CN103261409B - Mannanase, coding gene and production thereof - Google Patents

Abstract

The invention discloses a mannanase and the coding gene, a recombinant vector and a host cell comprising the gene, and expression of the recombinant vector in E.coli and production of the enzyme in yeast cells by using the recombinant vector. The present invention also provides a new wild type subspecies of Bacillus Megaterium, in particular, Bacillus Megaterium FB101.

Description

Its encoding gene of mannonase and production thereof

Technical field

The present invention relates to a kind of new mannase and encoding gene thereof, the recombinant vectors that contains this gene and host cell, and utilize recombinant vectors at this enzyme of expression in escherichia coli and utilize recombinant vectors in yeast cell, to produce this enzyme.The invention still further relates to a kind of newfound wild-type bacillus megaterium, specifically bacillus megaterium FB101, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation date is on September 13rd, 2010, and preserving number is CGMCC No.4162.

Background technology

Plant hemicellulose is the abundantest polysaccharide compound of nature after Mierocrystalline cellulose, mannosans and derivative thereof are the second largest components that forms hemicellulose, extensively be present in the endosperm, vegetable jelly (carob bean gum, locust bean gum, melon glue) of plant cell wall, seed, it is the chief component composition of all leguminous plants cell wallss, in other plant forage raw material, content is also very high, as corn, wheat, rapeseed meal and wheat bran etc.Mannosans, as a kind of component of hemicellulose, mainly exists with the form of glucomannan, polygalactomannan and gala glucomannan.

China's mannosans resource is very abundant, there is the mannosans of high-content in as konjaku, coconut and various plants glue (the alert glue of locust bean gum, field, melon glue) many plants, but people and most of domestic animals and fowls all do not secrete the enzyme of degraded mannosans, therefore only have small portion mannosans to use as food gel, major part is not yet developed effectively.

'beta '-mannase (is called again β-1,4-mannosans seminose lytic enzyme (β-1,4-mannan mannohydrolase)), referred to as 'beta '-mannase or β-1,4-D-mannase, EC3.2.1.78) be that a class can be hydrolyzed the mannooligo saccharide that contains β-Isosorbide-5-Nitrae-D-MANNOSE glycosidic bond, the inscribe lytic enzyme of mannocarolose (comprising mannosans, polygalactomannan, glucomannan etc.).

In recent years, along with the discovery of mannooligo saccharide physiological function, the enhancing of the rise of green feed and people's environmental consciousness, the research to 'beta '-mannase and utilization have entered a new stage.'beta '-mannase has been widely used in the numerous areas such as food, medicine, feed, papermaking, textile printing and dyeing, oil production, fine chemistry industry and biotechnology, is a kind of novel industrial enzyme, has very large potential using value.

In food service industry, beta-mannase enzyme liberating mannase generates manna oligosaccharide, have protect the liver, the physiological property such as antitumor, strengthening immunity, also can be used as sweeting agent; In paper-making industry, use 'beta '-mannase can obtain on the one hand, than simple chlorine bleaching, the better characteristics of pulp of alkaline extraction used, also can reducing the consumption of chlorine and alkali, reduce environmental pollution; Aspect oil production, 'beta '-mannase is the biological gel breaker of the high-quality of oil well petroleum fracturing liquid, have efficient, inexpensive, applied widely, to features such as low layer injury are little.

Mannosans has high-hydrophilic, in the digestive tube of monogastric animal, after a large amount of water suctions, has increased the viscosity of alimentary canal content, and opposing gastrointestinal peristalsis, directly affects animal digesting and assimilating nutritive substance.'beta '-mannase not only has the effect of general non-starch polysaccharide (NSP) enzyme---degraded NSP, reduce enteron aisle viscosity, promote digestion and the absorption of nutritive substance, and there is the secretion of promotion quasi-insulin growthing factor I GF-I, the effect of synthesizing, improving lean ratio of promotion protein.Therefore, 'beta '-mannase, as fodder additives, can be degraded to mannosans the oligose easily being absorbed by animal, helps digestion and the absorption of animal to nutritive substance, has greatly improved the utilization ratio of feed.

'beta '-mannase belongs to hemicellulose enzyme, is the wide spectrum induction type multifunctional enzyme with cellulase activity, is extensively present in animals and plants and microorganism.Because microorganism has the advantage such as easy cultivation, output height compared with animals and plants, the 'beta '-mannase of commercially producing all derives from microorganism mostly.The research of 'beta '-mannase at present mainly concentrates on the aspect such as the screening of zymogenic bacteria kind and the separation and purification of enzyme, the focus that is separated into the new 'beta '-mannase of searching of the clone of new 'beta '-mannase and highly active 'beta '-mannase.

The research of 'beta '-mannase has been obtained to larger progress in recent years both at home and abroad, but the research to 'beta '-mannase and application are mainly confined to the laboratory lab scale stage in China.Therefore, improve constantly Regulation Mechanism that bacterial classification produces enzyme level, research beta-mannase enzymic synthesis, enzyme gene clone and high expression level, carry out industrial application etc., be main from now on striving direction.

Due to constantly widening of 'beta '-mannase application, more and more higher to the requirement of the various 'beta '-mannases with special property in practice, although have been reported and obtain 'beta '-mannase from multiple-microorganism, but in industrial application, still lack high-quality enzyme source, particularly in feed industry, not only need the enzyme of high vigor, also need enzyme to keep enzymatic property under sour environment and hot conditions.Therefore, research and development there is good characteristic, be more suitable for practical application need efficient 'beta '-mannase significant.

Summary of the invention

For this reason, the invention provides following technical scheme, and obtained good technique effect.

The invention provides a kind of isolated polypeptide, it has β-Isosorbide-5-Nitrae-mannosans enzymic activity.In another embodiment, this polypeptide has β-Isosorbide-5-Nitrae-mannosans enzymic activity and following characteristic:

A) theoretical molecular is 39kDa;

B) theoretical pI value is 5.29;

C) optimal pH is 5.0-8.0, preferably 6.0-7.0, most preferably 6.5;

D) optimal reactive temperature is 35-60 DEG C, preferably 45-55 DEG C, most preferably 55 DEG C;

E), than vigor >11000U/mg, preferably >15000U/mg, most preferably is 16718.2U/mg; And

F) protease inhibitor is water-disintegrable, preferably stomach en-and trypsinase is had to resistance.

In another embodiment, the invention provides a peptide species, the aminoacid sequence of this polypeptide is SEQ ID NO:2.In another embodiment, the invention provides a peptide species, its comprise with the sequence homogeny of SEQ ID NO:2 be at least about 70%, aminoacid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%.In one embodiment, polypeptide of the present invention is by the polynucleotide encoding of hybridizing with the complementary strand of polynucleotide shown in SEQ ID NO.1 under rigorous condition.In one embodiment, polypeptide of the present invention is coded by allelotrope or the natural mutation of polynucleotide shown in SEQ ID NO.1.In one embodiment, polypeptide of the present invention comprises by process one or more amino acid whose replacements, the disappearance of aminoacid sequence shown in SEQ ID NO.2 and/or inserts derivative aminoacid sequence sequence.In a preferred implementation, described aminoacid sequence does not comprise the endogenous signal peptide sequence of the microorganism of originating.In a preferred embodiment, in described aminoacid sequence, the amino-acid residue corresponding to SEQ ID NO:2 position 153 is identical with the amino-acid residue of corresponding position in SEQ ID NO:2.

In one embodiment, the invention provides a kind of nucleic acid, its polypeptide of the present invention of encoding.In one embodiment, the invention provides a kind of nucleic acid, its encoding amino acid sequence is the polypeptide of SEQ ID NO:2.In one embodiment, the invention provides a kind of nucleic acid, its encoded packets containing the sequence homogeny of SEQ ID NO:2 be at least about 70%, the nucleic acid of aminoacid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%.In a preferred embodiment, in described aminoacid sequence, the amino-acid residue corresponding to SEQ ID NO:2 position 153 is identical with the amino-acid residue of corresponding position in SEQ ID NO:2.In another preferred embodiment, the invention provides a kind of nucleic acid, its nucleotides sequence is classified SEQ ID NO:1 as.In another preferred embodiment, the invention provides a kind of nucleic acid, it has the Nucleotide of 1-1008 position in nucleotide sequence SEQ ID NO:1.In another preferred embodiment, the invention provides a kind of nucleic acid, its comprise with the sequence homogeny of SEQ ID NO:1 be at least about 70%, nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%.In a preferred embodiment, in described nucleotide sequence, the nucleotide residue corresponding to SEQ ID NO:1 position 458 is identical with the nucleotide residue of corresponding position in SEQ ID NO:1.In another preferred embodiment, the invention provides a kind of nucleic acid, its comprise with SEQ ID NO:1 in the sequence homogeny of nucleotide sequence of 1-1008 position be at least about 70%, nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%.In a preferred embodiment, in described nucleotide sequence, the nucleotide residue corresponding to SEQ ID NO:1 position 458 is identical with the nucleotide residue of corresponding position in SEQ ID NO:1.In a preferred implementation, described nucleotide sequence does not comprise the endogenous signal peptide sequence of the microorganism of originating.

In one embodiment, the invention provides a kind of recombinant vectors, it comprises nucleic acid of the present invention.In one embodiment, recombinant vectors provided by the invention can comprise the carrier that is adapted at expressing in prokaryotic cell prokaryocyte.In one embodiment, recombinant vectors provided by the invention can comprise the carrier that is adapted at eukaryotic expression.In one embodiment, recombinant vectors provided by the invention can comprise the carrier that is adapted at expression in escherichia coli.In one embodiment, recombinant vectors provided by the invention can comprise and is adapted at the carrier of expressing in yeast or pichia yeast.Specifically, recombinant vectors provided by the invention can comprise pMD18-T carrier or pPIC9 carrier.

In one embodiment, the invention provides a kind of host cell, it comprises recombinant vectors of the present invention, and described recombinant vectors can be incorporated in the genome of described host cell.In one embodiment, host cell provided by the invention can be prokaryotic cell prokaryocyte.In one embodiment, host cell provided by the invention can be eukaryotic cell.In one embodiment, host cell provided by the invention can be colibacillary cell.In one embodiment, host cell provided by the invention can be the cell of yeast or pichia yeast.Specifically, host cell provided by the invention can be pichia yeast GS115.

On the other hand, the present invention also provides a kind of method of producing 'beta '-mannase, and it comprises the following steps successively:

I) cultivate host cell of the present invention,

Ii) induce it to express,

Iii) results expression product, and

Iv) optionally, purifying expression product.

On the other hand, the present invention also provides a kind of wild-type bacillus megaterium bacterial strain FB101, β-Isosorbide-5-Nitrae-mannase that its generation comprises aminoacid sequence SEQ ID NO:2, and/or comprise the nucleic acid that contains nucleotide sequence SEQ ID NO:1.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), and preservation date is on September 13rd, 2010, and preserving number is CGMCC No.4162.

On the other hand, the present invention also provides a kind of composition, in the polypeptide of the present invention that it contains significant quantity and bromatology or industrial acceptable vehicle or vehicle.Preferably, the composition described in the present invention is made up of the enzyme of one or more interpolations, and described enzyme can be selected from lower group: proteolytic enzyme, cellulase, beta-xylanase, powder-beta-dextrin enzyme, α-amylase, lipase, polygalacturonase or their mixture.Preferred, described composition can be used to feed, food, deinking and paper industry.

The present invention utilize genetic engineering means prepared can high efficient expression and secrete 'beta '-mannase recombinant production strain, realized the scale operation of 'beta '-mannase, and obtained the beta-mannase enzyme product of high-quality.The present invention is checked optimum temperature, Optimun pH, pH stability, the thermostability to enzyme and is analyzed than physico-chemical properties such as vigor by zymologic property, prove that β-Isosorbide-5-Nitrae-mannase of the present invention has good pH stability, good thermostability and protease inhibitor hydrolysis ability.

Brief description of the drawings

Fig. 1 is from nucleotide sequence SEQ ID NO:1 and the aminoacid sequence SEQ ID NO:2 of the beta-mannase gene BM-Man of bacillus megaterium FB101.

The recon structure iron of Fig. 2 BM-Man mannase gene on yeast plasmid pPIC9.

The optimal reaction pH of the bacillus megaterium FB101 mannase of Fig. 3 Pichia anomala expression, and with the comparison of subtilis (Bacillus subtilis) WL-7 mannase and subtilis MA139 mannase optimal reaction pH.

The optimal reactive temperature of bacillus megaterium FB101 mannase that Fig. 4 pichia yeast is expressed, and with the comparison of subtilis WL-7 mannase and subtilis MA139 mannase optimal reactive temperature.

The pH stability of bacillus megaterium FB101 mannase that Fig. 5 pichia yeast is expressed, and with the comparison of subtilis WL-7 mannase and subtilis MA139 mannase pH stability.

The thermostability of bacillus megaterium FB101 mannase that Fig. 6 pichia yeast is expressed, and with the comparison of subtilis WL-7 mannase and subtilis MA139 mannosans enzyme heat stability.

Enzyme activity and the bacterium weight in wet base of different time in the fermenting process of the 'beta '-mannase of Fig. 7 pichia yeast expression BM-Man gene source.

The SDS-PAGE electrophorogram of Fig. 8 fermented sample of different time in the fermenting process of the 'beta '-mannase of pichia yeast expression BM-Man gene source.

The SDS-PAGE electrophorogram of the purified 'beta '-mannase of the present invention of Fig. 9, molecular weight is about 39kDa.

The 'beta '-mannase of the BM-Man gene source that Figure 10 pichia yeast is expressed is to stomach en-and tryptic resistance.

The recombinate thermotolerance of 'beta '-mannase solid preparation of Figure 11 the present invention.

The recombinate package stability of 'beta '-mannase solid preparation of Figure 12 the present invention.

Embodiment

I. definition

Following used term is through whole specification sheets, and represents implication well-known to those skilled in the art, unless otherwise prescribed.

As wanted herein, " comprising " represents to exist as the feature of regulation mentioned in claim, entirety, step or composition, but do not get rid of existence or increase one or more features, entirety, step, composition or component.

As used herein, " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in viable cell and albumen do not have separation and purification, but same polynucleotide or albumen as from native state with in other materials that exist separately, for separation and purification.

As used herein, " 'beta '-mannase of separation " refers to that 'beta '-mannase does not basically contain natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be purified 'beta '-mannase with the purified technology of protein of standard.Substantially pure albumen can produce single master tape on non-reduced polyacrylamide gel.The purity of 'beta '-mannase can be used for amino acid sequence analysis.

" 'beta '-mannase " of the present invention is called again " β-Isosorbide-5-Nitrae-mannase ", can be recombinant protein (polypeptide), native protein, synthetic proteins, preferably recombinant protein.'beta '-mannase of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology for example, to produce from protokaryon or eucaryon host (, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to recombinant production scheme, 'beta '-mannase of the present invention can be glycosylated, can be maybe nonglycosylated.'beta '-mannase of the present invention also can comprise or not comprise initial methionine residues.

Term used herein " beta-mannase enzymic activity " refers to that one can cut off the ability of the β-Isosorbide-5-Nitrae-D-MANNOSE glycosidic bond on mannooligo saccharide, mannocarolose (comprising mannosans, polygalactomannan, glucomannan etc.) sugar chain at random.This enzyme has 4 substrate binding sites, is respectively W72, W172, W298 and W302; The binding site of metal ion is H1-H23-E336; Catalytic center is made up of 6 binding site structure rings, is respectively ring 1 (F37-M47), ring 2 (S103-A134), ring 3 (F162-N185), ring 4 (Y215-I236), ring 5 (P269-Y278) and ring 6 (W298-G309).

The present invention also comprises fragment, derivative and the analogue of 'beta '-mannase.As used herein, term " fragment ", " derivative " refer to " analogue " biological function or the active albumen that maintenance is substantially identical with the natural 'beta '-mannase of the present invention.Beta-mannase enzyme fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted albumen of non-conservation amino-acid residue (preferably conservative amino acid residue), and the amino-acid residue of such replacement can not be also to be encoded by genetic code, or (ii) in one or more amino-acid residues, there is the albumen of substituted radical, or (iii) maturation protein and another compound (such as extending the compound of albumen transformation period, for example polyoxyethylene glycol) merge the albumen that forms, or (iv) additional aminoacid sequence be fused to this protein sequence and the albumen that forms (as leader sequence or secretion sequence or be used for sequence or the proteinogen sequence of this albumen of purifying, or with the fusion rotein of the formation of antigen I gG fragment).According to instruction herein, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

The present invention also provides 'beta '-mannase and variant form thereof.In the present invention, term " 'beta '-mannase " refers to have polypeptide or the protein of beta-mannase enzymic activity, can be the albumen that comprises or have SEQ ID NO:2 sequence.This term also comprises having and variant form 'beta '-mannase identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): in SEQ ID NO.2 sequence disappearance, insert and/or replace and one or morely (be generally 1-30, preferably 1-20, more preferably 1-10,1-5 best) amino acid, and add one or several (being generally in 20 at its C-terminal and/or N-terminal, being preferably in 10, is more preferably in 5) amino acid obtain sequence.For example, in the art, while replacement with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, the function of adding or several amino acid and conventionally also can not change protein at C-terminal and/or N-terminal.These variant forms also can comprise with the sequence homogeny of SEQ ID NO.2 be at least about 70%, aminoacid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%; Preferably, in this series of variation, the amino-acid residue corresponding to SEQ ID NO:2 position 153 is identical with the amino-acid residue of corresponding position in SEQ ID NO:2.

The variant form of this 'beta '-mannase also comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, albumen that can be coded with the DNA of the DNA hybridization of 'beta '-mannase under high or low stringency condition and polypeptide or the albumen that utilizes the antiserum(antisera) of anti-'beta '-mannase to obtain.The present invention also provides other albumen, as the fusion rotein that comprises 'beta '-mannase or its fragment.Except the albumen of total length almost, the present invention has also comprised the soluble fragments of 'beta '-mannase.Conventionally, this fragment have beta-mannase enzyme sequence at least about 10 continuous amino acids, conventionally at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

The present invention also provides the analogue of 'beta '-mannase.The difference of these analogues and natural 'beta '-mannase can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These albumen comprise genetic variant natural or induction.Induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can obtain by site-directed mutagenesis method or the biological technology of other known moleculars.

This analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has the analogue of non-natural amino acid (as β, gamma-amino acid) that exist or synthetic.Should be understood that 'beta '-mannase of the present invention is not limited to the above-mentioned representative albumen of enumerating.(conventionally the not changing primary structure) form of modification comprises: in body or the chemically derived form of external albumen as acetylize or carboxylated.Modify and also comprise glycosylation, as those carry out albumen glycosylation modified and that produce in procedure of processing in the synthetic and processing of albumen or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and completes by albumen is exposed to.Modified forms also comprises the have phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the albumen that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

The present invention also provides isolated polypeptide or the albumen of aminoacid sequence shown in a kind of SEQ of comprising ID NO.2.Preferably, described polypeptide or albumen are coded by polynucleotide or its degenerate sequence shown in SEQ ID NO.1.In the present invention, " 'beta '-mannase conservative property variant protein (polypeptide) " refers to compared with the aminoacid sequence with SEQ ID NO.2, have 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid are replaced, are lacked and/or insert and obtain by the similar or close amino acid of character at the most best, and still have the albumen of beta-mannase enzymic activity.There is family's existing clearly definition in this area of the amino-acid residue of similar side chain.These families comprise amino acid (for example Methionin with basic side chain, arginine, Histidine), there is amino acid (for example aspartic acid of acid side-chain, L-glutamic acid), there is amino acid (for example glycine of uncharged polar side chain, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), there is amino acid (for example L-Ala of non-polar sidechain, α-amino-isovaleric acid, leucine, Isoleucine proline(Pro), phenylalanine, methionine(Met), tryptophane), there is amino acid (for example Threonine of β-branched building block, α-amino-isovaleric acid, Isoleucine) and there is amino acid (for example tyrosine of aromatic side chain, phenylalanine, tryptophane, Histidine).Therefore, in beta-mannase zymoprotein, use from another amino-acid residue of the same side chain class and replace one or several site, will can substantially not affect its mannosans enzymic activity.In addition, as well known to those skilled in the art, in gene clone operation, usually need to design suitable restriction enzyme site, this certainly will introduce one or more incoherent residues at expressed albumen end, and this does not affect the activity of target protein.And for example for construction of fusion protein, promote the expression of recombinant protein, obtain the recombinant protein being automatically secreted into outside host cell, or be beneficial to the purifying of recombinant protein, usually need the N-end to recombinant protein by some aminoacid addition, in other appropriate area in C-end or this albumen, for example, include but not limited to, applicable joint peptide, signal peptide, leading peptide, end extends, glutathione S-transferase (GST), maltose E is in conjunction with albumen, albumin A, as the label of 6His or Flag, or the proteolytic ferment site of Xa factor or zymoplasm or enteropeptidase.

Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature albumen can be identical with the coding region sequence shown in SEQ ID NO.1 or the varient of degeneracy.As used herein, " varient of degeneracy " refers to that encoded packets contains the protein of SEQ ID NO.2 in the present invention, but with the differentiated nucleotide sequence of coding region sequence shown in SEQ ID NO.1.

The polynucleotide of the maturation protein of coding SEQ ID NO.2 comprise: the encoding sequence of an encoding mature albumen; The encoding sequence of maturation protein and various additional code sequence; Encoding sequence (with optional additional code sequence) and the non-coding sequence of maturation protein.

Term " polynucleotide of proteins encoded " can be the polynucleotide that comprise this albumen of encoding, and can be also the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the albumen of identical aminoacid sequence or the fragment of albumen, analogue, derivative and variant form with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of albumen of its coding.

Again in one embodiment, the albumen with beta-mannase enzymic activity of the present invention comprise by as under rigorous condition with sequence table in SEQ ID NO.1 as shown in the nucleotide sequence coded aminoacid sequence of nucleotide sequence hybridization.As used herein, " under rigorous condition, to hybridize " be hybridization and the cleaning condition that the nucleotide sequence for describing typical at least 60% homology each other still can phase mutual cross to term.Preferably, rigorous condition is such condition, has each other with this understanding at least 65%, the sequence of more excellent at least 70% and even preferred at least 80% or higher homology generally still can phase mutual cross.This rigorous condition is that those of ordinary skill in the art are known.One of rigorous condition is preferably, and limiting examples is: (1) at the hybridization compared with under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, and 0.1%SDS, 0 DEG C; Or in (2) when hybridization, is added with denaturing agent, 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) only at the homogeny between two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the albumen of interfertile polynucleotide encoding has identical biological function and activity with the maturation protein shown in SEQ ID NO:2.

The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment ", containing 15 Nucleotide, is at least better at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding 'beta '-mannase.

Nucleic acid of the present invention can have nucleotide sequence SEQ ID NO:1, also can have the Nucleotide of 1-1008 position in nucleotide sequence SEQ ID NO:1.Nucleic acid of the present invention can comprise with the sequence homogeny of SEQ ID NO:1 be at least about 70%, nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%.In a preferred implementation, the nucleotide residue corresponding to SEQ ID NO:1 position 458 in this nucleotide sequence is identical with the nucleotide residue of corresponding position in SEQ ID NO:1.Nucleic acid of the present invention also can comprise with SEQ ID NO:1 in the sequence homogeny of nucleotide sequence of 1-1008 position be at least about 70%, nucleotide sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%.In a preferred implementation, the nucleotide residue corresponding to SEQ ID NO:1 position 458 in this nucleotide sequence is identical with the nucleotide residue of corresponding position in SEQ ID NO:1.

Protein in the present invention preferably provides with the form separating with polynucleotide, is more preferably purified to homogeneous.

'beta '-mannase Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.In the time that sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment amplifying for each time is stitched together by proper order.

Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.

In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic multiple small segments, and then connect and can obtain the fragment that sequence is very long.

At present, can be completely obtain the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA moleculars as known in the art (or as carrier) and cell.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.

Method (Saiki etc., the Science1985 of application round pcr DNA amplification/RNA; 230:1350-1354) be optimized for and obtain gene of the present invention.While being particularly difficult to obtain the cDNA of total length from library, can preferably use RACE method (RACE-cDNA end rapid amplifying method, Elizabeth Scotto – Lavino etc., NATURE PROTOCOLS2006, the 1st volume the 6th phase: 2555-2562. and Elizabeth Scotto – Lavino etc., NATURE PROTOCOLS2006, the 1st volume the 6th phase: 2742-2745), primer for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment increasing by gel electrophoresis separation and purifying.

In one embodiment, the inventor extracts the genomic dna of bacillus megaterium FB101, according to the conserved sequence design degenerated primer of genus bacillus (as bacillus megaterium) source 'beta '-mannase, obtain the full length sequence of coding 'beta '-mannase by PCR.Result obtains the nucleotide sequence as shown in SEQ ID NO.1.

The present invention also relates to the recombinant vectors that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with recombinant vectors of the present invention or 'beta '-mannase encoding sequence, and produce the method for albumen of the present invention through recombinant technology.

By conventional recombinant DNA technology (Science, 1984; 224:1431), polymerized nucleoside acid sequence of the present invention can be used to the 'beta '-mannase of expression or Restruction.In general there are following steps:

(1) with the polynucleotide (or its varient) of coding 'beta '-mannase of the present invention, or transform or the suitable host cell of transduceing with the recombinant expression vector that contains these polynucleotide;

(2) host cell of cultivating in suitable substratum;

(3) separation, protein purification from substratum or cell.

Again in one aspect, the carrier that the present invention relates to contain described beta-mannase gene, import the recombinant host cell of described carrier or described gene and in host cell, express the method for described 'beta '-mannase.Term used herein " recombinant expression vector " and " recombinant vectors " are used interchangeably, refer to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers, these carriers can copy and stablize in host, a key character of these recombinant vectorss is conventionally to contain replication orgin, promotor, marker gene and translation controlling elements, and applicable recombinant vectors includes but not limited in the present invention: yeast plasmid.Recombinant expression vector of the present invention comprises to be suitable for the nucleic acid of the present invention of nucleic acid expression-form in host cell, this means that recombinant expression vector comprises one or more condition sequences based on selecting for the host cell of expressing, itself and exercisable connection of nucleic acid of expressing.In recombinant expression vector, " exercisable connection " refers to that the nucleotide sequence of object is connected in the mode that allows nucleotide sequence to express with regulating sequence.Those skilled in the art knows and can be used for building containing 'beta '-mannase DNA sequences encoding and suitable transcribing/the translate method of the expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTR and some other known promotor of can controlling gene expressing in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

Those skilled in the art will appreciate that the design of recombinant expression vector can be depending on the factor such as selection, required protein expression level of the host cell transforming as wish.Expression vector of the present invention can be imported to host cell, to produce the albumen or the peptide that comprise fusion rotein.

In addition, recombinant expression vector preferably comprises one or more selected markers, with the phenotypic character of host cell that is provided for selecting transforming, as for eukaryotic Tetrahydrofolate dehydrogenase, neomycin resistance, or for colibacillary tsiklomitsin or amicillin resistance.

In one embodiment, the 'beta '-mannase encoding sequence of the present invention with endogenous signal coding sequence is not connected with the pPIC9 carrier of XhoI and NotI double digestion after NotI double digestion through XhoI, obtains yeast recombinant expression vector PIC9-BMMan.

Comprise above-mentioned suitable DNA sequence dna and the suitable recombinant vectors of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.Herein being called again recipient cell at term used " host cell ", referring to the cell that can receive and hold recombinant DNA molecules, is the place of recombination amplification, and desirable recipient cell should meet and is easy to obtain and breeds two conditions." host cell " of the present invention can comprise prokaryotic cell prokaryocyte and eukaryotic cell, specifically comprises bacterial cell, yeast cell, insect cell and mammalian cell.

Recombinant expression vector of the present invention can be designed at protokaryon or eukaryotic expression beta-mannase zymoprotein.Thereby, the present invention relates to import the host cell of recombinant expression vector of the present invention, preferred pichia spp.Host cell can be any protokaryon or eukaryotic cell, representative example has: intestinal bacteria, streptomyces, the bacterial cell of Salmonella typhimurium, fungal cell is as yeast, vegetable cell, the insect cell of fruit bat S2 or Sf9, the zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc., comprising but be not limited to those above-mentioned host cells.Described host cell is the various cells that are beneficial to gene product expression or fermentative production preferably, and this type of cell has been well known and conventional, for example various Bacillus coli cells and yeast cell.In an embodiment of the invention, select the host cell of Pichia pastoris GS115 construction expression 'beta '-mannase.

Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.

With recombinant DNA transformed host cell can be with transforming or the routine techniques well known to those skilled in the art such as transfection carry out.As used herein, term " conversion " and " transfection ", " joint " and " transduction " mean well known in the art various by exogenous nucleic acid (for example, linear DNA or RNA are (for example, linearized vector or DNAcarrier free independent gene construct)) or the nucleic acid of carrier format is (for example, plasmid, clay, phage, phasmid, phagemid, transposon or other DNA) import the technology of host cell, comprise calcium phosphate or calcium chloride co-precipitation, the transfection of DEAE-mannosans-mediation, fat transfection, natural competence, transfer or the electroporation of chemistry mediation.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl ₂method processing, step used is well-known in this area.Another kind method is to use MgCl ₂.If needed, transform and also can be undertaken by the method for electroporation.In the time that host cell is eukaryotic cell, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packaging etc.

The transformant obtaining can be cultivated by ordinary method, expresses 'beta '-mannase of the present invention.According to host cell used, in cultivation, substratum used can be various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.

Extracellular can be expressed or be secreted into recombinant protein in the above methods in cell or on cytolemma.If needed, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

In one embodiment, by yeast (routine Pichia pastoris GS115) the fermentative production 'beta '-mannase that comprises 'beta '-mannase encoding sequence of the present invention, and by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography have obtained the target protein of pure enzyme form.

The purposes of 'beta '-mannase of the present invention includes, but is not limited to: for being hydrolyzed mannosans, polygalactomannan or glucomannan etc.'beta '-mannase of the present invention has the effect of excellent hydrolysis mannosans, polygalactomannan or glucomannan etc., and has good thermostability and pH stability, and application prospect is good.

The present invention also provides a kind of composition, in the 'beta '-mannase that it contains significant quantity and bromatology or industrial acceptable vehicle or vehicle.This class vehicle comprises (but being not limited to): damping fluid, water etc.It can be made into solution or pulvis etc.Described " significant quantity " refers to can bring into play the function of 'beta '-mannase or active and can received amount.In use, described significant quantity can be determined easily according to the enzymic activity of described 'beta '-mannase in this area.

The present invention utilizes genetic engineering means to prepare the recombinant production strain that can express 'beta '-mannase, and has obtained the 'beta '-mannase of high-quality.The present invention is identified optimum temperature, Optimun pH, pH stability, the thermostability to enzyme and is analyzed than physico-chemical properties such as vigor by zymologic property, prove that 'beta '-mannase of the present invention has good pH stability, good thermostability and protease inhibitor hydrolysis ability.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.

The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as Sambrook etc., " molecular cloning: lab guide " (New York, United States: press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), 1989); Zhao Yongfang etc., Measurement for Biochemistry principle and application thereof (second edition) (Science Press, 2008); Zhu Jian etc., the condition described in Biochemistry Experiment [M] (Shanghai science tech publishing house, 1995), or the condition of advising according to manufacturer is carried out.Unless otherwise indicated, per-cent and umber are all calculated by weight.

I. experiment material and reagent

1. bacterial strain and carrier:

Bacillus coli DH 5 alpha and T carrier pMD18-T are purchased from Novartis's base (Novagen) company (Australia comes to obtain (NORTH RYDE NSW, AUSTRALIA) in north);

the light carrier system of-T ( -T Easy Vector systems) purchased from Pu Luomaige (Promega) company No. 2800, WH road, University of Wisconsin-Madison Xin Zhou Madison ((2800Woods Hollow Road, Madison, WI53711USA));

Pichia yeast GS115 and expression vector pPIC9 are all purchased from hero (Invitrogen) company (California, USA Carlsbad city (Carlsbad, CA, USA)).

2. enzyme and other biochemical reagents:

Restriction enzyme, DNA marker thing, protein label are all purchased from the (Fermentas (MBI) of Fu Meitaisi company (MBI) of No. 830, Burlinton city, Ontario, Canada Ha Lingdunlu, 830Harrington Court, Burlington, Ontario, Canada);

Beta-mannase enzyme activity determination substrate Viscogum BE is purchased from company of Sigma (Sigma) (St. Louis city (St.Louis, MO, USA));

Other conventional reagent are purchased from Chinese Shanghai biotechnology company limited or import.

3. substratum:

LB substratum, YPD, YPAD substratum are all with reference to the pichia yeast operational manual preparation of hero company.

4. method

4.1 'beta '-mannase vigour-testing methods

Enzyme work described in various embodiments of the present invention, enzyme activity, enzymic activity all refer to beta-mannase enzyme activity, and available Viscogum BE detects as substrate.Wherein, enzyme activity quantitative assay adopts international DNS (3,5-dinitrosalicylic acid) method (referring to Miller G.L., Apal.Chem., 1959,31:426).

Specifically, accurately pipette the beta-mannase substrate solution of 0.5ml0.5% (m/v) (with 0.5%NaOH solution dissolving Viscogum BE, after heating is dissolved it completely, regulate the solution of pH to 6.5 acquisition with glacial acetic acid) to 5ml scale test tube, put into 50 DEG C of water-bath preheating 5min.Enzyme liquid sample is done to 10 times of gradient dilutions with Sodium phosphate dibasic-citrate buffer solution of 50mM pH6.5, select the gratifying extent of dilution of color developing effect (absorbancy is between 0.2～0.4).The enzyme liquid having diluted according to selected extent of dilution (N) is put into 50 DEG C of water-bath preheating 5min, then get 0.5ml and join in the test tube that contains substrate solution, after concussion mixes, at 50 DEG C, be accurately incubated 10min.Add again 1.0ml DNS reagent (DNS reagent: take Seignette salt 182.0g, be dissolved in 800mL distilled water, heating (being no more than 50 DEG C), in hot solution, add successively 3,5-dinitrosalicylic acid 6.3g, NaOH20.96g, phenol 5.0g, sodium sulphite anhydrous 99.3 5.0g, stirs (heating) to dissolving completely, is coolingly settled to 1000mL with distilled water afterwards, if solution is not clarified, with water graceful (Whatman) l filter paper filtering, room temperature preservation, in brown bottle, is at room temperature placed and is used afterwards for 7 days.), after concussion mixes, boiling water heating 10min, is then cooled to rapidly room temperature, with termination reaction colour developing.With spectrophotometer in the absorbance A of 540nm place assaying reaction liquid (as occurred muddiness, need, by centrifugal reaction solution 4000rpm 10min, get supernatant liquor).

Blank is for first joining same 0.5ml dilution enzyme solution (preheating 5min in 50 DEG C of water-baths) in 5ml scale test tube, add 1.0ml DNS reagent, after concussion mixes, add again 0.5ml substrate solution (preheating 5min in 50 DEG C of water-baths), at 50 DEG C, be accurately incubated 10min, boiling water heating 10min, then be cooled to rapidly room temperature, in 540nm place assaying reaction liquid (as there is muddiness, need, by centrifugal reaction solution 4000rpm 10min, get supernatant liquor) absorbance A ₀.

The following development criteria curve of drawing enzymic hydrolysate (seminose): by the seminose of 1.6umol/ml Sodium phosphate dibasic-citrate buffer solution (50mM for reference liquid, pH6.5) be diluted to 1.60,1.28,0.96,0.64, the solution of 0.32umol/ml, react by the operation steps of above-mentioned sample determination one.Taking mannose concentration (C, umol/ml) as ordinate zou, taking absorbance (A) as X-coordinate, drawing standard curve, lists equation of linear regression (C=KA+b).

Beta-mannase unit of enzyme activity definition: it is 1 unit of activity (U) that the 'beta '-mannase per minute of unit vol decomposes mannosans (Viscogum BE) generation 1 μ mol seminose.

The beta-mannase enzyme activity U of sample calculates according to formula below:

$U=\frac{K\×(A-A_0)+b}{t\×V}\×V_0\×N$

In formula:

U---sample beta-mannase enzyme activity, U/mL;

The slope of K---typical curve;

The light absorption value of A---sample solution;

A ₀---the light absorption value of blank;

The intercept of b---typical curve;

T---the reaction times, min;

V---the add-on of enzyme liquid in reaction system, ml;

V ₀---the cumulative volume of reaction system, ml;

N---sample extension rate;

Each mensuration is all got the arithmetical av of two parallel test results, retains integer.

The formula that the ratio vigor of 'beta '-mannase is pressed face calculates:

Ｕ _ｃ＝Ｕ/ｃ

Wherein: U _c---sample beta-mannase specific enzyme activity, U/mg;

U---sample beta-mannase enzyme activity, U/ml;

C---the protein content in sample solution, mg/ml.

Purification X calculates according to formula below:

X＝U _C/U _C0

Wherein: X---purification;

U _c---the ratio vigor of 'beta '-mannase sample after purifying, U/mg;

U _c0---the ratio vigor of 'beta '-mannase sample before purifying, U/mg.

4.2 protein and nucleic acid electrophoresis method

Protein electrophorese and nucleic acid electrophoresis are conventional experimental technique, can be referring to conventional biological chemistry book of reference, as Zhao Yongfang etc., " Measurement for Biochemistry principle and application thereof " (second edition) (Science Press, 2008) and molecular biology book of reference, as people such as Sambrook, " molecular cloning: lab guide " (New York, United States: press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), 1989).

If not otherwise specified, protein electrophorese mentioned in this patent all refers to SDS-PAGE, and experiment parameter is: 12% separation gel, 5% concentrated glue; 120V constant voltage electrophoresis 30 minutes, and then 150V constant voltage electrophoresis 60 minutes; Running gel coomassie brilliant blue staining.

Nucleic acid electrophoresis, DNA electrophoresis etc. mentioned in this patent all refer to agarose gel electrophoresis, and experiment parameter is: 1% agarose, 120V constant voltage electrophoresis 40 minutes, adopts EB (ethidium bromide) dyeing in glue.

II. embodiment

The separation of embodiment 1 'beta '-mannase source strain

'beta '-mannase source strains separation is from the pedotheque of Zhongshan Park, Chinese Xiamen.

Concrete, in order to separate 'beta '-mannase source bacterial strain, gather the pedotheque of Zhongshan Park, Xiamen, be suspended in sterilized water, and dilution, then, the suspension of different samples is inoculated in the LB substratum without agar, then at 15 DEG C, 20 DEG C, 25 DEG C and 30 DEG C, cultivates 1-2 days respectively.Then utilize DNS method (concrete described in 4.1 " 'beta '-mannase vigour-testing methods ") to measure the beta-mannase activity under differing temps, in nutrient solution and the cytoclasis liquid of different samples.In nutrient solution, beta-mannase enzymic activity can be detected, illustrate that these bacterial strains have the 'beta '-mannase of secretor type.

First select the sample with beta-mannase enzymic activity, and judge that its suitable culture temperature, as 30 DEG C, is coated on after being diluted on the LB flat board that contains 1.5% agar, be placed in 30 DEG C of incubators and cultivate 1 day.Select the different bacterium colonies with various forms, be inoculated in incubated overnight in 5ml LB substratum.Measure the beta-mannase enzymic activity of each bacterium colony cell culture fluid, finally in selected bacterium colony, select the bacterial strain of the highest beta-mannase enzymic activity of demonstration, this bacterial strain be numbered FB101.

The signature analysis of embodiment 2 'beta '-mannase source bacterial strain FB101

By gram staining method, the bacillus megaterium FB101 bacterial strain separating in embodiment 1 is defined as to gram-positive microorganism.This bacterium is typical bacillus, has under the microscope the size similar to bacillus megaterium.Further study its some physiological-biochemical characteristics.

Result shows, this bacterial strain is Gram-positive aerobic bacteria, shaft-like, end round, single or be short chain and arrange, diameter is 1.2～1.5 × 2.0～4.0 microns, can move.Brood cell is 1.0～1.2 × 1.5～2.0 microns, ellipse, and middle life or inferior end are raw.Its optimum growth temp is 30 DEG C, aerobic, can be observed under opticmicroscope.The 16s rRNA of this bacterium has been carried out measuring (the raw work in Shanghai), and by using BLAST and Multiple Sequence Alignment program CLUSTAL W to compare the 16s rRNA sequence of this bacterium and the reference sequences in GenBank.Result shows, between the base sequence of its 16s rRNA and bacillus megaterium B188, CNR9 and UFV-E26 etc., has 99% consistence.The result of study of form, physio-biochemical characteristics and 16s rRNA based on selected bacterial strain, determines that this bacterial strain is new subspecies for bacillus megaterium.

According to the recording mechanism using in above result and research process, this bacterial strain is named as " bacillus megaterium FB101 " (Bacillus megaterium FB101), and submit to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) to give preservation on September 13rd, 2010, preserving number is CGMCC No.4162.

Clone and the acquisition of embodiment 3 bacillus megaterium β-Isosorbide-5-Nitrae-beta-mannase coding genes

The extraction of genomic dna: with nutrient broth medium (peptone 10.0g, extractum carnis 3.0g, sodium-chlor 5.0g, distilled water 1000ml, pH7.2-7.4) the bacillus megaterium FB101 bacterial strains that separate in 30 DEG C of cultivation embodiment 1 3～4 days, to cell concentration OD _600nmbe 0.5～0.8.Get this cultivation bacterium liquid 50ml, the centrifugal 10min of 10000rpm, gets 50mg thalline and adds 500 μ l sterile water wash, centrifuging and taking precipitation.Precipitation Eddy diffusion is in the lysozyme soln of 500 μ l1mg/ml, bathe after 30min in 37 DEG C of temperature, the N,O-Diacetylmuramidase liquid of adding again 100 μ l1mg/ml continues insulation 30min in 40-50 DEG C, after transparent to bacterium liquid, add 10%SDS to final concentration 2% (m/v), stir about 5min significantly declines to bacterium fluid viscosity, and the centrifugal 10min of 15000rpm removes fragment.Supernatant is used isopyknic phenol, phenol successively: chloroform (1:1), chloroform extracting.Get the Virahol normal temperature precipitation 10min that chloroform extracting gained upper solution adds 0.6-1 times of volume.The centrifugal 15min of 16000rpm.Precipitation is cleaned with 70% (v/v) ethanol, and low-speed centrifugal dries rear use 30 μ l sterilized waters by precipitation and dissolves, for subsequent use.

The clone of beta-mannase coding gene: comprehensively compare the DNA sequence dna of genus bacillus source beta-mannase coding gene in NCBI (National Center for Biotechnology Information) database, and design thus degenerated primer FI:5 '-TGAAGCGCATACTGTGKCKCCTG TRAATCCTAATGCC-3 ' (SEQ ID NO:3) and RI:5 '-TCAYTCAACGATTGGC GTTAAAGAATCRCCRYTCC-3 ' (SEQ ID NO:4).

Carry out pcr amplification taking bacillus megaterium FB101 bacterium genomic dna as template.PCR reaction parameter is: after 94 DEG C of sex change 3min, be cooled to 4 DEG C; Then 94 DEG C of sex change 30sec, 50 DEG C of annealing 30sec, 72 DEG C are extended 1min, 32 circulations; Last 72 DEG C of insulation 10min.Obtain the full-length gene fragment of 'beta '-mannase, carry out agarose gel electrophoresis qualification and reclaim test kit ((State of Georgia, US castor back of the body loop 1850-E (the 1850-E Beaver Ridge Circle of Omega (OMEGA) company with gel, Norcross, GA, USA)) E.Z.N.A. gel extraction kit) reclaim the target DNA fragment of 1000bp.

Get 4ul and reclaim the DNA fragmentation obtaining and be connected with plasmid pMD18-T, specific as follows:

Reaction conditions: connect 2hr(water-bath or metal bath at 16 DEG C).

The plasmid pMD18-T-BM Man connecting is proceeded in DH5 α, be coated with dull and stereotyped (LB-agar plate, containing 100ug/ml penbritin), just putting after 1h for 37 DEG C, be inverted overnight incubation, in the positive colony access 2ml LB substratum that picking is grown in resistant panel, (contain 100ug/ml penbritin, 10ml test tube), 37 DEG C, 200rpm are cultivated 4-6h, collect nutrient solution, the centrifugal 10min of 10000rpm room temperature collects thalline, extracts plasmid (the little test kit I that takes out of E.Z.N.A. plasmid of Omega company) with plasmid extraction kit, and uses BcaBEST ^tM(Taka is drawn the (large (Otsu of Jinshi City of Japan of (TaKaRa) company for sequencing primer and M13 primer, Shiga, 520-2193, Japan) the pMD18-T carrier of) producing) the PCR product after clone is carried out to determined dna sequence (Shanghai hero company), the total 1011bp of encoding sequence of the 'beta '-mannase that obtained thus.Wherein 1009-1011 position is terminator codon TGA, 1-1008 position is the encoding sequence (Fig. 1, SEQ ID NO:1) of 'beta '-mannase, and coding does not contain the mature protein of signal peptide, this mature protein contains 336 amino acid (Fig. 1, SEQ ID NO:2).

Sequence measurement used is specifically: adopt 4 kinds of fluorescence dyes respectively marks stop thing ddNTP or primer, after Sanger sequencing reaction, reaction product 3 ' end (mark stops thing method) or 5 ' hold (labeled primer method) with different fluorescent marks.By electrophoresis by each fluorescent mark fragment separately, laser detector synchronous scanning simultaneously, the fluorescence exciting is through grating beam splitting, to distinguish the fluorescence of the different colours that represents different base information, and on CCD (charge-coupled device) Kamera synchronous imaging, thereby obtain fluorescent absorption peak figure or base puts in order.

The structure of embodiment 4 yeast recombinant expression vectors

Encoding sequence design primer according to the 'beta '-mannase mature protein that does not contain signal peptide obtaining in embodiment 3:

ManF:5 '-CCG cTCGAGCcATACTGTGTCGCCTGTGAATC-3 ' (SEQ ID NO:5) and

ManR:5 '-AA gCGGCCGCtTATCACTCAACGATTGGCGTTAA-3 ' (SEQ ID NO:6), wherein inserts restriction enzyme site XhoI and NotI (shown in line part).

The DNA that cuts back to close gained taking enzyme in embodiment 2 is as template, utilizes above-mentioned primer to carry out pcr amplification to obtain the encoding gene of 'beta '-mannase.Pcr amplification condition is: 94 DEG C of 5min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 32 circulations; 72 DEG C of 10min.PCR product carries out after double digestion with XhoI and NotI, is connected on the carrier pPIC9 that carries out equally double digestion, obtains the recombinant plasmid pPIC9-BM Man (as shown in Figure 2) that contains beta-mannase coding gene.

Taking recombinant plasmid as template, with the Auele Specific Primer of original gene BM-Man (BMF:5 '-CTGTTCAGGCCGCTGCATGAAATGAACGGTG-3 ' (SEQ ID NO:7) and BMR:5 '-TCACTCAACGATTGGCGTTAAAGAATCG-3 ' (SEQ ID NO:8)), and comprise Insert Fragment and do pcr amplification at the Auele Specific Primer (a-factor primer: 5-' TACTATTGCCAGCATTGCTGC-3 ' (SEQ ID NO:9) and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' (SEQ ID NO:10)) of interior original plasmid.PCR product is carried out to agarose electrophoresis (1% agarose, 120v constant voltage, electrophoresis 40min) qualification, reclaim respectively the DNA fragmentation of 1000bp and 1200bp with glue recovery test kit, then carry out determined dna sequence (Shanghai hero company).In two sequence dna fragments, object is inserted the length of DNA and is 1011bp, consistent with sequence and other features of starting strain bacillus megaterium original gene, and insertion point, direction and the sequence of hence one can see that goal gene are correct.

Embodiment 5 pichia yeast fermentative production restructuring 'beta '-mannases

Recombinant plasmid pPIC9-BMMan prepared by embodiment 4 cuts through StuI or BglII enzyme, obtains linearization plasmid pPIC9-BMMan1.

Get the linear recombinant plasmid dna 50ug building, directly join still in sub-zero competent cell (pichia yeast GS115); Add 1.0ml to contain solution II (40% (w/v) cetomacrogol 1000 of the salmon sperm dna (company of Sigma) of 5ug/ml, 0.2M N, N-bicine N-, pH8.35), or first add the solution II of 1.0ml, then add the salmon sperm dna of 5ul1mg/ml try one's best both are mixed completely; More than 30 DEG C of water bath heat preservation 1h, every 15min mixing once gently; 42 DEG C of insulation 10min; The centrifugal 5min of room temperature 3000 × g, supernatant discarded, with solution III (0.15M NaCl, 10mM N, N-bicine N-, pH8.35) the Eddy diffusion thalline of 1.0ml; The centrifugal 5min of room temperature 3000 × g, removes 800ul supernatant, with remaining 200ul supernatant Eddy diffusion thalline; 200ul bacterium liquid is coated with to YPD flat board, and (YP (5% (w/v) yeast powder and 10% (w/v) peptone) and 20% (w/v) D (glucose) sterilizing separately, is down flat plate and in YP, adds 20%D by 1:9 before; Screening resistance is 80ug/ml penbritin), be inverted for 30 DEG C and cultivate 3-4 days, positive colony for containing recombinant plasmid of growing in resistant panel.

Get pichia yeast GS115 bacterial strain positive colony that recombinant plasmid pPIC9-BMMan1 transforms, be inoculated in 150ml YPD nutrient solution, 30 DEG C of 250rpm shaking culture are to OD600nm=0.3～0.5 (about 20hr), then be inoculated in 3L fermentation minimum medium (26.2ml/L phosphoric acid, 0.80g/L calcium sulfate, 18.7g/L potassium sulfate, 15.5g/L magnesium sulfate, 4.17g/L potassium hydroxide, 40g/L glucose) in, in 5L fermentor tank, ferment.

At initial period---the thalli growth stage, ammoniacal liquor with 25% in fermenting process regulates pH, make it maintain 6.5, and add PTM1 (30mM copper sulfate, 0.54mM sodium iodide, 17.6mM manganous sulfate, 0.80mM Sodium orthomolybdate, 0.32mM boric acid, 2.4mM cobalt chloride, 0.18mM zinc chloride, 0.24mM ferrous sulfate, 1.6mM vitamin H, 0.19M sulfuric acid) with the speed stream of 4.0ml/hr, carry out continuous flow feeding.Stir and aerated culture 20-24hr, in thalli growth process, dissolved oxygen drops to gradually lower than 100%, until carbon source exhausts, dissolved oxygen rises to gradually higher than 80% again, and now bacterium weight in wet base can reach 90g/L.

Entering carbon source feeds the stage, add the solution that contains 25% (w/v) glucose and 12ml/L PTM1 with distilled water configuration with the speed stream of 25ml/hr, continuous flow adds 4-6hr, and regulate air flow, dissolved oxygen is maintained 20% upper and lower, to the latter stage in this stage, bacterium weight in wet base can reach 160g/L.

At induction period, add with the speed stream of 10-15ml/hr the methyl alcohol that contains 12ml/L PTM1, make in substratum that the final concentration of methyl alcohol is the highest does not exceed 0.3% (v/v), and regulate air flow mixing speed, dissolved oxygen is maintained 20% upper and lower.In the fermenting process of induction period, every 8-16hr sampling 10ml, the centrifugal 5min of 10000rpm, collects supernatant liquor and measures beta-mannase enzyme activity and carry out SDS-PAGE analysis, and result respectively as shown in Figure 7 and Figure 8.When fermentation reaches 139hr, bacterium weight in wet base can reach 340g/L, the expression level (enzyme activity with fermented liquid supernatant represents) of 'beta '-mannase can reach 21458U/ml, and the beta-mannase gene in this explanation bacillus megaterium source has all obtained expressing and accumulation in pichia yeast.

The recombinate purifying of 'beta '-mannase of embodiment 6

By the centrifugal 10min of fermentation culture 10000rpm prepared embodiment 5 with remove thalline, get supernatant liquor as crude enzyme liquid, crude enzyme liquid is placed in to ice bath, slowly add while stirring ammonium sulfate to 80%, the centrifugal 15min of 13000rpm, gets precipitation, with damping fluid (Sodium phosphate dibasic-citric acid, pH6.5,50mM) again dissolve.

Precipitation after dissolving is above dialysed, to remove remaining ammonium sulfate, actual conditions is: selecting molecular weight cut-off is the dialysis tubing (the wide 5cm of bag) of 12-14kDa, extracellular fluid dialysis is Sodium phosphate dibasic-citric acid (pH6.5,10mM) damping fluid, the volume ratio of extracellular fluid dialysis and dialyzed solution (i.e. deposit sample after the above dissolving obtaining) is greater than 15, and in 4 DEG C of dialysis 24hr, in process, every 8hr changes an extracellular fluid dialysis.After end, collect dialyzed solution, after lyophilize, be stored at-80 DEG C stand-by.

Get appropriate above prepared freeze-drying sample, after dissolving with PBS (pH7.0,20mM) damping fluid (concentration is 5-10mg/ml), get the upper Sephacryl of 1-2ml ^tMs200 normal pressure gel-filtration column (Φ 1.6 × 200cm) ((the GE Healthcare of GE health care company of Uppsala, SWE SE-75184, SE-75184Uppsala, Sweden)), first use pH7.020mM PBS buffer solution elution balance pillar, then loading, with 1.5 column volumes of pH7.020mM PBS buffer solution elution, flow velocity is 1.0ml/min, collect with Fraction Collector, every pipe 6ml, then to collect sample determination beta-mannase enzyme activity and carry out protein electrophoresis.

Collect the Peak Activity after gel-filtration separates, after concentrated, desalination, freeze-drying, then with pH6.550mM Sodium phosphate dibasic-citrate buffer solution dissolving (concentration is 5-10mg/ml), upper Mono Q ^tM5/50GL high-efficiency anion exchange column (the GE health care company of Uppsala, SWE SE-75184).First use pH8.0,0.05mM Tris-HCl damping fluid balance pillar, then get the sample loading that 150ul dissolves, 5 column volumes of 0-1.0mol/L NaCl gradient elution that configure by same buffer again, flow velocity is 1ml/min, press peak collect, then to collect sample determination mannosans enzyme activity and carry out protein electrophoresis.

After purifying completes, the beta-mannase specific enzyme activity of BM-Man gene source is brought up to the 16718.2U/mg of pure enzyme from the 3427.8U/mg of crude enzyme liquid, and purification is 4.88.Result (Fig. 9) shows, only shows single band after the beta-mannase enzyme purification of BM-Man gene source in SDS-PAGE gel, and molecular weight is about 39kDa.

The recombinate zymologic property analysis of 'beta '-mannase of embodiment 7

Pure 'beta '-mannase prepared embodiment 6 enzyme is carried out to enzymatic reaction to measure its optimal pH under different pH.The pH scope of damping fluid used is that 3.0-10.0 (adopts 50mM Na within the scope of pH3.0-8.0 ₂hPO ₄-C ₆h ₈o ₇damping fluid, pH8.0-10.0 adopts 50mM Gly-NaOH damping fluid).With 'beta '-mannase, in the damping fluid of different pH, measure pH adaptive result at 50 DEG C, result shows that the optimal pH of the 'beta '-mannase of BM-Man gene source is 5.0-8.0 (seeing Fig. 3).Beta-mannase enzyme solution is processed to 60min in the damping fluid of different pH values under room temperature, then measure the pH stability of residual enzyme activity with research 'beta '-mannase.Result shows, processes 60min within the scope of pH3.0-4.0, and the 'beta '-mannase of BM-Man gene source has more than 70% residual enzyme activity; In the scope of pH4.5-10.0, process 60min, BM-Man 'beta '-mannase can keep more than 90% residual enzyme activity (seeing Fig. 5).The 'beta '-mannase of this explanation BM-Man gene source has the pH scope of application and well pH stability very widely.

Under Sodium phosphate dibasic-citric acid (pH6.5,50mM) buffer system and differing temps, (20 DEG C-80 DEG C) carry out enzymatic reaction, to measure optimal reactive temperature.Result shows, the optimal reactive temperature of BM-Man 'beta '-mannase is 45-55 DEG C (Fig. 4).

Under differing temps, after treat enzyme 60min, measure enzymic activity, to carry out THERMAL STABILITY.Result shows, within the scope of 20 DEG C-55 DEG C, processes 60min, and the residual enzyme activity of 'beta '-mannase maintains more than 90%; At 60 DEG C and 65 DEG C, be incubated 60min, the residual enzyme activity of 'beta '-mannase is respectively 88% and 78%; At 70 DEG C, be incubated 60min, the residual enzyme activity of 'beta '-mannase is 45% (Fig. 6).The 'beta '-mannase that BM-Man gene source is described has good thermostability.

In BM-Man gene source beta-mannase enzyme solution, add 0.05ml trypsin 0.1mg/ml, with the configuration of pH7.0PBS damping fluid) or stomach en-(0.1mg/ml, with the configuration of pH2.0 glycine-HCL damping fluid), in 37 DEG C of processing 120min, after dilution, measure again beta-mannase enzymic activity.Process respectively after 120min through trypsinase or stomach en-, 'beta '-mannase residual enzyme activity is more than 95% (Figure 10) all, illustrates that the 'beta '-mannase of BM-Man gene source has good protease inhibitor hydrolysis ability.

The recombinate comparison of 'beta '-mannase and other genus bacillus 'beta '-mannase of embodiment 8

By the ratio vigor of the mannase in the ratio vigor (referring to embodiment 6) of 'beta '-mannase of the present invention and subtilis (Bacillus subtilis) WL-7 source (referring to KWEUN, MIN A, the clone of MI-SUNGLEE and JOON HO CHOI.2004. subtilis WL-7 mannase gene and the qualification of gene product (Cloning of a Bacillus subtilis WL-7Mannanase Gene and Characterization of the Gene Product) .J.Microbiol.Biotechnol.14 (6): 1295-1302) compare, can find out that the ratio vigor (16718.2U/mg) of 'beta '-mannase of the present invention is significantly higher than the ratio vigor (10080U/mg) of the mannase in subtilis WL-7 source.

Also by restructuring 'beta '-mannase of the present invention and subtilis (Bacillus subtilis) WL-7 mannase (KWEUN, MIN A, the clone of MI-SUNGLEE and JOON HO CHOI.2004. subtilis WL-7 mannase gene and the qualification of gene product (Cloning of a Bacillus subtilis WL-7Mannanase Gene and Characterization of the Gene Product) .J.Microbiol.Biotechnol.14 (6): 1295-1302.) and subtilis MA139 mannase (Jiayun Qiao, Zhenghua Rao, Bing Dong, and Yunhe Cao.2010. expresses subtilis MA139 'beta '-mannase and enzyme qualification (Expression of Bacillus subtilis MA139 β-mannanase in pichia pastoris and the Enzyme Characterization) .Appl Biochem Biotechnol.160:1362-1370 and Wang Chunlin in pichia pastoris phaff, Cao Yunhe, a kind of beta-mannase coding gene and application thereof, Chinese patent (application number 200910084472.9) 2009) the optimal reaction pH of pure enzyme, optimal reactive temperature, the physico-chemical properties such as pH stability and thermostability compare, result as shown in Figures 3 to 6.Can find out, three kinds of restructuring 'beta '-mannases have similar pH stability and thermostability, but mannase of the present invention has the pH scope of application and temperature applicable range widely compared with other two kinds of mannases.

The recombinate stability of 'beta '-mannase drying agent of embodiment 9

The pure enzyme of restructuring 'beta '-mannase prepared embodiment 6 is carried out to preparation processing.Specifically, in enzyme liquid, add 25% (w/v) starch, 5% (w/v) sodium sulfate, 10% (w/v) dextrin, 2.5% (w/v) sodium-chlor, 1.5% (w/v) potassium sorbate and 1% (w/v) calcium sulfate, after mixing, carry out spray dried dry.Then obtained solid recombinant beta-mannase preparation is placed in to (50-100 DEG C) at different temperature and hatches 60min, measure remaining beta-mannase enzymic activity according to above-described method, investigating the thermotolerance of restructuring 'beta '-mannase solid preparation.At 50 DEG C-90 DEG C, process 60min, the residual enzyme activity of restructuring 'beta '-mannase solid preparation is all more than 90%; At 100 DEG C, process 60min, still can keep 78% residual enzyme activity (Figure 11).Illustrate that restructuring 'beta '-mannase solid preparation of the present invention has good thermotolerance, can be good at being applied to the industries such as granulated feed processing.

Above prepared restructuring 'beta '-mannase solid preparation is placed in to (25 DEG C of climatic chambers, relative humidity 50%) store 15 months, wherein the residual enzyme activity of fixing preparation is once measured in sampling every other month, investigates the package stability of restructuring 'beta '-mannase solid preparation.Store after 15 months, still maintain 94% residual enzyme activity (Figure 12), illustrate that 'beta '-mannase of the present invention has good package stability.

Can find out by above-mentioned experimental result, utilize the 'beta '-mannase of the prepared BM-Man gene source of bacterial strain of the present invention and method to there is prospects for commercial application widely, can be widely used in the numerous areas such as food, medicine, feed, papermaking, textile printing and dyeing, oil production, fine chemistry industry and biotechnology.

Claims (15)

1. an isolated polypeptide, is characterized in that, described polypeptide has β-Isosorbide-5-Nitrae-mannosans enzymic activity, and the aminoacid sequence of described polypeptide is SEQ ID NO:2.

2. the nucleic acid of polypeptide described in the claim 1 of encoding.

3. nucleic acid as claimed in claim 2, its nucleotides sequence is classified SEQ ID NO:1 as.

4. a recombinant vectors, it comprises the nucleic acid described in claim 2 or 3.

5. a host cell, it comprises recombinant vectors claimed in claim 4.

6. host cell as claimed in claim 5, is characterized in that, described host cell is eukaryotic cell.

7. host cell as claimed in claim 6, is characterized in that, described host cell is yeast cell.

8. host cell as claimed in claim 7, is characterized in that, described host cell is pichia yeast cell.

9. the host cell as described in any one in claim 5-8, is characterized in that, described recombinant vectors is integrated in the genome of described host cell.

10. a method of producing 'beta '-mannase, it comprises the following steps successively:

I) cultivate the host cell described in any one in claim 5-9,

Ii) induce it to express,

Iii) results expression product.

11. methods as claimed in claim 10, further comprising the steps of:

Iv) purifying expression product.

12. 1 kinds of compositions, in the polypeptide as claimed in claim 1 that it contains significant quantity and bromatology or industrial acceptable vehicle or vehicle.

13. 1 kinds of compositions, the polypeptide as claimed in claim 1 that it contains significant quantity and one or more are selected from the enzyme of lower group: proteolytic enzyme, cellulase, beta-xylanase, beta-glucanase, α-amylase, lipase, polygalacturonase; Or their mixture.

14. 1 kinds of compositions as described in claim 12 or 13, it is for feed and foodstuff additive, for deinking and association with pulp bleaching.

15. 1 kinds of wild-type bacillus megaterium bacterial strain FB101, is characterized in that, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation date is on September 13rd, 2010, and preserving number is CGMCC No.4162.