive to the effectively folding of albumen, thus making protein structure more stable, this is the maximum power-assisted that its thermostability improves.Thermotolerance improves the hydrolysis efficiency that can improve beta-glucosidase in the unit time, reduces enzyme dosage, improves the effect improving integral hydrolysis rate in composite middle enzyme equilibrium system of enzyme system.In addition, flavor precursors can be hydrolyzed to by beta-glucosidase has strong natural flavour mountaineous aroma substance, and the raising of thermotolerance can make it in beer, tealeaves, play better effect, is with a wide range of applications.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (10)
1. a composition, it contains a kind of enzyme, it is characterized in that, described enzyme has the activity of glucuroide, the activity of described glucuroide provides the function of degraded in cellulose degradation, and described enzyme is the protein of following (a) or (b):
(a) its aminoacid sequence as shown in SEQ ID NO:1,
B () aminoacid sequence in (a) passes through replacement, lacks or adds one or several amino acid and has the protein derivative by (a) of glucosidase activity.
2. composition as claimed in claim 1, is characterized in that, the Serine of the l-asparagine of the 335th that described enzyme is the aminoacid sequence shown in SEQ ID NO:1, the glutamine of the 341st and the 343rd is all substituted by tryptophane.
3. composition as claimed in claim 1, it is characterized in that, the Serine that described enzyme is the aminoacid sequence shown in SEQ ID NO:1 the 336th is substituted by phenylalanine, the glutamine of the 341st is substituted by tryptophane and the Serine of the 343rd is substituted by tryptophane.
4. composition as claimed in claim 1, it is characterized in that, the glutamine of the 341st that described enzyme is the aminoacid sequence shown in SEQ ID NO:1 and the Serine of the 343rd are all substituted by tryptophane.
5. a DNA molecular, is characterized in that, described DNA molecule encode is enzyme as described in any one of Claims 1 to 4.
6. a recombinant vectors, is characterized in that, its adjustment sequence for expressing containing DNA molecular according to claim 5 and be operably connected with described DNA molecular.
7. host cell, is characterized in that, described host cell contains DNA molecular according to claim 5 or recombinant vectors according to claim 6.
8. produce a method for the nucleotide sequence of codase mutant, it is characterized in that, comprising:
Step one, the nucleotide sequence that clone obtains as shown in SEQ ID NO:5 from Chinese juniper shape mould (Penicillium piceum) H16, and the adjustment sequence described polynucleotide sequence is connected to for expressing, build the connexon obtained with described nucleotide sequence, afterwards by being transformed in host cell by described connexon, obtain the recombinant vectors containing the nucleotide sequence shown in SEQ ID NO:5;
Step 2, utilize first pair of mutant primer, the recombinant vectors obtained with step one is template, carries out circulation extend amplification and obtain PCR primer by polymerase chain reaction method; And,
Step 3, utilize second pair of mutant primer and the 3rd pair of mutant primer respectively, with the PCR primer obtained in step 2 for template, again undertaken circulating by polymerase chain reaction method and extend the PCR primer that amplification obtains correspondence, afterwards by the PCR primer of the correspondence again obtained being transformed in host cell separately, to obtain the object recombinant vectors containing mutational site.
9. the method producing the nucleotide sequence of codase mutant as claimed in claim 8, it is characterized in that, in described step 3, before the PCR primer again obtained is transformed in host cell, also comprises and utilize DpnI restriction endonuclease to process 3h at temperature 37 DEG C the PCR primer again obtained.
10. the method producing the nucleotide sequence of codase mutant as claimed in claim 8, it is characterized in that, in described step 2, the base sequence of a wherein primer of described first pair of mutant primer is as shown in SEQ ID NO:11, the base sequence of another primer as shown in SEQ ID NO:12, the base sequence of SEQ IDNO:11 and the base sequence reverse complemental of SEQ ID NO:12;
In described step 3, the base sequence of a wherein primer of described second pair of mutant primer is as shown in SEQID NO:13, the base sequence of another primer as shown in SEQ ID NO:14, the base sequence of SEQ ID NO:13 and the base sequence reverse complemental of SEQ ID NO:14;
The base sequence of wherein one article of primer of described 3rd pair of mutant primer is as shown in SEQ ID NO:15, the base sequence of another primer as shown in SEQ ID NO:16, the base sequence of SEQ ID NO:15 and the base sequence reverse complemental of SEQ ID NO:16.
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