CN104293812B - Heat-resistance neutral cellulase Cel6C and encoding gene thereof and application - Google Patents
Heat-resistance neutral cellulase Cel6C and encoding gene thereof and application Download PDFInfo
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Abstract
The invention discloses heat-resistance neutral cellulase Cel6C and encoding gene thereof and application.The present invention is isolated heat-resistance neutral cellulase encoding genes from Humicola insolens, and its nucleotides sequence is classified as shown in SEQ ID No.3, and coded aminoacid sequence is shown in SEQ ID No.4；Invention further provides ripe heat-resistance neutral cellulase, its aminoacid sequence is shown in SEQ ID No.2, and the nucleotides sequence of its coding is classified as shown in SEQ ID No.1.The invention also discloses the recombinant expression carrier containing described encoding gene and recombinant host cell.Heat-resistance neutral cellulase good heat stability of the present invention, in alkaline range, there is high enzyme vigor neutral, can the material such as efficient degradation cellulose, hemicellulose, lichenin, sodium carboxymethyl cellulose, macromolecular polysaccharide, protein or lipid, it is possible to be applied to the fields such as wine brewing, weaving, energy industry.
Description
Technical field
The present invention relates to cellulase, particularly relate to from Humicola insolens (Humicola insolens) separate resistance to
Neutral cellulase Cel6C, the invention still further relates to encoding gene and the application thereof of this enzyme, belongs to the separation of cellulase and answers
Use technical field.
Background technology
Cellulose is the linear macromolecule polymer being formed by connecting with β-Isosorbide-5-Nitrae glycosidic bond by glucose, for being distributed at present
Wide natural carbohydrate, is also Renewable resource the abundantest in nature.Cellulase refer to hydrocellulose β-
L, 4 glucoside bonds, make cellulose become the general name of one group of enzyme of cellobiose and glucose.It is by endo-type β-1,4-Portugal
Dextranase (EC3.2.1.4, be called for short EG, CX enzyme, CMC enzyme), circumscribed-type β-Isosorbide-5-Nitrae-glucanase (EC3.2.1.91,
EC3.2.1.176, is called for short CBH, C1 enzyme, cellobiohydrolase) and cellobiase (EC3.2.1.21, abbreviation CB, β-Fructus Vitis viniferae
Glycosidase) 3 main component compositions play synergistic compound enzyme system (Juturu V and Wu J C 2014.Renew
Sust Energ Rev 33,188-203)。
Noncrystalline domain within cellulosic polymer is carried out by endo-type beta-1,4-glucan enzyme (EGs) with random form
Cutting, produces the oligosaccharide of different length and new chain end.Endo-type β-Isosorbide-5-Nitrae-glucanase is the industrial enzyme that a class is important, can
Be widely used in food, weaving, feedstuff, make wine, various fields (the Phitsuwan P et al.2013.Folia such as washing
Microbiol 58,163-176).Wherein, it is gentle that high temperature neutrality endoglucanase has action condition, resistant to elevated temperatures feature,
Particularly important for application, can be widely used for the various fields such as biomass energy, weaving, food, feedstuff and pharmaceutical industry.
In textile industry, neutral cellulase has important using value in denim biology granite-wash is processed.In pH neutral
In the range of, cellulase is the softest to the effect of fabric, will not destroy the hardness of fabric, and surfactant is at this pH value model
It is also maximally effective for enclosing, thus has saved the surfactant cost in the denim course of processing.It addition, at neutral cellulase
Action condition under, it is also possible to effectively prevent fabric from returning dye (Chen J et al.2007.Enzyme and Microbial
Technology 40,1651-1655).In medicated beer processing industry, 1,4 beta-glucanase can be efficiently by the beta glucan in Fructus Hordei Germinatus
It is hydrolyzed to glucose and oligosaccharide, destroying cells wall, makes cellular content discharge in large quantities, improve wheat juice yield, reduce
Mash viscosity thus shorten converted mash filtration time, obtained wheat juice is limpid, makes the beer color prepared shallow, and foam is the most lasting.
Therefore, 1,4 beta-glucanase is used for beer saccharification and sweat, grain can be economized on, reduce cost, improve saccharification filter device
Usefulness, the beneficially Quality advance of medicated beer and stable (Gong Chunbo thanks to beautiful source .2002. Guangzhou Food Industry science and technology).But,
In the beer saccharification stage, the dissolution of beta glucan increases along with the rising of temperature, causes the beta glucan ratio of chilling process dissolution
Example is relatively low, and the ratio of pyroprocess dissolution is higher, and resistant to elevated temperatures endo-type 1,4 beta-glucanase is the key solving this problem.
Therefore, it is thus achieved that the research of the novel cellulase with the good characteristic such as neutrality, Heat stability is good still has great
Meaning.
Summary of the invention
The technical problem to be solved is to provide one and divides from Humicola insolens (Humicola insolens)
From heat-resistance neutral cellulase Cel6C, this enzyme heat stability is excellent and has high enzyme vigor in neutral and alkaline range.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
First the present invention discloses the heat-resistance neutral cellulose separated from Humicola insolens (Humicola insolens)
The encoding gene of enzyme Cel6C, its nucleotides sequence is classified as shown in (a), (b), (c), (d) or (e):
Polynucleotide shown in (a), SEQ ID No.1；Or
Amino acid whose polynucleotide shown in (b), coding SEQ ID No.2；Or
C the complementary series of () and SEQ ID No.1 can carry out the polynucleotide hybridized at stringent hybridisation conditions, these are many
Protein coded by nucleotide still has the function of heat-resistance neutral cellulase Cel6C；Or
The polynucleotide of at least 90% or above homology of the polynucleotide shown in (d) and SEQ ID No.1, and should
Protein coded by polynucleotide still has the function of heat-resistance neutral cellulase Cel6C；Preferably, it is with SEQ ID No.1
The polynucleotide of shown polynucleotide at least 95% or above homology, and protein coded by these polynucleotide still has
The function of heat-resistance neutral cellulase Cel6C；Most preferably, it is with the polynucleotide shown in SEQ ID No.1 at least 98%
Or the polynucleotide of above homology, and protein coded by these polynucleotide still has heat-resistance neutral cellulase Cel6C's
Function；Or
(e), on the basis of the polynucleotide shown in SEQ ID No.1, carry out the disappearance of one or more base, replacement
Or the polynucleotide variant inserted, and this albumen coded by polynucleotide variant still has heat-resistance neutral cellulase Cel6C
Function.
The invention also discloses the heat-resistance neutral cellulase Cel6C of described encoding gene coding, its aminoacid sequence is
Shown in (a) or (b):
Aminoacid shown in (a), SEQ ID No.2；
(b), by the aminoacid shown in SEQ ID No.2 by replacement, the disappearance of one or more amino acid residues or/and
Insert and the derivative protein variant still with heat-resistance neutral cellulase Cel6C function obtained.
The present invention further discloses the heat-resistance neutral fiber separated from Humicola insolens (Humicola insolens)
The encoding gene of element enzyme Cel6C, its nucleotides sequence is classified as shown in (a), (b), (c), (d) or (e):
Polynucleotide shown in (a), SEQ ID No.3；Or
Amino acid whose polynucleotide shown in (b), coding SEQ ID No.4；Or
C the complementary series of () and SEQ ID No.3 can carry out the polynucleotide hybridized at stringent hybridisation conditions, these are many
Protein coded by nucleotide still has the function of heat-resistance neutral cellulase Cel6C；Or
The polynucleotide of at least 90% or above homology of the polynucleotide shown in (d) and SEQ ID No.3, and should
Protein coded by polynucleotide still has the function of heat-resistance neutral cellulase Cel6C；Preferably, it is with SEQ ID No.3
The polynucleotide of shown polynucleotide at least 95% or above homology, and protein coded by these polynucleotide still has
The function of heat-resistance neutral cellulase Cel6C；Most preferably, it is with the polynucleotide shown in SEQ ID No.3 at least 98%
Or the polynucleotide of above homology, and protein coded by these polynucleotide still has heat-resistance neutral cellulase Cel6C's
Function；Or
(e), on the basis of the polynucleotide shown in SEQ ID No.3, carry out the disappearance of one or more base, replacement
Or the polynucleotide variant inserted, and this albumen coded by polynucleotide variant still has heat-resistance neutral cellulase Cel6C
Function.
The heat-resistance neutral cellulase Cel6C of described encoding gene coding, its aminoacid sequence is shown in (a) or (b):
Aminoacid shown in (a), SEQ ID No.4；
(b), by the aminoacid shown in SEQ ID No.4 by replacement, the disappearance of one or more amino acid residues or/and
Insert and the derivative protein variant still with heat-resistance neutral cellulase Cel6C function obtained.
Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these operational approach are the most originally
Field is understood.Such as, can by the sudden change of DNA prepare heat-resistance neutral cellulase Cel6C amino acid sequence variation or
Fragment, wherein the method for mutation or change polynucleotide is known by this area.Wherein, conservative replacement is by one ammonia
Base acid residue replaces to the another kind of aminoacid with similar quality.Heat-resistance neutral cellulase Cel6C of the present invention and
Its encoding gene includes naturally occurring sequence and two kinds of forms of variant." variant " means the sequence of basic simlarity, for multinuclear
Thuja acid, variant comprises in native polynucleotide the disappearance of the one or more nucleotide of one or more site, insertion or/and replace
Change.For polynucleotide, conservative variant includes not changing the aminoacid sequence of coding due to the degeneracy of genetic code
Those variants.Naturally occurring variant like this can be identified by existing Protocols in Molecular Biology.Variant polynucleotides
Also include the polynucleotide in synthesis source, for example with still encoding SEQ ID No.2 or SEQ ID obtained by direct mutagenesis
Amino acid whose polynucleotide variant shown in No.4, or the method (such as DNA reorganization) by restructuring.
The present invention passes through the method separating clone of the PCR encoding gene of heat-resistance neutral cellulase Cel6C, DNA total order
Row analysis result shows, cDNA sequence total length 1149bp of the heat-resistance neutral cellulose enzyme gene containing signal peptide sequence, coding
382 aminoacid and a termination codon, its nucleotides sequence is classified as shown in SEQ ID No.3, its coded aminoacid sequence
It is classified as shown in SEQ ID No.4；18 aminoacid of its N end are the signal peptide sequence of its prediction, and its aminoacid sequence is SEQ ID
Shown in No.6, the coded sequence of signal peptide is shown in SEQ ID No.5.Do not contain the ripe heat-resistance neutral cellulose of signal peptide
Enzyme Cel6C, its aminoacid sequence is shown in SEQ ID No.2, and the nucleotides sequence of its coding is classified as shown in SEQ ID No.1.Cause
This, the theoretical molecular of ripe heat-resistance neutral cellulase Cel6C is 40.0kDa.
By the cDNA sequence (shown in SEQ ID No.1) of heat-resistance neutral cellulose enzyme gene and the aminoacid sequence derived
(shown in SEQ ID No.2) carries out BLAST comparison in GenBank, this gene with derive from Chaetomium
The glycoside hydrolase family 6protein Amino acid sequence identity of atrobrunneum is 83%.Explanation
The heat-resistance neutral cellulase Cel6C that the present invention separates is a kind of new cellulase.
The invention also discloses the recombinant expression carrier of encoding gene containing described heat-resistance neutral cellulase Cel6C,
Described recombinant expression carrier is preferably pPIC-cel6C.
The heat-resistance neutral cellulase Cel6C encoding gene of the present invention is inserted into the suitable restricted enzyme action of expression vector
Between site so that it is nucleotide sequence is exercisable to be connected with expression regulation sequence.One as the present invention most preferred
Embodiment, be preferably EcoR I that the heat-resistance neutral cellulase encoding genes of the present invention is inserted on plasmid pPIC9 and
Between Not I restriction enzyme site, make this nucleotide sequence be positioned at the downstream of AOX1 promoter and be regulated and controled by it, recombinated
Expression plasmid of yeast pPIC-cel6C.
It addition, the polynucleotide shown in SEQ ID No.1 or SEQ ID No.3 can be carried out excellent by those skilled in the art
Change to strengthen the expression efficiency in recombinant host cell or recombinant bacterial strain.Such as, the preference that can use target host cell is close
Numeral is optimized synthetic polyribonucleotides to strengthen the expression efficiency in target host cell.
The invention also discloses the recombinant host cell containing described recombinant expression carrier or recombinant bacterial strain.
Wherein, described recombinant host cell can be prokaryotic cell or eukaryotic cell；Preferably, described recombinant host cell is
Any one in Pichia pastoris, beer yeast cells or many types of inferior yeast cells, most preferably Pichia pastoris；
Described recombinant bacterial strain is selected from saccharomyces, genus Kluyveromyces, Schizasaccharomyces or methylotrophic yeast
Any one in bacterial strain；Wherein, described methylotrophic yeast strain preferably pichia belongs to bacterial strain.
The appropriate method that described polynucleotide or polypeptide introduce recombinant host cell is included: electrotransformation, protoplast
Method, chemical transformation etc., these operational approach are usually this area and are understood.
The invention also discloses a kind of method preparing described heat-resistance neutral cellulase Cel6C, comprise the following steps:
(1) with the recombinant expression carrier transformed host cell containing described heat-resistance neutral cellulase Cel6C encoding gene,
Obtain recombinant bacterial strain；
(2) cultivating recombinant bacterial strain, induction heat-resistance neutral cellulase Cel6C expresses；
(3) the heat-resistance neutral cellulase Cel6C also expressed by purification is reclaimed.
The present invention builds the recombiant plasmid pPIC-cel6C obtained containing ripe heat-resistance neutral cellulase encoding genes also
Convert Pichia pastoris GS115, it is thus achieved that recombinant pichia yeast strain GS115/cel6C.Heat-resistance neutral cellulase Cel6C is complete red
Having obtained secreting, expressing in yeast, expression is 37.5U/mL.The ratio of heat-resistance neutral cellulase Cel6C is lived as 378U/mg.
The optimum pH of heat-resistance neutral cellulase Cel6C and the measurement result of pH stability show, heat-resistance neutral cellulose
The optimum pH of enzyme Cel6C is 6.5, has the relative activity of more than 60% at pH5.0～9.0.Heat-resistance neutral cellulase Cel6C
Between pH 5.0-11.0 the most stable, in the range of this pH, process after 60min residual enzyme activity about 80%, this explanation
This enzyme has preferable pH stability neutral in alkaline range.The optimum temperature of heat-resistance neutral cellulase Cel6C and heat
Stability Determination result shows, the optimum temperature of heat-resistance neutral cellulase Cel6C is 70 DEG C, has good heat stability,
Incubation 1h at 60 DEG C, enzyme activity still retains more than 90%.
After measured, with barley for the K of heat-resistance neutral cellulase Cel6C during substratemValue is 1.285mg/mL,
Big response speed VmaxIt is 752 μm ol/min mg.
The measurement result that affects that heat-resistance neutral cellulase Cel6C enzyme is lived by different metal ion chemistry reagent shows, greatly
Most ions and chemical reagent vigor of heat-resistance neutral cellulase Cel6C when concentration is 1mM or 10mM the most substantially become
Change.But Mn2+, its vigor of CTAB strong inhibition；SDS can partly suppress its activity；Cu2+、Fe3+、Pb2+Hanker resistance to when 1mM
Property cellulase Cel6C enzyme activity impact inconspicuous, and can partly suppress its vigor when concentration is 10mM.
The heat-resistance neutral cellulase Cel6C that the present invention separates is in addition to may act on glucosan, for lichenin, carboxylic first
Base sodium cellulosate also has certain Degradation.The degradation capability of lichenin relative to barley is by it
60.5%, its to the degradation capability of sodium carboxymethyl cellulose relative to barley for 13.6%.
Add heat-resistance neutral cellulase Cel6C and barley malt juice viscosity and the rate of filtration affected measurement result table
Bright, the heat-resistance neutral cellulase Cel6C enzyme liquid adding 100U processes, its with compare, the rate of filtration improves 19.5%, glutinous
Degree reduction by 7.69%.
Heat-resistance neutral cellulase Cel6C of the present invention has good heat stability, in neutrality and alkaline range
Still keep high enzyme live, it is possible to degraded cellulose, hemicellulose, lichenin, sodium carboxymethyl cellulose, macromolecular polysaccharide,
The material such as protein or lipid；Heat-resistance neutral cellulase Cel6C or its encoding gene can be applicable to wine brewing, weaving, energy work
Industry, fibrous matter is converted into the process of fermentable sugars.
Such as in textile industry, heat-resistance neutral cellulase Cel6C of the present invention can be used to process the fibres such as cotton, fiber crops, viscose glue
Dimension cellulose fabric, cuts off young pilose antler, makes fabric table and bright and clean, bright in colour, can improve anti-pilling, it is possible to carry out soft finish, wind
Lattice become privileged arrangement etc..
In medicated beer processing industry, the beta glucan in Fructus Hordei Germinatus can be hydrolyzed to by heat-resistance neutral cellulase Cel6C efficiently
Glucose and oligosaccharide, destroying cells wall, make cellular content discharge in large quantities, improve wheat juice yield, reduce mash and glue
Spending thus shorten converted mash filtration time, obtained wheat juice is limpid, makes the beer color prepared shallow, and foam is the most lasting.
At feed industry, when preparing low fiber feedstuff, heat-resistance neutral cellulase Cel6C of the present invention can convert roughage such as
Wheat straw, residual wood, wheat bran, Caulis et Folium Oryzae, corn cob etc., be converted into monomer sugar, small molecular protein, low fat a portion crude fibre
Fat etc., reduce crude fiber content in feedstuff, are more digestible small-molecule substance macromolecules degradation.
In terms of paper-making industry, process hot paper pulp with heat-resistance neutral cellulase Cel6C of the present invention, carry out burr process, can
Improve the quality of paper.
Other the most many application of heat-resistance neutral cellulase Cel6C of the present invention also have a lot, will not enumerate.
Technical solution of the present invention compared with prior art has the advantages that
The optimum pH of the heat-resistance neutral cellulase Cel6C of the present invention is 6.5, has the phase of more than 60% at pH5.0～9.0
To enzymatic activity；Between pH 5.0-11.0 the most stable, in the range of this pH, process residual enzyme activity after 60min left 80%
The right side, illustrates that this enzyme has preferable pH stability neutral in alkaline range.The thermophilic of heat-resistance neutral cellulase Cel6C
Degree is 70 DEG C, has good heat stability, incubation 1h at 60 DEG C, and enzyme activity still retains more than 90%.The resistance to of the present invention is hankered
Property cellulase Cel6C can degraded cellulose, hemicellulose, lichenin, sodium carboxymethyl cellulose, macromolecular polysaccharide, egg
The material such as white matter or lipid, can be applicable to wine brewing, weaving or energy industry etc..
The term definition that the present invention relates to
Unless otherwise defined, all technology the most used herein and scientific terminology all have with of the art
Those of ordinary skill is generally understood identical implication.
Term " polynucleotide " or " nucleotide " mean sub-thread or the deoxyribonucleotide of bifilar form, deoxyribose core
Glycosides, ribonucleotide or ribonucleotide and polymer thereof.Unless specific restriction, the most described term is contained containing natural nucleotide
The nucleic acid of known analog, described analog has and is similar to the binding characteristic of reference nucleic acid naturally-produced to be similar to
The mode of nucleotide carries out metabolism.Unless other specific restriction, the most described term also means oligonucleotide analogs, and it includes
PNA (peptide nucleic acid(PNA)), DNA analog (thiophosphate, phosphamide acid esters etc.) used in antisense technology.Unless additionally referred to
Fixed, otherwise specific nucleic acid sequence is the most impliedly contained its conservative variant modified and (is included, but is not limited to degenerate codon take
Generation) and complementary series and the sequence clearly specified.Particularly, can by produce one of them or more than one selected by (or
All) the 3rd blended base of codon and/or the substituted sequence of deoxyinosine residue realize degenerate codon and replace
(Batzer et al., Nucleic Acid Res.19:5081 (1991)；Ohtsuka et al., J.Biol.Chem.260:2605-
2608(1985)；With Cassol et al., (1992)；Rossolini et al., Mol Cell.Probes 8:91-98 (1994)).
Term " stringent hybridisation conditions " means known low ionic strength and the condition of high temperature in the art.Generally,
Under high stringency conditions, probe and its target sequence hybridize can detection level than with other sequence hybridization can detection level higher
(such as exceeding background at least 2 times).Stringent hybridisation conditions is sequence dependent, will be different under different environmental conditions,
Longer sequence specific hybrid at relatively high temperatures.The preciseness hybridized by control or wash conditions can be identified and probe
The target sequence of 100% complementation.Detailed guidance for nucleic acid hybridization refers to relevant document (Tijssen, Techniques in
Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,"
Overview of principles of hybridization and the strategy of nucleic acid
assays.1993).More specifically, described high stringency conditions is typically selected to be less than distinguished sequence under regulation ionic strength pH
Heat fusion joint (Tm) about 5-10 DEG C.Tm is that 50% probe complementary with target hybridizes to during target sequence residing in the state of the equilibrium
Temperature (under specifying ionic strength, pH and nucleic acid concentration) (because target sequence is present in excess, so in equilibrium-like under Tm
Under state, the probe of 50% is occupied).High stringency conditions can be following condition: wherein in 7.0 to 8.3 times salinity of pH below about
1.0M Na ion concentration, typically about 0.01 arrives 1.0M Na ion concentration (or other salt), and temperature (includes for short probe
10 to 50 nucleotide of (but not limited to)) for be at least about 30 DEG C, and long probe (is included, but is not limited to more than 50
Nucleotide) for be at least about 60 DEG C.High stringency conditions also can realize by adding the destabilizing agent of such as Methanamide.For choosing
For selecting property or specific hybrid, positive signal can be the background hybridization of at least twice, is optionally 10 times of background hybridizations.Exemplary
Stringent hybridisation conditions can be as follows: 50% Methanamide, 5 × SSC and 1%SDS, cultivates at 42 DEG C；Or 5 × SSC, 1%SDS,
Cultivate at 65 DEG C, washing and washing in 0.1%SDS at 65 DEG C in 0.2 × SSC.Described washing can carry out 5,15,30,
60,120 minutes or the longer time.
Term " recombinant host cell strain " or " host cell " mean to comprise the cell of polynucleotide of the present invention, regardless of making
By which kind of method carry out inserting to produce recombinant host cell, the most directly absorb, transduce, known in f pairing or art
Other method.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated into host genome
In.Host cell can be prokaryotic cell or eukaryotic cell.
Term " promoter " means to be present in the upstream of genes of interest coded sequence, it is provided that RNA polymerase and correctly transcribing
The recognition site of initial necessary other factors, starts or instructs genes of interest to be transcribed into mRNA.
Term " exercisable connection " means functional connection between two or more elements, exercisable connection
Element can be adjacent or non-adjacent.
Term " converts " and means to be incorporated into heterologous DNA sequence host cell or organic method.
Term " is expressed " and is meant the transcribing and/or translating in cell of endogenous gene or transgenic.
Term " Rate activity (property) " is measuring of enzyme purity, it is intended that the vigor list of had enzyme in the protein of Unit Weight
Figure place, typically with enzyme activity unit/mg Representation of Proteins；In general, the Rate activity of enzyme is the highest, and enzyme is the purest.
Accompanying drawing explanation
Fig. 1 is recombinant pichia yeast strain GS115/cel6C fermentation weight in wet base and enzyme activity determination；
Fig. 2 is the heat-resistance neutral cellulase Cel6C of SDS-PAGE detection purification；Wherein, M is albumen Marker；Cel6C
For heat-resistance neutral cellulase Cel6C；
Fig. 3 is the optimum pH of heat-resistance neutral cellulase Cel6C；
Fig. 4 is the pH stability of heat-resistance neutral cellulase Cel6C；
Fig. 5 is the optimum temperature of heat-resistance neutral cellulase Cel6C；
Fig. 6 is the heat stability of heat-resistance neutral cellulase Cel6C.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
1, test material and reagent
1.1 bacterial strains and carrier
Humicola insolens (Humicola insolens Y1CGMCC 4573) is from Microbiological Culture Collection management committee of China
Member can distribute at common micro-organisms center, and its preserving number is: CGMCC No.4573.Yeast expression vector pPIC9 and bacterial strain
GS115 is purchased from Invitrogen company.
1.2 enzymes and other biochemical reagents
Restriction endonuclease is purchased from Fermentas company, and ligase is purchased from NEB company.Barley is purchased from Sigma company, its
It is all domestic reagent (all can be commercially available from common biochemical Reagent Company).
1.3 culture medium
(1) Humicola insolens produces spore culture medium is potato culture: 1000mL 200g Rhizoma Solani tuber osi liquor, 10g
Glucose, 25g agar, pH5.0.
(2) Humicola insolens cellulase induction culture medium: 30g/L Testa Tritici, 30g/L maize cob meal, 5g/L
(NH4)SO4、1g/L KH2PO4、0.5g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.2g/L CaCl2。
(3) Escherichia coli culture medium LB:1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0.
(4) Pichia sp. fermentation medium:
PTM trace salt: 0.6%CuSO4, 0.008%NaI2, 0.3%MnSO4, 0.02%Na2MoO4, 0.002%H3BO3,
0.05%CoCl2, 2%ZnCl2, 6.5%FeSO4, 0.5% sulphuric acid (v/v).
Culture propagation basal salt media (FBSM): 0.5%KH2PO4, 5%NH4H2PO4, 1.485%MgSO4, 1.82%
K2SO4, 0.093%CaSO4, 0.15%KOH, 0.00011%Biotin, 0.44%PTM trace salt, 2% glucose.
Culture propagation basis Salt treatment culture medium (FBIM): 0.5%KH2PO4, 5%NH4H2PO4, 1.485%MgSO4,
1.82%K2SO4, 0.093%CaSO4, 0.15%KOH, 0.00011%Biotin, 0.44%PTM trace salt, 0.5% methanol.
Na2HPO4-citrate buffer solution (0.1mol/L pH5.2): 0.2mol/L disodium hydrogen phosphate 536ml, 0.1mol/L
Citric acid 464ml, adjusts pH to 5.2 after mixing.
Illustrate: following example are not made the experimental methods of molecular biology illustrated, all with reference to " Molecular Cloning: A Laboratory
Guide " concrete grammar listed in (third edition) J. Pehanorm Brooker one book carries out, or according to test kit and product description
Carry out.
The clone of embodiment 1 Humicola insolens (Humicola insolens) cellulase encoding genes cel6C cDNA
Extract the total serum IgE of thalline when Humicola insolens grows 2d in cellulase induction culture medium, by its reverse transcription
After obtaining cDNA, as template, utilize specific primer cel6CF (5 '-GGGAATTCGCTCCCAGCCCCAAGAGC-3 ')
With cel6CR (5 '-GCGGCCGCTTAGTGGTGGTGGTGGTGGTGCCAGAACTTGAAGATGG-3 '), obtain through PCR amplification
Remove the cDNA fragment of the heat-resistance neutral cellulose enzyme gene cel6C of natural signals peptide, and be cloned into pEASY-Blunt carrier,
Sequence verification.
DNA complete sequence analysis result shows, the cDNA sequence of the heat-resistance neutral cellulose enzyme gene containing signal peptide sequence
Total length 1149bp, encodes 382 aminoacid and a termination codon, and its nucleotides sequence is classified as shown in SEQ ID No.3, its institute
The aminoacid sequence of coding is shown in SEQ ID No.4；18 aminoacid of its N end are the signal peptide sequence of its prediction, its amino
Acid sequence is shown in SEQ ID No.6, and the coded sequence of signal peptide is shown in SEQ ID No.5.Do not contain the maturation of signal peptide
Heat-resistance neutral cellulase Cel6C, its aminoacid sequence is shown in SEQ ID No.2, its coding nucleotides sequence be classified as SEQ
Shown in ID No.1.Therefore, the theoretical molecular of ripe heat-resistance neutral cellulase Cel6C is 40.0kDa.
By the cDNA sequence (shown in SEQ ID No.1) of heat-resistance neutral cellulose enzyme gene and the aminoacid sequence derived
(shown in SEQ ID No.2) carries out BLAST comparison in GenBank, this gene with derive from Chaetomium
The glycoside hydrolase family 6protein Amino acid sequence identity of atrobrunneum is 83%.Explanation
Heat-resistance neutral cellulase Cel6C is a kind of new cellulase.
The preparation of embodiment 2 heat-resistance neutral cellulase Cel6C
CDNA sequence (the SEQ of the heat-resistance neutral cellulose enzyme gene not containing signal peptide sequence that embodiment 1 is cloned
Shown in ID No.1) it is inserted on yeast expression vector pPIC9, it is allowed to be positioned at α-factor signal peptide downstream, builds restructuring table
Reach plasmid pPIC-cel6C.
Expression vector pPIC9 is carried out double digestion (EcoR I+Not I), connection is had without signal peptide sequence simultaneously
The cloning vehicle double digestion (EcoR I+Not I) of the cDNA sequence of heat-resistance neutral cellulose enzyme gene, cuts out purpose fragment and table
Reach carrier pPIC9 to connect, it is thus achieved that recombiant plasmid pPIC-cel6C also converts Pichia pastoris GS115, it is thus achieved that recombinant yeast pichia pastoris bacterium
Strain GS115/cel6C.
Take the GS115 bacterial strain containing recombiant plasmid, first after FBSM culture medium culturing makes its fast-growth 48h, then warp
FBIM culture medium inducing culture 108h.Sampling and measuring thalline weight in wet base and the vigor of cellulase in sweat, result is shown in Fig. 1.
The expression of heat-resistance neutral cellulase Cel6C is 37.5U/mL.Fermentation supernatant is with 0.1 μm membrane filtration impurity, then with match
Tuo Lisi 10kD filter membrane concentrates, and concentrated solution directly crosses the His-Trap post of Akta, collects enzyme peak alive.SDS-PAGE result shows,
Heat-resistance neutral cellulase Cel6C has obtained secreting, expressing (Fig. 2) in Pichia sp..
DNS method is used to carry out the activity analysis of heat-resistance neutral cellulase Cel6C: at pH6.0, under the conditions of 60 DEG C, 1mL's
Reaction system includes 100 dilution enzyme liquid (the heat-resistance neutral cellulase Cel6C of embodiment 2 preparation) suitable for μ L, 900 μ L Fructus Hordei Vulgaris
Dextran substrate (1%), reacts 10min, adds 1.5mL DNS and terminates reaction, boiling water boiling 5min.After cooling, 540nm measures OD
Value.1 enzyme unit (U) alive is defined as the enzyme amount discharging 1 μm ol reducing sugar the most per minute.Experimental result table
Bright, the ratio of heat-resistance neutral cellulase Cel6C is lived as 378U/mg.
The property testing of embodiment 3 heat-resistance neutral cellulase Cel6C
With the heat-resistance neutral cellulase Cel6C of embodiment 2 purification as object of study, the relevant nature of this enzyme is surveyed
Fixed.
1, the optimum pH of heat-resistance neutral cellulase Cel6C and the mensuration of pH stability
The heat-resistance neutral cellulase Cel6C of embodiment 2 purification is carried out enzymatic reaction to measure it under different pH
Optimum pH.Substrate barley, carries out fibre at 70 DEG C in the 0.2mol/L citrate-phosphate disodium hydrogen buffer of different pH
Dimension element enzyme activity determination.Result (Fig. 3) shows, the optimum pH of heat-resistance neutral cellulase Cel6C is 6.5, at pH5.0～9.0
There is the relative activity of more than 60%.Cellulase is 37 DEG C of process 60min in the buffer of above-mentioned various different pH, then
PH6.5 buffer solution system measures enzymatic activity, with the pH patience of studying enzyme at 70 DEG C.Result (Fig. 4) shows, heat-resistance neutral fiber
Enzyme Cel6C is the most stable between pH 5.0-11.0 for element, and after processing 60min in the range of this pH, residual enzyme activity is left 80%
The right side, this illustrates that this enzyme has preferable pH stability neutral in alkaline range.
2, the optimum temperature of heat-resistance neutral cellulase Cel6C and thermal stability determination
Being determined as at citrate-phosphate disodium hydrogen buffer (pH6.5) buffer solution system of the optimum temperature of cellulase
And under different temperatures, carry out enzymatic reaction.Temperature tolerance is determined as cellulase and locates under different temperatures (60 DEG C, 65 DEG C and 70 DEG C)
Reason different time, then at 70 DEG C, carry out enzyme assay.Enzyme reaction optimum temperature measurement result (Fig. 5) shows, heat-resistance neutral
The optimum temperature of cellulase Cel6C is 70 DEG C.The heat stability of enzyme test result indicate that (Fig. 6), heat-resistance neutral cellulase
Cel6C has good heat stability, incubation 1h at 60 DEG C, and enzyme activity still retains more than 90%.
3, the K of heat-resistance neutral cellulase Cel6CmThe mensuration of value
It is substrate with the glucosan of variable concentrations, at citrate-phosphate disodium hydrogen buffer (pH6.5) buffer solution system
In, measure enzymatic activity at 70 DEG C, calculate its KmValue.
After measured, with barley for K during substratemValue is 1.285mg/mL, maximum reaction velocity VmaxIt is 752 μ
mol/min·mg。
4, the impact that heat-resistance neutral cellulase Cel6C enzyme is lived by different metal ion chemistry reagent measures
In enzymatic reaction system, add different metal ions and the chemical reagent of variable concentrations, study it to enzymatic activity
Impact, final concentration of 1mM or 10mM of various materials.70 DEG C, measure enzymatic activity under the conditions of pH6.5.
Result shows (table 1), most of ions and the chemical reagent heat-resistance neutral cellulose when concentration is 1mM or 10mM
The vigor of enzyme Cel6C does not has significant change.But Mn2+, its vigor of CTAB strong inhibition；SDS can partly suppress its activity；Cu2 +、Fe3+、Pb2+When 1mM, the enzyme activity of heat-resistance neutral cellulase Cel6C is affected inconspicuous, and can when concentration is 10mM
Part suppresses its vigor.
The impact that heat-resistance neutral cellulase Cel6C enzyme is lived by table 1 different metal ion chemistry reagent
5, the substrate specificity of heat-resistance neutral cellulase Cel6C
Heat-resistance neutral cellulase Cel6C is in addition to may act on glucosan, and for lichenin, sodium carboxymethyl cellulose is also
There is certain Degradation.Its to the degradation capability of lichenin relative to barley for 60.5%, it is to carboxymethyl
The degradation capability of sodium cellulosate relative to barley for 13.6% (table 2).
The substrate specificity analysis of table 2 heat-resistance neutral cellulase Cel6C
*It is not detected by.
6, heat-resistance neutral cellulase Cel6C is added on barley malt juice viscosity and the impact of the rate of filtration
The size-reduced machine of barley malt processes, and crosses 0.2mm screen cloth, is dissolved in 100mL citrate-phosphate disodium hydrogen buffer.Add
Add 100 or the heat-resistance neutral cellulase Cel6C of 200U.Processing 30min at 45,50 DEG C the most respectively, 60 DEG C process 60min,
70 DEG C process 30min, finally boil 5min and terminate reaction.Experiment contrast is without cellulase.Measure it with filter paper to filter
Speed.Filtrate viscosity agent measures its viscosity numerical value.
Result (table 3) shows, add 100U enzyme liquid process, its with compare, the rate of filtration improve 19.5%, viscosity
Reduce by 7.69%.
Table 3 heat-resistance neutral cellulase Cel6C is on barley malt juice viscosity and the impact of the rate of filtration
Claims (11)
1. the encoding gene of the heat-resistance neutral cellulase Cel6C separated from Humicola insolens (Humicola insolens),
It is characterized in that, its nucleotides sequence is classified as shown in (a) or (b):
Polynucleotide shown in (a), SEQ ID No.1；Or
Amino acid whose polynucleotide shown in (b), coding SEQ ID No.2.
2. the heat-resistance neutral cellulase Cel6C of encoding gene coding described in claim 1, it is characterised in that its aminoacid sequence
It is classified as shown in SEQ ID No.2.
3. the encoding gene of the heat-resistance neutral cellulase Cel6C separated from Humicola insolens (Humicola insolens),
It is characterized in that, its nucleotides sequence is classified as shown in (a) or (b):
Polynucleotide shown in (a), SEQ ID No.3；Or
Amino acid whose polynucleotide shown in (b), coding SEQ ID No.4.
4. the heat-resistance neutral cellulase Cel6C of encoding gene coding described in claim 3, it is characterised in that its aminoacid sequence
It is classified as shown in SEQ ID No.4.
5. contain the recombinant expression carrier of encoding gene described in claim 1 or 3.
6. according to the recombinant expression carrier described in claim 5, it is characterised in that: described recombinant expression carrier is pPIC-
cel6C。
7. contain the recombinant host cell of recombinant expression carrier described in claim 5.
8. according to the recombinant host cell described in claim 7, it is characterised in that: described recombinant host cell is that Pichia sp. is thin
Any one in born of the same parents, beer yeast cells or many types of inferior yeast cells.
9. prepare the method for heat-resistance neutral cellulase Cel6C described in claim 2 or 4 for one kind, it is characterised in that include following
Step:
(1), with the recombinant expression carrier transformed host cell described in claim 5, it is thus achieved that recombinant bacterial strain；
(2), cultivating recombinant bacterial strain, induction heat-resistance neutral cellulase Cel6C expresses；
(3) the heat-resistance neutral cellulase Cel6C also expressed by purification, is reclaimed.
10. the encoding gene of heat-resistance neutral cellulase Cel6C described in claim 1 or 3 degraded cellulose, hemicellulose,
Application in lichenin, sodium carboxymethyl cellulose, macromolecular polysaccharide, protein or lipid material.
Described in 11. claim 2 or 4, heat-resistance neutral cellulase Cel6C is at degraded cellulose, hemicellulose, lichenin, carboxylic
Application in sodium carboxymethylcellulose pyce, macromolecular polysaccharide, protein or lipid material.
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| WO2008008070A2 (en) * | 2006-07-13 | 2008-01-17 | Dyadic International (Usa), Inc. | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
| WO2008025164A1 (en) * | 2006-08-31 | 2008-03-06 | Iogen Energy Corporation | Family 6 cellulases with increased thermostability, thermophilicity, and alkalophilicity |
| WO2011153516A2 (en) * | 2010-06-03 | 2011-12-08 | Mascoma Corporation | Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose |
| CN103074317A (en) * | 2012-11-08 | 2013-05-01 | 中国农业科学院饲料研究所 | High temperature neutral xylanase XYNA used for brewing beer and its gene and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008008070A2 (en) * | 2006-07-13 | 2008-01-17 | Dyadic International (Usa), Inc. | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
| WO2008025164A1 (en) * | 2006-08-31 | 2008-03-06 | Iogen Energy Corporation | Family 6 cellulases with increased thermostability, thermophilicity, and alkalophilicity |
| WO2011153516A2 (en) * | 2010-06-03 | 2011-12-08 | Mascoma Corporation | Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose |
| CN103074317A (en) * | 2012-11-08 | 2013-05-01 | 中国农业科学院饲料研究所 | High temperature neutral xylanase XYNA used for brewing beer and its gene and application |
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