CN103261409A - 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

Mannase, its encoding gene 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 be bacillus megaterium FB101, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms 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 constitutes 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 main moiety of all leguminous plants cell wallss, content is also very high in other plant forage raw material, as corn, wheat, rapeseed meal and wheat bran etc.Mannosans is as a kind of component of hemicellulose, and mainly the form with glucomannan, polygalactomannan and gala glucomannan exists.

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

'beta '-mannase (is called β-1 again, 4-mannosans seminose lytic enzyme (β-1,4-mannan mannohydrolase)), abbreviate 'beta '-mannase or β-1 as, the 4-D-mannase, EC3.2.1.78) be that a class can hydrolysis contain β-1, the inscribe lytic enzyme of the mannooligo saccharide of 4-D-seminose glycosidic bond, 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 is to the research of 'beta '-mannase with utilize and entered a new stage.'beta '-mannase has been widely used in 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 big potential using value.

In food service industry, beta-mannase enzyme liberating mannase generates manna oligosaccharide, have protect the liver, 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 also can reducing the consumption of chlorine and alkali than simple chlorine bleaching, the better characteristics of pulp of alkaline extraction used, 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 characteristics such as the low layer injury are little.

Mannosans has high-hydrophilic, after a large amount of suctions, has increased the viscosity of alimentary canal content in the digestive tube of monogastric animal, and the opposing gastrointestinal peristalsis directly influences 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 have the secretion that promotes quasi-insulin growthing factor I GF-I, the effect of synthesizing, improving lean ratio that promotes protein.Therefore, 'beta '-mannase can be degraded to mannosans the oligose that is easily absorbed by animal as fodder additives, helps animal to digestion and the absorption of nutritive substance, has improved the utilization ratio of feed greatly.

'beta '-mannase belongs to the hemicellulose enzyme, is the wide spectrum induction type multifunctional enzyme with cellulase activity, extensively is present in animals and plants and the microorganism.Because microorganism has advantages such as easy cultivation, output height than 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 screening of zymogenic bacteria kind and the aspects such as 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.

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

Because what 'beta '-mannase was used constantly widens, more and more higher to the requirement of various 'beta '-mannases with special property in the practice, though existing report obtains 'beta '-mannase from multiple microorganism, but on industrial application, still lack high-quality enzyme source, particularly in the feed industry, the enzyme that not only needs high vigor also needs enzyme to keep enzymatic property under sour environment and hot conditions.Therefore, research and development has good characteristic, to be more suitable for the efficient 'beta '-mannase that practical application needs significant.

Summary of the invention

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

The invention provides a kind of isolated polypeptide, it has β-1,4-mannosans enzymic activity.In another embodiment, this polypeptide has β-1,4-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, preferred 6.0-7.0, most preferably 6.5;

D) optimal reactive temperature is 35-60 ℃, preferred 45-55 ℃, and most preferably 55 ℃;

E) than vigor〉11000U/mg, preferred 15000U/mg, most preferably be 16718.2U/mg; And

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

In another embodiment, the invention provides a peptide species, this amino acid sequence of polypeptide is SEQ ID NO:2.In another embodiment, the invention provides a peptide species, it comprises, and sequence homogeny with SEQ ID NO:2 is at least about 70%, the 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 the 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 the SEQ ID NO.1.In one embodiment, polypeptide of the present invention comprises the aminoacid sequence sequence of being derived through one or more amino acid whose replacements, disappearance and/or insertion by aminoacid sequence shown in the SEQ ID NO.2.In a preferred implementation, described aminoacid sequence does not comprise the endogenous signal peptide sequence of the microorganism of originating.In a preferred embodiment, the amino-acid residue corresponding to SEQ ID NO:2 position 153 is identical with the amino-acid residue of corresponding position among the SEQ ID NO:2 in the described aminoacid sequence.

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, the sequence homogeny that its coding comprises SEQ ID NO:2 is at least about 70%, the nucleic acid of the 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, the amino-acid residue corresponding to SEQ ID NO:2 position 153 is identical with the amino-acid residue of corresponding position among the SEQ ID NO:2 in the described aminoacid sequence.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 among the nucleotide sequence SEQ ID NO:1.In another preferred embodiment, the invention provides a kind of nucleic acid, it comprises, and sequence homogeny with SEQ ID NO:1 is at least about 70%, the 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, the nucleotide residue corresponding to SEQ ID NO:1 position 458 is identical with the nucleotide residue of corresponding position among the SEQ ID NO:1 in the described nucleotide sequence.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, the nucleotide residue corresponding to SEQ ID NO:1 position 458 is identical with the nucleotide residue of corresponding position among the SEQ ID NO:1 in the described nucleotide sequence.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 the prokaryotic cell prokaryocyte.In one embodiment, recombinant vectors provided by the invention can comprise the carrier that is adapted at expressing in the eukaryotic cell.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 the 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 may further comprise the steps successively:

I) cultivate host cell of the present invention,

Ii) induce its expression,

Iii) gather in the crops expression product and

Iv) randomly, purifying expression product.

On the other hand, the present invention also provides a kind of wild-type bacillus megaterium bacterial strain FB101, and its generation comprises the β-1 of aminoacid sequence SEQ ID NO:2, the 4-mannase, 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 common micro-organisms center (No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city), 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, and it contains on the polypeptide of the present invention of significant quantity and the 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 down group: proteolytic enzyme, cellulase, beta-xylanase, powder-beta-dextrin enzyme, α-Dian Fenmei, lipase, polygalacturonase or their mixture.Preferred, described composition can be used to feed, food, deinking and paper industry.

The present invention utilizes genetic engineering means to prepare the recombinant production strain that can efficiently express the justacrine 'beta '-mannase, has realized the scale operation of 'beta '-mannase, and has obtained the beta-mannase enzyme product of high-quality.The present invention analyzes to the optimum temperature of enzyme, the suitableeest action pH value, pH stability, thermostability and than physico-chemical properties such as vigor by zymologic property check, prove β of the present invention-1, the 4-mannase has good pH stability, good thermostability and protease inhibitor hydrolysis ability.

Description of 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 the 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 the 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 the 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 'beta '-mannase of the present invention that Fig. 9 is purified, 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

Below employed term through whole specification sheets, and represent implication well-known to those skilled in the art, unless otherwise prescribed.

Want as this paper, there be feature, integral body, step or the composition as regulation mentioned in the claim in " comprising " expression, but does not get rid of existence or increase one or more features, integral body, step, composition or component.

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

As used herein, " 'beta '-mannase of separation " refers to that 'beta '-mannase is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying 'beta '-mannase of standard.Basically 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 " β-1,4-mannase " again, can be recombinant protein (polypeptide), native protein, synthetic proteins, preferred recombinant protein.'beta '-mannase of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, 'beta '-mannase of the present invention can be glycosylated, maybe can be 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 a kind of β-1 that can cut off at random on mannooligo saccharide, mannocarolose (comprising mannosans, polygalactomannan, the glucomannan etc.) sugar chain, the ability of 4-D-seminose glycosidic bond.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 " and " analogue " refer to the biological function or the active albumen that keep identical with the natural 'beta '-mannase of the present invention basically.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 (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has the albumen of substituted radical, or (iii) maturation protein and another compound (such as the compound that prolongs the albumen transformation period, polyoxyethylene glycol for example) merges formed albumen, or (iv) additional aminoacid sequence is 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 the instruction of this paper, 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 the 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 at its C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) sequence that obtains of amino acid.For example, in the art, when replacing with the close or similar amino acid of performance, can not change the function of protein usually.Again such as, add the function that or several amino acid also can not change protein usually at C-terminal and/or N-terminal.These variant forms can comprise also that sequence homogeny with SEQ ID NO.2 is at least about 70%, the 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, the amino-acid residue corresponding to SEQ ID NO:2 position 153 is identical with the amino-acid residue of corresponding position among the SEQ ID NO:2 in this series of variation.

The variant form of this 'beta '-mannase also comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of the DNA hybridization of 'beta '-mannase and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-'beta '-mannase to obtain.The present invention also provides other albumen, as comprises the fusion rotein of 'beta '-mannase or its fragment.Except the albumen of total length almost, the present invention has also comprised the soluble fragments of 'beta '-mannase.Usually, this fragment have the beta-mannase enzyme sequence at least about 10 continuous amino acids, usually 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 the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These albumen comprise genetic variant natural or that induce.The 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 the analogue with that non-natural exists or synthetic amino acid (as β, gamma-amino acid).Should be understood that 'beta '-mannase of the present invention is not limited to the above-mentioned representative albumen of enumerating.(the not changing primary structure usually) form of modification comprises: chemically derived form such as the acetylize or carboxylated of the albumen that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and albumen that produce in the procedure of processing as those in the synthetic and processing of albumen or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by albumen is exposed to.Modified forms also comprises have the 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 the SEQ ID NO.1.In the present invention, " 'beta '-mannase conservative property variant protein (polypeptide) " refers to compare with the aminoacid sequence of 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 similar performance or close amino acid at the most best, and still have the albumen of beta-mannase enzymic activity.Family's existing clearly definition in this area with amino-acid residue of similar side chain.These families comprise amino acid with basic side chain (Methionin for example, arginine, Histidine), amino acid (aspartic acid for example with acid side-chain, L-glutamic acid), amino acid (glycine for example with uncharged polar side chain, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), amino acid (L-Ala for example with non-polar sidechain, Xie Ansuan, leucine, the Isoleucine proline(Pro), phenylalanine, methionine(Met), tryptophane), amino acid (Threonine for example with β-branched building block, Xie Ansuan, Isoleucine) and have the amino acid (tyrosine for example of aromatic side chain, phenylalanine, tryptophane, Histidine).Therefore, in the 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 influence its mannosans enzymic activity.In addition, as well known to those skilled in the art, in the gene clone operation, usually need to design proper restriction site, this certainly will introduce one or more incoherent residues at expressed albumen end, and this does not influence the activity of target protein.And for example for construction of fusion protein, the purifying that promotes Recombinant Protein Expression, obtain to be secreted into the outer recombinant protein of host cell automatically or be beneficial to recombinant protein, usually need with some aminoacid addition to the N-end of recombinant protein, C-is terminal or this albumen in other appropriate area in, for example, include but not limited to, the joint peptide that is fit to, signal peptide, leading peptide, terminal extension, glutathione S-transferase (GST), maltose E are 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 the SEQ ID NO.1 or the varient of degeneracy.As used herein, " varient of degeneracy " refers in the present invention encode and comprises the protein of SEQ ID NO.2, but with the differentiated nucleotide sequence of coding region sequence shown in the 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; The encoding sequence of maturation protein (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of proteins encoded " can be the polynucleotide that comprise this albumen of encoding, and also can be 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 fragment, analogue, derivative and the variant form of albumen with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.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 the function of its encoded protein in fact.

Again in one embodiment, the albumen with beta-mannase enzymic activity of the present invention comprise by as under the rigorous condition with sequence table in SEQ ID NO.1 shown in the nucleotide sequence coded aminoacid sequence of nucleotide sequence hybridization.As used herein, term " is hybridized under rigorous condition " but is nucleotide sequence still hybridization and the cleaning condition of phase mutual cross of describing typical at least 60% homology each other.Preferably, rigorous condition is such condition, but has at least 65% with this understanding each other, more excellent at least 70% and even preferred at least 80% or the generally still phase mutual cross of sequence of higher homology.This rigorous condition is that those of ordinary skills are known.One of rigorous condition is preferred, and limiting examples is: (1) than the hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 0 ℃; Or (2) hybridization the time is added with denaturing agent, 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the 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 the 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 " contains 15 Nucleotide at least, better is 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 among the nucleotide sequence SEQ ID NO:1.Nucleic acid of the present invention can comprise that sequence homogeny with SEQ ID NO:1 is at least about 70%, the 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 among the 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 among the SEQ ID NO:1.

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

'beta '-mannase Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the 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.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

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

Use method (Saiki etc., the Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA, Elizabeth Scotto – Lavino etc., NATURE PROTOCOLS2006, the 1st volume the 6th phase: 2555-2562. and Elizabeth Scotto – Lavino etc., NATURE PROTOCOLS2006, the 1st the 6th phase of volume: 2742-2745), the primer that is used 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 by gel electrophoresis separation and purifying amplification.

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.The result obtains the nucleotide sequence shown in SEQ ID NO.1.

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

Recombinant DNA technology (Science, 1984 by routine; 224:1431), polymerized nucleoside acid sequence of the present invention can be used to express or produce the 'beta '-mannase of reorganization.In general following steps are arranged:

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

(2) host cell of in suitable medium, cultivating;

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

Again in one aspect, the method that the present invention relates to contain the carrier of described beta-mannase gene, has imported the recombinant host cell of described carrier or described gene and in host cell, expressed 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 in host and stablize, a key character of these recombinant vectorss is to contain replication orgin, promotor, marker gene and translation controlling elements usually, and the recombinant vectors of Shi Yonging 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 of selecting based on the host cell of be used for expressing, itself and exercisable connection of nucleic acid of expression.In recombinant expression vector, " exercisable connection " refers to that the nucleotide sequence of purpose is connected in the mode that allows nucleotide sequence to express with the adjusting sequence.Those skilled in the art knows be used for to make up and contains '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 effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; But eukaryotic promoter comprises LTR and some other known controlling gene expression promoter 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.

The design that those skilled in the art will appreciate that recombinant expression vector can be depending on as the selection of desiring transformed host cells, required factors such as protein expression level.Expression vector of the present invention can be imported host cell, comprise albumen or the peptide of fusion rotein with generation.

In addition, recombinant expression vector preferably comprises one or more selected markers, being provided for selecting the phenotypic character of transformed host cells, as being used for eukaryotic Tetrahydrofolate dehydrogenase, neomycin resistance, or be used for colibacillary tsiklomitsin or amicillin resistance.

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

The recombinant vectors that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence can be used for transforming appropriate host cell, so that it can marking protein.This paper is called recipient cell again at used term " host cell ", refers to receive and to hold the cell of recombinant DNA molecules, is the place of recombination amplification, and desirable recipient cell should satisfy 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 for and express the beta-mannase zymoprotein in protokaryon or eukaryotic cell.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 such 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.The preferred various cells that are beneficial to gene product expression or fermentative production of described host cell, this type of cell has been well known and commonly used, for example various Bacillus coli cells and yeast cell.In an embodiment of the invention, select the host cell of pichia spp GS115 construction expression 'beta '-mannase for use.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniquess well known to those skilled in the art such as conversion or transfections with the recombinant DNA transformed host cell.As used herein, term " conversion " and " transfection ", " joint " and " transduction " mean well known in the art various with 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 transfer or electroporation that transfection, fat transfection, natural competence, the chemistry of calcium phosphate or calcium chloride co-precipitation, DEAE-mannosans-mediation mediate.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When host cell is eukaryotic cell, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

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

The extracellular be expressed or be secreted into to recombinant protein in the above methods can in cell or at cytolemma.If desired, 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 handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, 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 spp 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: be used for hydrolysis 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, and it contains on the 'beta '-mannase of significant quantity and the 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 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 identifies to the optimum temperature of enzyme, the suitableeest action pH value, pH stability, thermostability and than physico-chemical properties such as vigor by zymologic property and analyzes, 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 only to be used for explanation the present invention and be not used in and limit the scope of the invention.

The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition such 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 the Biochemistry Experiment [M] (Shanghai science tech publishing house, 1995), or carry out according to the condition that manufacturer advises.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 available from Novartis's base (Novagen) company (Australia north come (NORTH RYDE NSW, AUSTRALIA));

The light carrier system of-T (

-T Easy Vector systems) available from Pu Luomaige (Promega) company (No. 2800, WH road, Madison city, the hot state of University of Wisconsin-Madison (2800Woods Hollow Road, Madison, WI53711USA));

Pichia yeast GS115 and expression vector pPIC9 are all available 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 available from the (Fermentas (MBI) of rich enzyme Tai Si company (MBI) of No. 830, Ontario, Canada Burlinton city Ha Lingdunlu, 830Harrington Court, Burlington, Ontario, Canada);

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

Other conventional reagent are available 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 method

Enzyme work described in the various embodiments of the present invention, enzyme activity, enzymic activity all refer to the beta-mannase enzyme activity, and available Viscogum BE detects as substrate.Wherein, the enzyme activity quantitative assay adopt 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 fully, regulate the solution that pH to 6.5 obtains with glacial acetic acid) to 5ml scale test tube, put into 50 ℃ of water-bath preheating 5min.Sodium phosphate dibasic-citrate buffer solution with 50mM pH6.5 is done 10 times of gradient dilutions with enzyme liquid sample, selects the gratifying extent of dilution of color developing effect (absorbancy is between 0.2～0.4).To dilute good enzyme liquid according to selected extent of dilution (N) and put into 50 ℃ of water-bath preheating 5min, and get 0.5ml then to join in the test tube that contains substrate solution, after concussion mixes, accurately be incubated 10min down in 50 ℃.Add 1.0ml DNS reagent (DNS reagent: take by weighing Seignette salt 182.0g again, be dissolved in the 800mL distilled water, heating (being no more than 50 ℃), in hot solution, add 3 successively, 5-dinitrosalicylic acid 6.3g, NaOH20.96g, phenol 5.0g, sodium sulphite anhydrous 99.3 5.0g stirs (heating) to dissolving fully, and the cooling back is settled to 1000mL with distilled water, if solution is not clarified, with water graceful (Whatman) l filter paper filtering, room temperature preservation is at room temperature placed after 7 days and is used in brown bottle.), after concussion mixed, boiling water heating 10min was cooled to room temperature then rapidly, with termination reaction and colour developing.With the absorbance A of spectrophotometer in 540nm place assaying reaction liquid (as muddiness occurring, need the centrifugal 10min of reaction solution 4000rpm, get supernatant liquor).

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

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

The definition of beta-mannase unit of enzyme activity: 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 following formula:

$U=\frac{K\&times;(A-A_0)+b}{t\&times;V}\&times;{\mathrm{<figure-callout\; id="0"\; label="V"\; filenames="HDA00003306125500041.png,HDA00003306125500061.png"\; state="\{\{state\}\}">V</figure-callout>}}_0\&times;N$

In the 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 the reaction system, ml;

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

N---sample extension rate;

Each mensuration is all got two parallel test results' arithmetical av, keeps integer.

The ratio vigor of 'beta '-mannase calculates by following formula:

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 the sample solution, mg/ml.

Purifying multiple X calculates according to following formula:

X＝U _C/U _C0

Wherein: X---the purifying multiple;

U _C---the ratio vigor of 'beta '-mannase sample behind the purifying, U/mg;

U _C0---the ratio vigor of 'beta '-mannase sample before the 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 biological chemistry book of reference commonly used, as Zhao Yongfang etc., " Measurement for Biochemistry principle and application thereof " (second edition) (Science Press, 2008) and the 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, and 5% concentrates glue; 120V constant voltage electrophoresis 30 minutes, and then 150V constant voltage electrophoresis 60 minutes; The running gel coomassie brilliant blue staining.

Mentioned nucleic acid electrophoresis, DNA electrophoresis etc. all refer to agarose gel electrophoresis in this patent, and experiment parameter is: 1% agarose, 120V constant voltage electrophoresis 40 minutes adopts EB (ethidium bromide) dyeing in the 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, gathered the pedotheque of Zhongshan Park, Xiamen, it is suspended in the sterilized water, and dilution, then, the suspension of different samples is inoculated in the LB substratum of no agar, then cultivated 1-2 days down at 15 ℃, 20 ℃, 25 ℃ and 30 ℃ respectively.Then utilize DNS method (concrete described in 4.1 " 'beta '-mannase vigour-testing methods ") to measure under the differing temps, in the nutrient solution of different samples and the beta-mannase activity in the cytoclasis liquid.In nutrient solution, can detect the beta-mannase enzymic activity, illustrate that these bacterial strains have the 'beta '-mannase of secretor type.

At first select the sample with beta-mannase enzymic activity, and judge that its suitable culture temperature is 30 ℃, will be coated on the LB flat board that contains 1.5% agar after its dilution, place incubator to cultivate 1 day for 30 ℃.Select the different bacterium colonies with various forms, be inoculated in incubated overnight in the 5ml LB substratum.Measure the beta-mannase enzymic activity of each bacterium colony cell culture fluid, in selected bacterium colony, select at last 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 '-mannases source bacterial strain FB101

By gram staining method, the bacillus megaterium FB101 bacterial strain that separates among the embodiment 1 is defined as gram-positive microorganism.This bacterium is typical bacillus, has the size similar to bacillus megaterium at microscopically.Its part physio-biochemical characteristics have further been studied.

The result shows that this bacterial strain is the Gram-positive aerobic bacteria, and shaft-like, terminal round, single or be the short chain arrangement, diameter is 1.2～1.5 * 2.0～4.0 microns, can move.The brood cell is 1.0～1.2 * 1.5～2.0 microns, ellipse, and middle life or inferior end are given birth to.Its optimum growth temp is 30 ℃, and is aerobic, can be observed under opticmicroscope.16s rRNA to this bacterium has carried out measuring (worker is given birth in Shanghai), and by using BLAST and multisequencing comparison program CLUSTAL W to compare the 16s rRNA sequence of this bacterium and the reference sequences in GenBank.The result shows to have 99% consistence between the base sequence of its 16s rRNA and bacillus megaterium B188, CNR9 and the UFV-E26 etc.Based on the result of study of form, physio-biochemical characteristics and the 16s rRNA of selected bacterial strain, determine that this bacterial strain is the new subspecies of bacillus megaterium.

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

Embodiment 3 bacillus megaterium β-1, the clone of 4-beta-mannase coding gene and acquisition

The extraction of genomic dna: the usefulness 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 strain that separates in 30 ℃ of cultivation embodiment 1 is 3～4 days, to cell concentration OD _600nmBe 0.5～0.8.Get this cultivation bacterium liquid 50ml, the centrifugal 10min of 10000rpm gets the 50mg thalline and adds 500 μ l sterile water wash, the centrifuging and taking precipitation.Precipitation is suspended in the lysozyme soln of 500 μ l1mg/ml again, after 37 ℃ of temperature are bathed 30min, the N,O-Diacetylmuramidase liquid of adding 100 μ l1mg/ml again continues insulation 30min in 40-50 ℃, after transparent to bacterium liquid, add 10%SDS to final concentration 2% (m/v), stir about 5min significantly descends to the 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 the back with the dissolving of 30 μ l sterilized waters with precipitation, and is standby.

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

Be that template is carried out pcr amplification with bacillus megaterium FB101 bacterium genomic dna.The PCR reaction parameter is: be cooled to 4 ℃ behind 94 ℃ of sex change 3min; 94 ℃ of sex change 30sec then, 50 ℃ of annealing 30sec, 72 ℃ are extended 1min, 32 circulations; Last 72 ℃ of insulation 10min.Obtain the full-length gene fragment of 'beta '-mannase, carry out the agarose gel electrophoresis evaluation 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 obtain and be connected with plasmid pMD18-T, specific as follows:

Reaction conditions: connect 2hr(water-bath or metal bath down in 16 ℃).

The plasmid pMD18-T-BM Man that connects is changed among the DH5 α, be coated with flat board (LB-agar plate, contain the 100ug/ml penbritin), 37 ℃ just putting 1h after, be inverted overnight incubation, picking inserts in the 2ml LB substratum at positive colony that resistant panel is grown and (contains the 100ug/ml penbritin, the 10ml test tube), 37 ℃, 200rpm are cultivated 4-6h, collect nutrient solution, the centrifugal 10min of 10000rpm room temperature collects thalline, extracts plasmid (the E.Z.N.A. plasmid of Omega company is taken out test kit I for a short time) with plasmid extraction kit, and uses BcaBEST ^TM(Taka is drawn the (big (Otsu of Jinshi City of Japan of (TaKaRa) company for sequencing primer and M13 primer, Shiga, 520-2193, Japan)) the pMD18-T carrier of Sheng Chaning) the PCR product behind the clone is carried out determined dna sequence (Shanghai hero company), thus the total 1011bp of the encoding sequence of resulting 'beta '-mannase.Wherein the 1009-1011 position is terminator codon TGA, the 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).

Used sequence measurement is specifically: adopt 4 kinds of fluorescence dyes respectively marks stop thing ddNTP or primer, behind the Sanger sequencing reaction, reaction product 3 ' end (mark stops the thing method) or 5 ' hold (labeled primer method) to have different fluorescent marks.By electrophoresis with each fluorescent mark fragment separately, laser detector synchronous scanning simultaneously, excited fluorescent 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

Design primer according to the resulting encoding sequence that does not contain the 'beta '-mannase mature protein of signal peptide among the 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 the line part).

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

Be template with the recombinant plasmid, 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)) with comprise and 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.The PCR product is carried out agarose electrophoresis (1% agarose, 120v constant voltage, electrophoresis 40min) identify, reclaim the dna fragmentation that test kit reclaims 1000bp and 1200bp respectively with glue, carry out determined dna sequence (Shanghai hero company) then.In two sequence dna fragments, the length that purpose is inserted DNA is 1011bp, and is consistent with sequence and other features of starting strain bacillus megaterium original gene, and insertion site, direction and the sequence of hence one can see that goal gene are correct.

Embodiment 5 pichia yeast fermentative production reorganization 'beta '-mannase

The recombinant plasmid pPIC9-BMMan of embodiment 4 preparations is cut through StuI or BglII enzyme, obtain linearization plasmid pPIC9-BMMan1.

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

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

At initial period---the thalli growth stage, ammoniacal liquor with 25% in the fermenting process is regulated pH, make it maintain 6.5, and the velocity flow with 4.0ml/hr adds 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), carries out continuous flow feeding.Stir and aerated culture 20-24hr, dissolved oxygen drops to gradually and is lower than 100% in the thalli growth process, exhausts until carbon source, and dissolved oxygen rises to gradually again and is higher than 80%, and this moment, the bacterium weight in wet base can reach 90g/L.

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

At induction period, add the methyl alcohol that contains 12ml/L PTM1 with the velocity flow of 10-15ml/hr, make that the final concentration of methyl alcohol is the highest in the substratum surpasses 0.3% (v/v), and regulate the air flow mixing speed, dissolved oxygen is maintained about in the of 20%.Every 8-16hr sampling 10ml, the centrifugal 5min of 10000rpm collects supernatant liquor and measures the beta-mannase enzyme activity and carry out the SDS-PAGE analysis in the fermenting process of induction period, and the result respectively as shown in Figure 7 and Figure 8.When fermentation reaches 139hr, the bacterium weight in wet base can reach 340g/L, the expression level of 'beta '-mannase (enzyme activity with fermented liquid supernatant is represented) can reach 21458U/ml, and the beta-mannase gene in this explanation bacillus megaterium source has all obtained expressing in pichia yeast and accumulation.

The purifying of embodiment 6 reorganization 'beta '-mannases

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

Precipitation after the above dissolving is dialysed, to remove the residual sulfuric acid ammonium, actual conditions is: selecting molecular weight cut-off for use 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 resulting dissolving) is greater than 15, and in 4 ℃ of dialysis 24hr, every 8hr changes an extracellular fluid dialysis in the process.After the end, collect dialyzed solution, after the lyophilize, preserve in-80 ℃ stand-by down.

Get an amount of above prepared freeze-drying sample, (pH7.0,20mM) damping fluid dissolving back (concentration is 5-10mg/ml) are got 1-2ml and are gone up Sephacryl with PBS ^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)), with pH7.020mM PBS buffer solution elution balance pillar, go up sample then, with 1.5 column volumes of pH7.020mM PBS buffer solution elution earlier, flow velocity is 1.0ml/min, collect with Fraction Collector, every pipe 6ml is then to the sample determination beta-mannase enzyme activity collected and carry out the protein electrophorese analysis.

Collect the active peak after gel-filtration separates, concentrate, after the desalination, freeze-drying, dissolve (concentration is 5-10mg/ml), last Mono Q with pH6.550mM Sodium phosphate dibasic-citrate buffer solution again ^TM5/50GL high-efficiency anion exchange column (the GE health care company of Uppsala, SWE SE-75184).Use earlier pH8.0,0.05mM Tris-HCl damping fluid balance pillar, get sample on the sample of 150ul dissolving then, again with 5 column volumes of 0-1.0mol/L NaCl gradient elution of same buffer configuration, flow velocity is 1ml/min, press the peak and collect, then to the sample determination mannosans enzyme activity collected and carry out the protein electrophorese analysis.

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

The zymologic property analysis of embodiment 7 reorganization 'beta '-mannases

The embodiment 6 prepared pure enzymes of 'beta '-mannase are carried out enzymatic reaction to measure its optimal pH under different pH.The pH scope of used damping fluid is that 3.0-10.0 (adopts 50mM Na in the pH3.0-8.0 scope ₂HPO ₄-C ₆H ₈O ₇Damping fluid, pH8.0-10.0 adopt 50mM Gly-NaOH damping fluid).With 'beta '-mannase in the damping fluid of different pH, 50 ℃ measure down the suitable property results of pH, the result shows that the optimal pH of the 'beta '-mannase of BM-Man gene source is the 5.0-8.0 (see figure 3).The beta-mannase enzyme solution is handled 60min in the damping fluid of different pH values, measure the residual enzyme activity again with the pH stability of research 'beta '-mannase under room temperature.The result shows, handles 60min in the pH3.0-4.0 scope, and the 'beta '-mannase of BM-Man gene source has the residual enzyme activity more than 70%; Handle 60min in the scope of pH4.5-10.0, the BM-Man 'beta '-mannase can keep the residual enzyme activity (see figure 5) more than 90%.The 'beta '-mannase of this explanation BM-Man gene source has the pH scope of application and well pH stability very widely.

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

Under differing temps, measure enzymic activity behind the treat enzyme 60min, to carry out THERMAL STABILITY.The result shows, handles 60min in 20 ℃ of-55 ℃ of scopes, and the residual enzyme activity of 'beta '-mannase maintains more than 90%; Be incubated 60min down at 60 ℃ and 65 ℃, the residual enzyme activity of 'beta '-mannase is respectively 88% and 78%; Be incubated 60min down at 70 ℃, the residual enzyme activity of 'beta '-mannase is 45% (Fig. 6).The 'beta '-mannase that the 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 pH2.0 glycine-HCL damping fluid configuration), in 37 ℃ of processing 120min, measure the beta-mannase enzymic activity after the dilution again.After trypsinase or stomach en-were handled 120min respectively, the 'beta '-mannase residual enzyme activity illustrated that all at (Figure 10) more than 95% the 'beta '-mannase of BM-Man gene source has protease inhibitor hydrolysis ability preferably.

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

With the ratio vigor of the ratio vigor (referring to embodiment 6) of 'beta '-mannase of the present invention and the mannase in 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 evaluation 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, 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 as can be seen.

Also with reorganization '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 evaluation of gene product (Cloning of a Bacillus subtilis WL-7Mannanase Gene and Characterization of the Gene Product) .J.Microbiol.Biotechnol.14 (6): 1295-1302.) with subtilis MA139 mannase (Jiayun Qiao, Zhenghua Rao, Bing Dong, and Yunhe Cao.2010. expresses subtilis MA139 'beta '-mannase in pichia pastoris phaff and enzyme is identified (Expression of Bacillus subtilis MA139 β-mannanase in pichia pastoris and the Enzyme Characterization) .Appl Biochem Biotechnol.160:1362-1370 and Wang Chunlin, Cao Yunhe, a kind of beta-mannase enzyme coding gene and application thereof, the optimal reaction pH of pure enzyme Chinese patent (application number 200910084472.9) 2009), optimal reactive temperature, physico-chemical properties such as pH stability and thermostability compare, and result such as Fig. 3 are to shown in Figure 6.As can be seen, three kinds of reorganization '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 than other two kinds of mannases.

The stability of embodiment 9 reorganization 'beta '-mannase drying agents

The embodiment 6 prepared pure enzymes of reorganization 'beta '-mannase are carried out preparation processing.Specifically be, 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, carry out spraying drying after mixing.Then resulting solid recombinant beta-mannase preparation is placed under the different temperature (50-100 ℃) to hatch 60min, measure remaining beta-mannase enzymic activity according to above-described method, investigating the thermotolerance of reorganization 'beta '-mannase solid preparation.Handle 60min down at 50 ℃-90 ℃, the residual enzyme activity of reorganization 'beta '-mannase solid preparation is all more than 90%; Handle 60min down at 100 ℃, still can keep 78% residual enzyme activity (Figure 11).Illustrate that reorganization 'beta '-mannase solid preparation of the present invention has good heat endurance, can be good at being applied to industries such as granulated feed processing.

Above prepared reorganization 'beta '-mannase solid preparation is placed (25 ℃ of climatic chambers, relative humidity 50%) stored 15 months, wherein the fixedly residual enzyme activity of preparation is once measured in sampling every other month, investigates the package stability of reorganization '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.

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

The PCT/RO/134 table

Claims (22)

1. an isolated polypeptide is characterized in that, described polypeptide has β-1,4-mannosans enzymic activity.

2. polypeptide as claimed in claim 1 is characterized in that, described polypeptide has following feature:

A) theoretical molecular is 39kDa;

B) theoretical pI value is 5.29;

C) optimal pH is 5.0-8.0;

D) optimal reactive temperature is 45-55 ℃;

E) than vigor〉11000U/mg, and

F) very high protease hydrolysis resistance is arranged, especially to stomach en-and trypsinase.

3. polypeptide as claimed in claim 1 or 2 is characterized in that, described amino acid sequence of polypeptide is SEQ ID NO:2.

4. polypeptide as claimed in claim 1 or 2, it is characterized in that described polypeptide comprises that sequence homogeny with SEQ ID NO:2 is at least about 70%, the 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%.

5. polypeptide as claimed in claim 1 or 2 is characterized in that, described polypeptide is by the polynucleotide encoding of hybridizing with the complementary strand of polynucleotide shown in the SEQ ID NO.1 under rigorous condition.

6. polypeptide as claimed in claim 1 or 2 is characterized in that, described polypeptide is coded by allelotrope or the natural mutation of polynucleotide shown in the SEQ ID NO.1.

7. polypeptide as claimed in claim 1 or 2 is characterized in that, described polypeptide comprises the aminoacid sequence sequence of being derived through one or more amino acid whose replacements, disappearance and/or insertion by aminoacid sequence shown in the SEQ ID NO.2.

8. the nucleic acid of each described polypeptide among the claim 1-6 that encodes.

9. nucleic acid as claimed in claim 8, it comprises, and sequence homogeny with SEQ ID NO:1 is at least about 70%, the 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%.

10. nucleic acid as claimed in claim 8, its nucleotides sequence is classified SEQ ID NO:1 as.

11. a recombinant vectors, it comprises each described nucleic acid among the claim 8-10.

12. a host cell, it comprises the described recombinant vectors of claim 11.

13. host cell as claimed in claim 12 is characterized in that, described host cell is eukaryotic cell.

14. host cell as claimed in claim 13 is characterized in that, described host cell is yeast cell.

15. host cell as claimed in claim 14 is characterized in that, described host cell is the pichia yeast cell.

16. as each described host cell among the claim 12-15, it is characterized in that described recombinant vectors is integrated in the genome of described host cell.

17. a method of producing 'beta '-mannase, it may further comprise the steps successively:

I) cultivate each described host cell among the claim 12-16,

Ii) induce its expression,

Iii) gather in the crops expression product.

18. method as claimed in claim 17 is further comprising the steps of:

Iv) purifying expression product.

19. a composition, its contain significant quantity as on each described polypeptide and the bromatology among the claim 1-7 or industrial acceptable vehicle or vehicle.

20. a composition, what it contained significant quantity is selected from down the enzyme of group as each described polypeptide among the claim 1-7 and one or more: proteolytic enzyme, cellulase, beta-xylanase, beta-glucanase, α-Dian Fenmei, lipase, polygalacturonase; Or their mixture.

21. one kind as claim 19 and 20 described compositions, it is used for feed and foodstuff additive, is used for deinking and association with pulp bleaching.

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

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