CN102732494B - Beta-mannanase and preparation method thereof - Google Patents
Beta-mannanase and preparation method thereof Download PDFInfo
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Abstract
The invention discloses beta-mannanase and a preparation method thereof. The beta-mannanase provided in the invention is a protein which is a protein 1, a protein 2 or a protein 3, wherein the protein 1 is composed of a 22-416 amino acid sequence shown in a sequence 1 in a sequence table; the protein 2 is composed of an amino acid sequence represented by the sequence 1 in the sequence table; and the protein 3 is obtained through substituting and/or deleting and/or adding one or more amino acid residues to an amino acid residue sequence of the protein 1 or the protein 2, has activities of the beta-mannanase, and is derived from the protein 1 or the protein 2. Engineering bacteria formed by introducing protein coding gen into Pichia pastoris are fermented in a 5L fermenting tank in a high density manner, and the enzymatic activity of a fermenting solution can reach 50029.6U/mL (the protein content is 6.1mg/mL), so efficient express is realized. The beta-mannanase of the invention has application potentials in the food industry, the medicine industry, the papermaking industry, the forage industry, the petroleum exploitation industry, the fine chemical engineering industry and the like.
Description
Technical field
The present invention relates to 'beta '-mannase and preparation method thereof.
Background technology
'beta '-mannase (endo-1,4-β-D-mannanmanno hydroase EC 3.2.1.78) be the inscribe lytic enzyme of the mannosans (glucomannan, polygalactomannan etc.) of hydrolysis taking β-Isosorbide-5-Nitrae-D-mannopyranose as main chain.Mannosans is the wire polymer being formed by connecting by β-Isosorbide-5-Nitrae-D-mannopyranose, is the second largest component of hemicellulose, is extensively present in occurring in nature, and it is the chief component composition of various plants cell walls.'beta '-mannase is widely used in the industries such as feed, food, papermaking, bio-transformation, be a kind of important industrial enzyme (Cui Xu, Cao Yunhe, Li Ruiguo. 'beta '-mannase progress. Chinese herding magazine, 2010,46 (15): 69-72).'beta '-mannase exists widely at occurring in nature.Plant, animal and most microorganism that some are low etc., can both produce mannase.And microorganism is because the condition of its growth is relatively simple, yield of enzyme is large, character enzyme is various, it is the main source of mannase.Wherein study many microbes producing cellulases, there is the genus bacillus in bacterium, aspergillus in fungi, Trichodermareesei, and (the Mao Shaoming such as streptomycete in actinomycetes, Zhang Huaiyun. 'beta '-mannase Progress on Molecular Biology. biotechnology communication, 2006,17 (6): 995-997).
Tradition 'beta '-mannase comes from natural strain enzyme-producing, produces production of enzyme low, and the character of enzyme is not ideal enough.In the last few years, due to the development of needs and the biotech development, particularly genetic engineering technique of industrial application, many investigators had done a large amount of work in the research of the screening of new enzyme resource, high efficient expression, catalytic mechanism and the transformation of enzyme molecule.Chinese invention patent ZL 200510081649.1 discloses a kind of by the method for Bacillus licheniformis mannase high efficient expression in intestinal bacteria or genus bacillus, improves expression level and the quality of existing mannase.ZL200510103125.8 discloses a kind of by the method for subtilis mannase high efficient expression in pichia spp, fermentor tank yield of enzyme reaches 200,000IU/mL (being calculated as 1,100U/mL by μ mol), higher than original genus bacillus yield of enzyme, manufacturing cost is low.ZL 200910084472.9 discloses subtilis by the method for expressing in pichia spp after codon optimized, and the average enzyme work of fermentor tank reaches 2100U/mL.Chinese patent ZL200610033307.7 discloses the mannase gene of armillariella tabescens, and has built Pichia anomala expression system.ZL200610073160.4 discloses sulfuraspergillus mannase gene, and its optimal pH is 2.4.ZL201010022054.X discloses aspergillus niger 26 family's mannase genes, and 40 DEG C of its optimal pH 5.0, optimal reactive temperatures are processed 1 hour residual enzyme for 90 DEG C and lived 66.39%.The patent of expressing about beta-mannase gene is relatively also few, and the source of enzyme is less, can not adapt to the needs of multiple industry; And expression amount is not high yet, be difficult to cheap being applied in the middle of industrial production.
Chaetomium (Chaetomium) is common mould rotten bacterium, and great majority have stronger cellulose-decomposing ability.The research of domestic cellulase to chactomium globosum, zytase is many, but there are no the research that it is produced to mannase, also has no external relevant report (BRENDA
http:// www.brenda-enzymes.org/).Chactomium globosum CQ31 is the fungal strain that this laboratory is sieved to from soil, and it can utilize Rhizoma amorphophalli powder for carbon source, and a large amount of secretion mannases is an important resource of mannase.Pichia spp is the albumen of secreting, expressing foreign protein, particularly originated from fungus efficiently.Pichia anomala expression, has and cultivates the feature simple, expression amount is large, is suitable for industrialized production.
Summary of the invention
The object of the present invention is to provide a kind of albumen, derive from chactomium globosum (Chaetomium sp.) CQ31.Bacterial strain CQ31 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City) on October 15th, 2009, and preserving number is CGMCC № 3341.
Albumen provided by the invention, called after CsManA, specifically following 1) or 2) or 3) protein:
1) protein being formed by the aminoacid sequence shown in the 22-416 position of sequence in sequence table 1;
2) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
3) by 1) or 2) amino acid residue sequence through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have beta-mannase enzymic activity by 1) or 2) derivative protein.
SEQ ID No:1 in sequence table is made up of 416 amino-acid residues, is signal peptide from aminoterminal (N end) 1-21 amino acids residue, is maturation protein from aminoterminal 22-416 amino acids residue.
In order to make 1) in albumen be convenient to purifying, can be in by sequence table 1) N-terminal or the C-terminal of protein connect upper label as shown in table 1.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned 3) albumen in can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned 3) encoding gene of the albumen in can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in the 98-1285 position of sequence in sequence table 2 or 35-1285 position, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.
Within the encoding gene (called after CsManA) of above-mentioned albumen also belongs to protection scope of the present invention.
Above-mentioned encoding gene specifically can be following 1)-5) in arbitrary described gene:
1) its encoding sequence be in sequence table sequence 2 from 5 ' end 98-1285 position;
2) its nucleotide sequence be sequence 2 in sequence table from 5 ' end 35-1285 position;
3) gene shown in sequence 2 in sequence table;
4) under stringent condition with 1) or 2) or 3) gene recombination and coding claim 1 described in the gene of albumen;
5) with 1) or 2) or 3) gene there is the gene of albumen described in more than 90% homology and coding claim 1.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS is hybridized at 65 DEG C and washes film in DNA or RNA hybrid experiment.
SEQ ID No:2 in sequence table is by 1495 based compositions, encoding sequence is from 5 ' end 35-1285 bit base, coding has the protein of the aminoacid sequence of SEQ ID No:1 in sequence table, be 5 ' end non-translational region from 5 ' end 1-34 bit base, be signal coding sequence from 5 ' end 35-97 bit base, being mature protein coding sequence from 5 ' end 98-1285 bit base, is 3 ' end non-translational region from 5 ' end 1286-1495 bit base.
The primer pair of amplification said gene total length or its arbitrary fragment also belongs to protection scope of the present invention.
Within the expression cassette that contains said gene or recombinant vectors or transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.
Above-mentioned recombinant vectors can be specifically between the multiple clone site of pPIC9K, to insert the recombinant expression vector that said gene obtains.
Above-mentioned recombinant bacterium is that above-mentioned recombinant vectors is imported to the recombinant yeast pichia pastoris that pichia spp obtains.
Above-mentioned pichia spp can be GS115.
Another object of the present invention is to provide a kind of method of producing the above-mentioned albumen of claim.
Aforesaid method is by above-mentioned recombinant bacterium fermentation culture, obtains described albumen.
Described fermentation culture is passed through following steps successively:
1) cultivate on basis: the recombinant bacterium described in claim 4 or 6 is cultivated in BMGY substratum, treated that glycerine consumption flows the glycerol adding stage completely;
2) the glycerol adding stage: in step 1) basis on, glycerine is added in described substratum, treat that glycerine consumption carries out stream completely and add the methyl alcohol stage;
3) add the methyl alcohol stage: in step 2) basis on, methyl alcohol is added in described substratum and is cultivated.
Wherein: step 1), the solvent of described BMGY substratum is water, solute is the material of following final concentration: the yeast extract that mass percentage concentration is 1%, mass percentage concentration is 2% peptone, 100mmol/L pH 6.0 phosphoric acid buffers, mass percentage concentration is 1.34% YNB, and mass percentage concentration is 4 × 10
-5% vitamin H, and the glycerine of volume percent 1%; The temperature of described cultivation is that 30 DEG C, pH are 5.0, dissolved oxygen is greater than 20%;
Step 2) in, described glycerine is aqueous glycerin solution soluble in water and 500g/L that form; The speed that described glycerine is added described substratum is 30mL/h/1L starting fermentation liquid, and the time is 4h; Step 2) temperature is that 30 DEG C, pH are 5.0, dissolved oxygen is greater than 20%;
Step 3) in, temperature is that 30 DEG C, pH are 6.0, dissolved oxygen is greater than 20%; The flow acceleration of described methyl alcohol is 6mL/h/1L starting fermentation liquid.
Within above-mentioned albumen also belongs to protection scope of the present invention as the application in 'beta '-mannase.Described application is proteolysis locust bean gum and/or Rhizoma amorphophalli powder described in claim 1 preferably.
The invention provides a kind of 'beta '-mannase CsManA and encoding gene thereof that derives from chactomium globosum CQ31.The present invention constructs the Pichia yeast engineering of 'beta '-mannase.By this bacterial strain, in the fermentation of 5L fermentor tank middle-high density, the enzyme activity of fermented liquid can reach 50029.6U/mL (protein content is 6.1mg/mL), has realized high efficient expression.Zymologic property is expressed, and the optimal pH of CsManA is 5.0, and optimum temperuture is 65 DEG C.CsManA has than the live feature of high, Heat stability is good of enzyme, can within the scope of pH widely, stablize and bring into play katalysis.'beta '-mannase of the present invention has the potentiality of application in industries such as food, medicine, papermaking, feed, oil production and fine chemistry industries.
Brief description of the drawings
Fig. 1 is the product enzyme course of 5L fermentor tank, and wherein (■) is the protein concentration in fermented liquid, and (▲) is that the enzyme of 'beta '-mannase in fermented liquid is lived.
Fig. 2 is the SDS-PAGE of 'beta '-mannase purge process.Wherein swimming lane mark 1 is ferment tank liquid supernatant, and 2 is the pure enzyme after Q-sepharose column chromatography purification, and 3 is with the result after N-deglycosylating enzyme desaccharification base (in swimming lane, the protein band at 30kDa place is N-deglycosylating enzyme).
Fig. 3 is 'beta '-mannase (a) optimal pH and (b) pH stability.Wherein (◆) citrate buffer solution (pH3.5-6.0), (■) acetate buffer solution (pH 4.0-5.5), (▲) MOPS damping fluid (pH 6.0-8.0), (×) phosphoric acid buffer (pH 6.0-8.0), (△) CHES damping fluid (pH 8.0-10.0), () CAPS damping fluid (pH 9.0-11.0), (zero) Glycine-NaOH (pH 9.0-12.0).
Fig. 4 is 'beta '-mannase (a) optimum temperuture and (b) temperature stability.
Fig. 5 is 'beta '-mannase (a) hydrolysis locust bean gum, (b) the TLC result of hydrolysis Rhizoma amorphophalli powder.
Fig. 6 is 'beta '-mannase (a) hydrolysis mannobiose, (b) the TLC result of hydrolysis mannotriose.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but the present invention is not limited to following examples.
In following embodiment, method therefor is ordinary method if no special instructions, and the solvent in described substratum is water, and the synthetic and examining order of described primer completes by Shanghai Sheng Gong biotechnology company limited (Beijing company).
Embodiment 1: the clone of the beta-mannase gene of chactomium globosum CQ31
The acquisition of the full-length gene order of the 'beta '-mannase of chactomium globosum CQ31 comprises the following steps:
1, the clone of beta-mannase gene fragment
According to the aminoacid sequence of announcing fungi 'beta '-mannase in GenBank, pass through compare of analysis conserved sequence, use Codehop software (
http:// bioinformatics.weizmann.ac.i1/blocks/codehop.html) designing online degenerated primer, the sequence of degenerated primer and corresponding conserved amino acid is as follows:
DP1 (upstream primer): CCTGCGCGTCTGGGGNTTYAA (LRVWGF)
DP2 (downstream primer): CGTTGGCCAGCTCCCANGCRAA (FAWELANE)
Wherein Y:A/G, N:A/T/G/C, R:C/T
PCR reacts taking the total DNA of chactomium globosum CQ31 as template, and DP1, DP2 are primer, uses Ex taq archaeal dna polymerase (Takara company) amplification.Program is: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 cyclic amplifications; Extend 10min.PCR product reclaims and is connected to pMD-18 T carrier (Takara company) after detecting with 1% agarose gel electrophoresis, and heat shock method transforms intestinal bacteria, selects single bacterium colony order-checking.Amplified fragments is long is 445bp.
2, the acquisition of RACE reaction and full length cDNA sequence
CQ31 is inoculated into (1% yeast extract paste, 1% peptone, 2% Rhizoma amorphophalli powder, 0.03%MgSO in culture medium
47H
2o, 0.03%CaCl
2, 0.03%FeSO
47H
2o), 37 DEG C of shaking tables are cultivated 3d.Mycelia is collected by the method for suction filtration, and is placed in liquid nitrogen and grinds.Get about 100mg and grind rear thalline, be placed in 1.5mL centrifuge tube, add 1mL Trizol reagent (Invitrogen company), extract total RNA.Use paramagnetic particle method (Omega company) purified mRNA, as the template of reverse transcription.
According to amplification sequence fragment out, design 5 ', 3 ' RACE primer.Primer sequence is as follows:
5’SP1：CTAACTCCCACGCGAAGATGGTG
5’SP2：GTAGCGGGTCACCATCTCTTTGAC
3’SP1：TGGTGACCCGCTACAAGGATTCTC
3’SP2：ATCTTCGCGTGGGAGTTAGCCAAC
With the mRNA of purifying as template,, 3 ' RACE-Ready eDNA synthetic 5 ' according to SMART RACE eDNA Amplification Kit (Takara company) kit method reverse transcription.Taking above-mentioned 5 ', 3 ' eDNA as template, corresponding SP1, SP2 are gene-specific primer, carry out respectively 2 and take turns nested PCR reaction.Amplified fragments is connected on pMD-18T carrier and checks order.The product that wherein obtains 619bp by 5 ' RACE, 3 ' RACE obtains 861bp product.After sequence assembly, obtain eDNA sequence, long 1495bp (SEQ ID No:2), wherein contains the open reading frame of 1251bp.This gene translation obtains the peptide section being made up of 416 amino-acid residues, through SignalP 3.0 (
http:// www.cbs.dtu.dk/services/SignalP/) predict to be signal peptide from aminoterminal (N end) 1-21 amino acids residue, be maturation protein from aminoterminal 22-416 amino acids residue.Use BlastP (
http:// blast.ncbi.nlm.nih.gov/Blast.cgi) with NCBI on protein sequence comparison, the homogeny of the mannosans enzyme sequence of this albumen and Aspergillus nidulans FGSC A4, Aspergillus terreus NIH2624 is only 74% and 72%, has certain novelty.
Embodiment 2: the structure of the expression engineering bacteria of chactomium globosum CQ31 'beta '-mannase and high efficient expression
1, the structure of beta-mannase expression of enzymes engineering bacteria
Express primer according to the sequences Design of Yeast expression carrier and 'beta '-mannase, upstream and downstream primer adds respectively upper EcoR I and Not I restriction enzyme site upstream and downstream primer is as follows respectively:
Upstream primer:
gAATTCcCAAGCCGAGCTGTGCAGG (EcoR I)
Downstream primer:
gCGGCCGCtTAGTCACTTCTCGCCTCGCTA (Not I).
The eDNA obtaining from total RNA reverse transcription of the chactomium globosum CQ31 of embodiment 1 with above-mentioned primer pair, increase, PCR product is after agarose gel electrophoresis detects, recovery is connected to pMD-18T carrier, be transformed in E.coli JM109 (purchased from Bo Maide biotech firm), select single bacterium colony order-checking, the nucleotide sequence of amplified production as shown in the 98-1285 position of sequence in sequence table 2, the protein of aminoacid sequence in its codified sequence table shown in the 22-416 position of sequence 1 composition.In sequence table, the 35-97 position of sequence 2 is signal peptide gene sequence, the signal peptide of the 1-21 position of encoding sequence 1.
By transformant correct sequence, enzyme is connected on the pPIC9K carrier that corresponding enzyme was cut in advance (purchased from Invitrogen company) after cutting (EcoR I/Not I).Use PCR and double digestion authentication sequence, correct being connected to finished to red expression vector called after pPIC-CsmanA.
Pichia pastoris GS115 (purchased from Invitrogen company) competence is according to the method preparation in pichia spp operation instruction (Invitrogen company).With restriction enzyme Sal I linearization plasmid pPIC-CsmanA, and adjust concentration to 1ug/uL.Get 80uL yeast competence, mix with the plasmid of 10uL linearity, be placed in the electric shock cup (Bio-rad company) of the 0.2cm of precooling, electric shock transforms.After electric shock, add rapidly the 1M Sorbitol Solution USP of 1.0mL precooling, coat on auxotrophy screen plate, cultivate 3-4d, collect thalline with sterilized water.The thalline of collecting is suitably coated with on different concns G418 screen plate after dilution, and the concentration of G418 is respectively 1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL, selects respectively well-grown single bacterium colony under different G418 concentration, carries out shaking flask and sieves again.Select by the following method the bacterial strain that expression amount is high, then carry out the fermentor tank high density fermentation checking of step 2:
Select pichia spp transformant list bacterium colony, be inoculated in 25mL BMGY substratum, 30 DEG C, 200rpm shaking culture is to OD
600nmbe 12 left and right, centrifugal collection thalline, is transferred in the 500mL triangular flask that the improved BMGY substratum of 100mL (glycerine being replaced with to 0.5% methyl alcohol) is housed, and makes OD
600nmreach 8, same culture conditions is cultivated, and every 24h adds 100% methyl alcohol to final concentration 1%, induction 5d.Select a strain pichia spp (obtaining from 8mg/mL concentration G418 screening), yield of enzyme is up to 567.8U/ml (enzyme activity determination method and enzyme are lived definition with reference to embodiment 3), called after GS115/pPIC-CsmanA.
2, fermentor tank high density fermentation (5L ferment tank)
Seed culture: the recombinant pichia yeast strain GS115/pPIC-CsmanA that experimental example 2 steps 1 are obtained, be inoculated in the 500mL triangular flask that 100mL BMGY substratum is housed, 30 DEG C, 200 turn shaking culture 24h more than, obtain OD
600nmbe about 3.0 seed liquor.
Cultivate on basis: be inoculated in 5L fermentor tank and (2L BMGY substratum be housed, this substratum solvent is water, solute is the material of following final concentration: the yeast extract that mass percentage concentration is 1%, mass percentage concentration is 2% peptone, 100mmol/L pH 6.0 phosphoric acid buffers, mass percentage concentration is 1.34% YNB, and mass percentage concentration is 4 × 10
-5% vitamin H, and the glycerine of volume percent 1%).In process, temperature is 30 DEG C, with ammoniacal liquor and phosphorus acid for adjusting pH to 5.0, is greater than 20% (relative value by adjusting rotary speed and air flow control dissolved oxygen, the present invention is with 30 DEG C of fermentation conditions, pH 5.0, rotating speed 700rpm is defined as 100%, taking saturated sodium bisulfite solution dissolved oxygen as 0).Treat glycerine consumption (dissolved oxygen DO value rises rapidly) completely, enter the stream glycerol adding stage.
The stream glycerol adding stage: glycerine (50%W/V is 500g/L aqueous glycerin solution) flow velocity is 30mL/h/1L starting fermentation liquid, and stream adds 4h.After glycerine consumption completely, stop stream and add, enter the methanol feeding stage; Wherein this phase temperature is that 30 DEG C, pH are 5.0, dissolved oxygen is greater than 20%.
The methanol feeding stage: temperature is 30 DEG C, pH is adjusted to 6.0, control methyl alcohol flow velocity and be about 6mL/h/1L starting fermentation liquid, stream adds until fermentation ends, keep dissolved oxygen (as can not make dissolved oxygen remain on more than 20%, suitably reduce flow acceleration) more than 20% simultaneously.Fermenting process is shown in Fig. 1, and wherein (■) is protein content in supernatant in fermented liquid, and (▲) is that in fermented liquid supernatant, 'beta '-mannase enzyme is lived.It is maximum that enzyme work reached at the 6th day, and the highest enzyme work reaches 50029.6U/mL (enzyme activity determination method and enzyme are lived definition with reference to embodiment 3), and the protein content in fermented liquid reaches 6.1mg/mL simultaneously.Empty carrier pPIC9K is imported in GS115 according to the method for the present embodiment step 1, then carry out the fermentation of step 2, result shows in fermented liquid without above-mentioned albumen generation.(shaking flask checking yeast itself does not produce 'beta '-mannase).
Embodiment 3: the purifying of chactomium globosum 'beta '-mannase and zymologic property
1, enzyme is lived definition and measuring method
Enzyme activity determination adopts DNS method.100 μ L are the enzyme liquid of dilution suitably, joins 0.5% locust bean gum (LBG) of 900 μ L 50mM pH 5.0 citrate buffer solutions preparations, 55 DEG C of reaction 10min.After reaction, with DNS reagent termination reaction and reducing sugar reaction.Taking seminose as graticule, it is 1U that definition per minute generates the needed enzyme amount of 1 μ mol seminose.
2, the purifying of 'beta '-mannase
The fermented liquid supernatant of 10mL fermentor tank, after dialysis equilibrium, the Q-Sepharose post of the phosphoric acid buffer balance of 20mM pH 6.5 for loading.After the clean unconjugated albumen of damping fluid with 5 column volumes, 0-500mM NaCl linear elution fraction collection.After the pure enzyme obtaining is merged, for subsequent use with the citrate buffer solution dialysis of pH 5.0.Whole purge process is in table 2; SDS-PAGE electrophorogram is shown in Fig. 2, wherein swimming lane 1 is ferment tank liquid supernatant, 2 is the pure enzyme after Q-Sepharose column chromatography purification, and 3 is by the result (in swimming lane, the protein band at 30kDa place is N deglycosylating enzyme) after N deglycosylating enzyme (NEB company) desaccharification base.
The purge process of table 2 'beta '-mannase
3, 'beta '-mannase optimal reaction pH and pH stability
The mensuration of optimal reaction pH: be in the different buffer systems of 3.5 to 11.0 scopes at pH, measure enzyme at 55 DEG C and live, live taking high enzymatic activity as the relative enzyme of 100% calculating.Result is as Fig. 3 a, and buffer system is (◆) citrate buffer solution (pH 3.5-6.0), (■) acetate buffer solution (pH 4.0-5.5), (▲) MOPS damping fluid (pH6.0-8.0), (×) phosphoric acid buffer (pH 6.0-8.0), (△) CHES damping fluid (pH 8.0-10.0), () CAPS damping fluid (pH 9.0-11.0), (zero) Glycine-NaOH (pH 9.0-12.0).
The detection of pH stability: enzyme liquid, respectively by above-mentioned buffer system dilution suitable multiple, is placed in to 50 DEG C of water-baths and is incubated 30min, and ice bath 30min measures remaining enzyme work immediately, taking untreated contrast as 100%, the results are shown in Figure 3b.
The optimal pH that experiment records this enzyme is 5.0, has the more than 50% of the highest enzyme work in the time of pH 4.0, still has the more than 40% of the highest enzyme work for 9.5 times at pH.This enzyme still keeps the more than 70% of enzyme activity in the scope of pH 5.0-11.0 after thermal treatment 30min, stablizing widely within the scope of pH.This enzyme of description of test belongs to acid mannase, and can under very wide pH scope, have higher response capacity.
4, 'beta '-mannase optimal reactive temperature and temperature stability
The mensuration of optimal reactive temperature: pH 5.0 citric acid buffer systems, react under the differing temps within the scope of 30 DEG C-90 DEG C, taking high enzymatic activity as 100%.The results are shown in Figure 4a, the optimum temperuture of this enzyme is 65 DEG C.
The detection of temperature stability: after the citrate buffer solution of enzyme liquid use pH 5.0 is suitably diluted, be placed under differing temps and be incubated 30min, ice bath 30min measures remaining enzyme work immediately, thinks that treated contrast is 100%.The results are shown in Figure 4b, it is stable that this enzyme keeps in the time of≤55 DEG C, and enzyme is lived and lost hardly.
5, the substrate specificity of enzyme and the mensuration of kinetic parameter
Hydrolysis specificity: select different substrates, measure enzyme and live, concentration of substrate is 0.5%.The results are shown in Table 3.This enzyme has higher activity to locust bean gum, guar gum, Rhizoma amorphophalli powder, and xylan is also had to a small amount of activity, and starch and Mierocrystalline cellulose are not had to activity.
The substrate specificity of table 3 chactomium globosum CQ31 'beta '-mannase
Kinetic parameter (K
m: Michaelis-Menton constant, V
max: maximum reaction velocity) mensuration: prepare a series of locust bean gums, guar gum, Rhizoma amorphophalli powder with pH 5.0 citric acids, concentration is respectively substrate from 1.5mg/mL to 17.5mg/mL, 50 DEG C reaction 5min, measure produce reducing sugar amount.Use " Grafit " software, analyze K
mand V
max.The results are shown in Table 4.This mannase has higher V to locust bean gum and guar gum
max(be respectively 5974 μ mol min
-1mg
-1with 5693 μ mol min
-1mg
-1), reflect that this mannase has higher catalytic activity to gala mannase.But this 'beta '-mannase is stronger to the substrate avidity of locust bean gum, K
mfor 3.1mg/mL, much smaller than the avidity to guar gum.
The kinetic parameter of table 4 chactomium globosum CQ31 'beta '-mannase
6, the hydrolysis properties analysis of 'beta '-mannase
Above-mentioned mannase is joined in the different substrate of pH 5.0 citric acid preparations 1% to 50 DEG C of insulation 24h, at regular intervals samplings.Substrate comprises locust bean gum, Rhizoma amorphophalli powder, mannobiose, mannotriose, and enzyme concentration is 5U/mL, TLC assay products.Experimental result demonstration, the product of this enzymic hydrolysis locust bean gum is mainly mannobiose and seminose, the amount of monose and disaccharides increases gradually with the prolongation in reaction times; The product of hydrolysis Rhizoma amorphophalli powder is that between seminose, mannobiose, mannotriose and mobility, the one sugar in mannobiose and trisaccharide (is shown in Fig. 5 a, Fig. 5 b).In addition, in the process of two kinds of substrate hydrolysis, do not observe the generation of mannotetrose, pentasaccharides (yet can't see trisaccharide taking locust bean gum as substrate), but have all the time the oligosaccharides that molecular weight is very large to occur in the bottom of TLC plate.Taking mannobiose and mannotriose as substrate, (see Fig. 6 a, Fig. 6 b), result shows that this mannase can not decompose mannobiose, generates mannobiose and seminose but can decompose mannotriose.
Claims (12)
1. an albumen is following 1) or 2) protein:
1) protein being formed by the aminoacid sequence shown in the 22-416 position of sequence in sequence table 1;
2) protein being formed by the aminoacid sequence shown in sequence in sequence table 1.
2. the encoding gene of albumen described in claim 1.
3. encoding gene according to claim 2, is characterized in that: described encoding gene is following 1)-2) in arbitrary described gene:
1) its encoding sequence be in sequence table sequence 2 from 5 ' end 98-1285 position;
2) its nucleotide sequence be sequence 2 in sequence table from 5 ' end 35-1285 position.
4. contain the expression cassette of gene described in claim 2 or 3.
5. contain the recombinant vectors of gene described in claim 2 or 3.
6. recombinant vectors according to claim 5, is characterized in that: described recombinant vectors is between the multiple clone site of pPIC9K, to insert the recombinant expression vector that gene obtains described in claim 2 or 3.
7. contain the recombinant bacterium of gene described in claim 2 or 3.
8. recombinant bacterium according to claim 7, is characterized in that: described recombinant bacterium is that the recombinant vectors described in claim 5 or 6 is imported to the recombinant yeast pichia pastoris that pichia spp obtains.
9. a method of producing albumen described in claim 1, is by the recombinant bacterium fermentation culture described in claim 7 or 8, obtains described albumen.
10. method according to claim 9, is characterized in that: described fermentation culture is passed through following steps successively:
1) cultivate on basis: the recombinant bacterium described in claim 7 or 8 is cultivated in BMGY substratum, treated that glycerine consumption flows the glycerol adding stage completely;
2) the glycerol adding stage: on the basis of step 1), glycerine is added in described substratum, treated that glycerine consumption carries out stream completely and add the methyl alcohol stage;
3) add the methyl alcohol stage: in step 2) basis on, methyl alcohol is added in described substratum and is cultivated.
11. methods according to claim 10, it is characterized in that: in step 1), the solvent of described BMGY substratum is water, solute is the material of following final concentration: the yeast extract that mass percentage concentration is 1%, mass percentage concentration is 2% peptone, 100mmol/L pH6.0 phosphoric acid buffer, the YNB that mass percentage concentration is 1.34%, mass percentage concentration is 4 × 10
-5% vitamin H, and the glycerine of volume percent 1%; The temperature of described cultivation is that 30 DEG C, pH are 5.0, dissolved oxygen is greater than 20%;
Step 2) in, described glycerine is aqueous glycerin solution soluble in water and 500g/L that form; The speed that described glycerine is added described substratum is 30mL/h/1L starting fermentation liquid, and the time is 4h; Step 2) temperature is that 30 DEG C, pH are 5.0, dissolved oxygen is greater than 20%;
In step 3), temperature is that 30 DEG C, pH are 6.0, dissolved oxygen is greater than 20%; The flow acceleration of described methyl alcohol is 6mL/h/1L starting fermentation liquid.
Described in 12. claims 1, albumen is as the application in 'beta '-mannase, and described application is proteolysis locust bean gum and/or Rhizoma amorphophalli powder described in claim 1.
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| CN104762243A (en) * | 2014-01-08 | 2015-07-08 | 中国科学院微生物研究所 | Recombinant strain and method for preparing alkaline beta-mannanase |
| CN104046645A (en) * | 2014-06-11 | 2014-09-17 | 四川农业大学 | Method for enhancing gene expression level of beta-mannase by co-expressing chaperonin |
| CN108396017A (en) * | 2017-10-20 | 2018-08-14 | 山东奥博生物科技有限公司 | A kind of industrial fermentation process of mannase |
| CN107936100A (en) * | 2017-11-17 | 2018-04-20 | 四川大学 | Common calanthe herb mannose-binding protein |
| CN108165540B (en) * | 2018-02-11 | 2020-09-01 | 中国农业大学 | Rhizomucor miehei alpha-amylase and coding gene and application thereof |
| CN112029751B (en) * | 2019-06-03 | 2022-05-03 | 中国农业大学 | Production method and application of thermophilic fungus mannase |
| CN113151216B (en) * | 2021-04-21 | 2022-09-30 | 中国农业大学 | Heat-resistant xylanase with high arabinoxylan activity, and coding gene and application thereof |
| CN118086251A (en) * | 2024-04-24 | 2024-05-28 | 中国科学院天津工业生物技术研究所 | Beta-mannase high-enzyme activity mutant M-Q7S, recombinant bacterium and application thereof |
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