CN103275954A - High temperature and alkali resisting mannanase Man5XZ7, gene and application thereof - Google Patents
High temperature and alkali resisting mannanase Man5XZ7, gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of gene engineering, concretely relates to high temperature and alkali resisting mannanase Man5XZ7, gene and application thereof. The amino acid sequence is shown in the SEQ ID No.1 or SEQ ID No.2. The invention provides a new excellent high temperature and alkali resisting mannanase which is suitable for foodstuff and weaving industry, and solves the technology problem and overcomes the prior art. The optimized pH of mannanase of the present invention is pH 5.0, and the mannanase has a higher enzyme activity at pH 8.0-9.0; the product has a good pH stability; the product has a good antitrypsin capability.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of high temperature alkali resistant mannase Man5XZ7 and gene and application.
Background technology
Plant cell wall polysaccharides is mainly by Mierocrystalline cellulose, and this three major types polysaccharide of hemicellulose and xylogen is formed.Wherein, the content of hemicellulose is only second to Mierocrystalline cellulose, accounts for 35% of plant dry weight, is the abundantest reproducible biomass resource of occurring in nature.Hemicellulose mainly comprises multiple polysaccharide composition such as xylan, mannosans, xylan, arabogalactan, dextran.Mannosans is with linear polysaccharide that 1,4-β-D-mannopyranose glycosidic bond is formed by connecting.The structural analysis of mannosans finds, on its main chain residue can be replaced by glucose or semi-lactosi by α-1, the 6-glycosidic link formation branch that links to each other with mannose residue then is called different mannosans.Mainly contain polygalactomannan (galactomannan), glucomannan (glucomannan), gala glucomannan (galactoglucomannan).Therefore digesting mannosans mainly is to rely on the synergy of beta-mannase excision enzyme and mannoside restriction endonuclease to be decomposed into seminose.Comprising: β-D-mannase (β-mannanase), beta-Mannosidase (β-mannosidase), beta-glucosidase (glucosidase), alpha-galactosidase (α-galactosidase) and take off acetyl esterification enzyme side chain enzymes such as (deacytle esterase).
(β-mannanase is the hemicellulase of a class energy hydrolysis mannosans EC3.2.1.78) to 'beta '-mannase, extensively is present in microorganism, the animal and plant.Protein structure analysis revealed, 'beta '-mannase mainly belong to glycosyl hydrolase family 5 and 26.The glycosyl hydrolase that comprises multiple different sources in the glycosyl hydrolase family 5.Be mainly derived from microorganism, comprise fungi, bacterium and actinomycetes, as reporting wooden mould, mould, aspergillus, yeast and the shuttle spore bacterium in the fungi, the streptomycete in the pseudomonas in the bacterium, bud pole bacterium, vibrios and the actinomycetes etc. all is the main monoid of 'beta '-mannase.Because microbe-derived 'beta '-mannase has lot of advantages, as the enzymic activity height, Heat stability is good and extraction are convenient, cost is low, compares with the 'beta '-mannase of plant and animal material, has broader temperature action scope, action pH, outstanding features such as Substratspezifitaet, therefore, microbe-derived 'beta '-mannase is widely used in actual production and fundamental research
Along with the further investigation to 'beta '-mannase, this enzyme has all obtained at aspects such as feed, papermaking, protective foods and biotechnology research using widely.Wherein the mannase of originated from fungus is beneficial to downstream pichia spp external source and efficiently expresses, and more is applicable to industrial production.At present, warm acidicenzym during the mannase of most of originated from fungus mostly is is beneficial at feedstuff industry and uses.But food, textile industry need the high-temperature alkaline enzyme to be fit to its processing condition.Among the present invention, derive from the mannase Man5XZ7 optimal pH 5.0 of the mould Thielavia terrestris of Tai Ruisisuo spore shell XZ7CGMCC6544, under pH9.0, still have 25.6% activity, and have satisfactory stability at pH3-10; 75 ℃ of optimum temperutures; And at the pichia yeast expression system efficient secretory expression.Because above advantageous property, Man5XZ7 can be used under needs alkalescence processing condition, has broad application prospects.
Summary of the invention
The high temperature alkali resistant mannase that the purpose of this invention is to provide a kind of energy efficient application.
A further object of the present invention provides the gene of the above-mentioned high temperature alkali resistant mannase of coding.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides the recombinant bacterial strain that comprises said gene.
Another object of the present invention provides a kind of gene engineering method for preparing above-mentioned high temperature alkali resistant mannase.
Another object of the present invention provides the application of above-mentioned high temperature alkali resistant mannase.
Another object of the present invention provides the mould XZ7 of Tai Ruisisuo spore shell.
The present invention (was stored in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on September 10th, 2012 from the mould XZ7 of Tai Ruisisuo spore shell (Thielavia terrestris XZ7CGMCC6544), Institute of Microorganism, Academia Sinica, 100101), its preserving number is: separate obtaining a kind of new high temperature alkali resistant mannase Man5XZ7 CGMCC6544).
The invention provides a kind of high temperature alkali resistant mannase Man5XZ7, its aminoacid sequence is shown in SEQ ID NO.1.
SEQ?ID?NO.1：
MVRNIVATGLSLLSTAGVFLGGAAAQMPTANGTRFTIDGKTGYFAGTNSYWISFLTNNRDVDLVLDHISSSGLRILRVWGFNDVNRRPSSGTVWFQLLSSSGSQINTGADGLQRLDYVVQSAEKRGVKLIINFVNNWNDYGGMQAYVSAFGGSKESWYTNTRAQDQYKKYIAAVVSRYVNLPAVFAWELANEPRCKGCNTDVIFKWATDISAYIRSLDPKHMITLGDEGFGLPGQTTYPYQYGEGTDFVKNLRIKNLDFGTFHMYPSSWGVPNSFGPGWIRDHAAACKAAGKPCLLEEYGVTSDQCNVERTWQAASREQAANGMAGDLFWQWGDQLSTGQTHNDGHTIYYGSSTATCLVTDHVRAINAM*
Wherein, 369 amino acid of this enzyme genes encoding, 25 amino acid of N end are signal peptide sequence " MVRNIVATGLSLLSTAGVFLGGAAA " (SEQ ID NO.3).
Therefore, the theoretical molecular of ripe high temperature alkali resistant mannase Man5XZ7 is 38.3kDa, and its aminoacid sequence is shown in SEQ ID NO.2:
QMPTANGTRFTIDGKTGYFAGTNSYWISFLTNNRDVDLVLDHISSSGLRILRVWGFNDVNRRPSSGTVWFQLLSSSGSQINTGADGLQRLDYVVQSAEKRGVKLIINFVNNWNDYGGMQAYVSAFGGSKESWYTNTRAQDQYKKYIAAVVSRYVNLPAVFAWELANEPRCKGCNTDVIFKWATDISAYIRSLDPKHMITLGDEGFGLPGQTTYPYQYGEGTDFVKNLRIKNLDFGTFHMYPSSWGVPNSFGPGWIRDHAAACKAAGKPCLLEEYGVTSDQCNVERTWQAASREQAANGMAGDLFWQWGDQLSTGQTHNDGHTIYYGSSTATCLVTDHVRAINAM*
Mannase Man5XZ7 of the present invention has good thermostability simultaneously and also at high temperature all has high reactivity in neutrality and the alkaline range.The present invention screens the mannase that Thielavia terrestris XZ7CGMCC6544 produces, and its optimum pH is 5.0, also keeps enzymic activity more than 25% at pH9.0; Optimum temperuture is 75 ℃, at 85 ℃ of enzyme activities that still have about 50%.
The invention provides the above-mentioned high temperature alkali resistant mannase man5XZ7 of coding.Particularly, the genome sequence of this gene is shown in SEQ ID NO.4:
Atggtcagaaacatcgtggccactgggctcagcctgctctcgacggctggagtttttcttggaggagcagcagcacaaatgcccactgccaacgggacacgcttcaccattgacggcaagacgggctactttgcgggcacaaactcgtactggatcagcttcctcaccaacaacagggacgttgatctcgtcttggaccacatctcgtcctccggcctcaggatcctccgagtgtggggattcaacgacgtcaaccgccgcccctcttcgggcaccgtgtggtttcaactgctctcgtcgtccggttcgcagatcaacaccggcgctgacggtctgcagagactcgactatgtcgtccagtcggccgagaagcgcggggtcaagctgattatcaactttgtcaacaactggaacgactacggcggcatgcaagcctacgtttcggcgtttggcggctccaaggaaagctggtacaccaacacgcgggcccaggaccaatacaagaagtacattgcagccgttgtcagccgctatgtcaacttgcccgccgtctttgcctgggaactggccaacgagccccgctgcaaagggtgcaataccgatgtcattttcaagtgggcaaccgatatctcggcttacatccgcagtctggatcccaaacatatgatcacgctgggtgacgagggattcggcctgccaggacaaaccacttacccttaccaatacggcgagggtaccgacttcgtcaagaacctgcgtatcaaaaatctggatttcggcactttccacatgtatcctagtagctggggcgtgccgaacagctttggtcctggttggatccgcgatcacgccgcagcctgcaaagccgctggcaagccttgtcttctggaggagtatggcgtcacatcggaccagtgcaatgtggagcggacgtggcaggcggcttcacgagagcaagctgccaacgggatggcaggcgacctcttctggcagtggggagaccagctgagcaccgggcagacacacaacgacggccacaccatctactatggatctagcaccgccacgtgtttggtgactgatcacgtcagggctattaacgccatgtaa
The method separating clone of the present invention by RT-PCR mannase gene man5XZ7, the cDNA complete sequence analysis is the result show, mannase Man5XZ7 gene man5XZ7 total length 1110bp.Wherein, the base sequence of signal peptide is:
ATGGTCAGAAACATCGTGGCCACTGGGCTCAGCCTGCTCTCGACGGCTGGAGTTTTTCTTGGAGGAGCAGCAGCA(SEQ?ID?NO.6)。
The gene order of ripe mannase Man5XZ7 is shown in SEQ ID NO.5.
SEQ?ID?NO.5
caaatgcccactgccaacgggacacgcttcaccattgacggcaagacgggctactttgcgggcacaaactcgtactggatcagcttcctcaccaacaacagggacgttgatctcgtcttggaccacatctcgtcctccggcctcaggatcctccgagtgtggggattcaacgacgtcaaccgccgcccctcttcgggcaccgtgtggtttcaactgctctcgtcgtccggttcgcagatcaacaccggcgctgacggtctgcagagactcgactatgtcgtccagtcggccgagaagcgcggggtcaagctgattatcaactttgtcaacaactggaacgactacggcggcatgcaagcctacgtttcggcgtttggcggctccaaggaaagctggtacaccaacacgcgggcccaggaccaatacaagaagtacattgcagccgttgtcagccgctatgtcaacttgcccgccgtctttgcctgggaactggccaacgagccccgctgcaaagggtgcaataccgatgtcattttcaagtgggcaaccgatatctcggcttacatccgcagtctggatcccaaacatatgatcacgctgggtgacgagggattcggcctgccaggacaaaccacttacccttaccaatacggcgagggtaccgacttcgtcaagaacctgcgtatcaaaaatctggatttcggcactttccacatgtatcctagtagctggggcgtgccgaacagctttggtcctggttggatccgcgatcacgccgcagcctgcaaagccgctggcaagccttgtcttctggaggagtatggcgtcacatcggaccagtgcaatgtggagcggacgtggcaggcggcttcacgagagcaagctgccaacgggatggcaggcgacctcttctggcagtggggagaccagctgagcaccgggcagacacacaacgacggccacaccatctactatggatctagcaccgccacgtgtttggtgactgatcacgtcagggctattaacgccatgtaa
The maturation protein theoretical molecular is 38.3kDa, and mannase gene man5XZ7 sequence and the aminoacid sequence derived are carried out the BLAST comparison in GenBank, and this gene is a kind of new mannase gene.
The present invention also provides the recombinant vectors that comprises above-mentioned high temperature alkali resistant mannase gene man5XZ7, is preferably pPIC-man5XZ7.Mannase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably mannase gene of the present invention is inserted between the EcoR I and Not I restriction enzyme site on the plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control by it, obtain expression of recombinant yeast plasmid pPIC9-man5XZ7.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned high temperature alkali resistant mannase gene man5XZ7, and preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain GS115/man5XZ7.
The present invention also provides a kind of method for preparing high temperature alkali resistant mannase Man5XZ7, may further comprise the steps:
1) with above-mentioned recombinant vectors transformed host cell, gets recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce reorganization mannosans expression of enzymes;
3) reclaim the also expressed mannase Man5XZ7 of purifying.
Wherein, preferred described host cell is pichia spp cell, cerevisiae or many types of inferior yeast cell, preferably the expression of recombinant yeast plasmid is transformed pichia spp cell (Pichia pastoris) GS115, obtains recombinant bacterial strain GS115/Man5XZ7.
The present invention also provides the application of above-mentioned high temperature alkali resistant mannase Man5XZ7, and for example, alkali mannase Man5XZ7 is used for the application of degraded polygalactomannan and glucomannan.
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, provide a kind of character good, be suitable in food, textile industry using new high temperature alkali resistant mannase.Mannase optimal pH of the present invention is 5.0, in pH8.0～9.0 higher enzymic activity is arranged; The pH good stability; Has good antitryptic ability.In addition, Man5XZ7 can effectively degrade various types of polygalactomannan and glucomannan etc., and not degradation of xylan and Microcrystalline Cellulose sodium, the mannosans part in the bleached pulp of can effectively degrading and do not influence Mierocrystalline cellulose.Therefore, the application of this mannase in industry also demonstrates its great potential.
Description of drawings
The recombinate optimal pH of mannase of Fig. 1.
The recombinate pH stability of mannase of Fig. 2.
The recombinate optimum temperuture of mannase of Fig. 3.
The recombinate thermostability of mannase of Fig. 4.
The mould XZ7 of Tai Ruisisuo spore shell (Thielavia terrestris XZ7CGMCC6544) (was stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on September 10th, 2012, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC6544).
Embodiment
Test materials and reagent
1, bacterial strain and carrier: the present invention has separated the mould XZ7 of Tai Ruisisuo spore shell (Thielavia terrestris XZ7CGMCC6544).Yeast expression vector pPIC9 and bacterial strain GS115 are available from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Carob bean gum is available from Sigma company, and other all is domestic reagent (all can buy from common biochemical reagents company and obtain).
3, substratum:
(1) Thielavia terrestris XZ7CGMCC6544 substratum is the potato juice substratum: 1000mL potato juice, 10g glucose, 25g agar, pH6.0.
(2) intestinal bacteria substratum LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
(3) BMGY substratum: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V).
(4) BMMY substratum: replace glycerine divided by 0.5% methyl alcohol, all the other compositions are all identical with BMGY, pH4.0.
Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book, perhaps carry out according to test kit and product description.
Embodiment 1 Thielavia terrestris XZ7CGMCC6544 produces the enzyme characteristic
To derive from sand top layer, Guangxi sample (enrichment medium: (NH after enrichment culture
4)
2SO
45g/L, KH
2PO
41g/L, MgSO
47H
2O0.5g/L, FeSO
47H
2O0.01g/L, CaCl
20.2g/L, corn cob meal 0.5%, wheat bran 0.5% pH2.5), is coated after the dilution routinely and is produced enzyme substratum ((NH
4)
2SO
45g/L, KH
2PO
41g/L, MgSO
47H
2O0.5g/L, FeSO
47H
2O0.01g/L, CaCl
20.2g/L carob bean gum 0.5%, 1.5% agarose pH6.0) on the flat board, is cultivated 3～5d for 45 ℃, picking produces the transparent circle bacterium colony and is producing enzyme culture medium flat plate line separation, and the sepn process 3 that repeats to rule is taken turns, and makes the bacterial strain purifying.Screen the bacterial strain of this secretion mannase by this method.The bacterial strain of product transparent circle maximum is named after the line separation and purification and is XZ7.
Thalline after cultivating 5d respectively on the PDA of the differing temps substratum, 45 ℃ down growth rapidly, 30 ℃ of following poor growths are not grown under 19 ℃.The bacterium colony circle, white, the fine hair shape, back side color milk yellow, a large amount of pores appear in the later stage, are ascoma.The ascoma Vandyke brown, spherical no aperture, frangible, the appendage mycelioid.The thecaspore Vandyke brown, smooth, unicellular, ellipse, conidiophore are closely vertical, and adnation has separation on mycelium, rare branch, conidiogenous cell taper.Conidium is smooth, and water white transparency falls bar-shaped or melon seeds shape more.Identify that through 18S rDNA this bacterial strain is that Tai Ruisisuo spore shell is mould, name Thielavia terrestris XZ7.Cultivate 3d for 45 ℃ on the liquid nutrient medium that with the carob bean gum is sole carbon source, mannase is lived and is 4.6U/mL.
The clone of embodiment 2 Thielavia terrestris XZ7CGMCC6544 beta-mannase coding gene man5XZ7
Extract Thielavia terrestris XZ7CGMCC6544 genomic dna:
3 days mycelium of liquid culture is put into mortar with the aseptic filter paper filtration, add the 2mL extracting solution, grind 5min, lapping liquid is placed the 50mL centrifuge tube, 65 ℃ of water-bath cracking 20min, every the 10min mixing once, at 4 ℃ of centrifugal 5min of following 12000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, get supernatant again and add the equal-volume Virahol, after room temperature leaves standstill 5min, 4 ℃ of centrifugal 10min of following 12000rpm.Abandon supernatant, precipitation is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ standby.
Conservative (NNWSDYGG and AWELGNEP) sequences Design according to the 5th family's mannase gene has been synthesized degenerated primer P1, P2
P1:5'-AAYAAYTGGGAYGAYTWYGGNGG-3'；
P2:5'-GGYTCRYYNSCNARYTCCCANGC-3'。
Be that template is carried out pcr amplification with the total DNA of Thielavia terrestris XZ7CGMCC6544.The PCR reaction parameter is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30sec then, 45 ℃ of (0.5 ℃ of each circulation landing) annealing 30sec of 50 ℃ of landing, 72 ℃ are extended 30s, then 94 ℃ of sex change 30s after 10 circulations, 45 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations altogether, 72 ℃ of insulation 10min.Obtain an about 176bp fragment, this fragment recovery back is linked to each other with the pEASY-T3 carrier send the order-checking of three rich Bioisystech Co., Ltd.
According to the nucleotide sequence that order-checking obtains, each three TAIL-PCR Auele Specific Primer of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1, and sp3 is positioned at the inboard of sp2.Distance between per two primers does not have strict regulation, the general 22～30nt of primer length, and annealing temperature is at 60～65 ℃.And with they difference called after usp1, usp2, usp3 (upstream Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees Table 1.
Table 1. mannase Man5XZ7TAIL-PCR Auele Specific Primer
Obtain the flanking sequence of known sequence by reverse TAIL-PCR, amplification obtains sending after product reclaims the order-checking of three rich Bioisystech Co., Ltd.Splicing back Man5XZ7 mannase gene total length 1247bp.Utilize RNA to extract test kit (SV Total RNA Isolation System, Promega) total RNA of the thalline of extracting liq inducing culture.By the cDNA of RT-PCR method amplification gene, obtaining its full-length cDNA through the order-checking of three rich Bioisystech Co., Ltd is 1110bp, 369 amino acid of encoding.Carry out 25 amino acid of analysis revealed N end with SignalP (http://www.cbs.dtu.dk/services/SignalP) and be the signal peptide of prediction.The theoretical molecular of predicting the maturation protein of this coded by said gene is 38.3kDa.
The preparation of embodiment 3 recombined xylanases
Expression vector pPIC9 is carried out double digestion (EcoR I+Not I), to encode the simultaneously gene M an5XZ7 double digestion (EcoR I+Not I) of mannase, gene sheet (the not containing signal peptide sequence) section that cuts out the encoding mature mannase is connected with expression vector pPIC9, the recombinant plasmid pPIC-man5XZ7 that acquisition contains mannase gene man5XZ7 is after Bgl II enzyme linearity and transform pichia spp GS115, obtains recombinant pichia yeast strain GS115/man5XZ7.
Get the GS115 bacterial strain that contains recombinant plasmid, be inoculated in the 300mL BMGY nutrient solution, behind 30 ℃ of 250rpm shaking culture 48h, centrifugal collection thalline.Resuspended in 100mL BMMY substratum then, 30 ℃ of 250rpm shaking culture.After inducing 72h, centrifugal collection supernatant.Measure the vigor of mannase.The expression amount of reorganization mannase is 15.8U/mL.SDS-PAGE result shows that the reorganization mannase has obtained expression in pichia spp, and apparent molecular weight is 42.0kDa, and molecular weight becomes about 38.0 after EndoH handles.The ratio of reorganization mannosans is lived and is 705U/mg.
The gene M an5XZ7 sequence that will comprise the mannase of signal peptide is connected with expression vector pPIC9, and other testing sequence is the same, and SDS-PAGE result shows that the reorganization mannase has obtained expression in pichia spp.
The activation analysis of embodiment 4 reorganization mannases
The DNS method: concrete grammar is as follows: at Ph5.0, under 50 ℃ of conditions, the reaction system of 1mL comprises 100 μ L suitable dilution enzyme liquid, 900 μ L substrates, and reaction 10min adds 1.5mL DNS termination reaction, and boiling water boils 5min.Cooling back 540nm measures the OD value.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition per minute discharges 1 μ mol reducing sugar.
The property testing of embodiment 5 reorganization mannase Man5XZ7
The reorganization mannase Man5XZ7 that embodiment 3 is obtained carries out following property testing
1, the measuring method of the optimal pH of reorganization mannase Man5XZ7 and pH stability is as follows:
The reorganization mannase of embodiment 3 purifying is carried out enzymatic reaction to measure its optimal pH under different pH.The substrate carob bean gum carries out Xylanase activity mensuration under in 0.1mol/L citric acid-Sodium phosphate dibasic damping fluid of different pH 50 ℃.Result (Fig. 1) shows that the optimal pH of recombinase Man5XZ7 is 5.0, at pH9.0 relative activity more than 25% is arranged.Mannase is 37 ℃ of processing 60min in the damping fluid of above-mentioned various different pH, measure enzymic activity again under 75 ℃ in the pH5.0 buffer solution system, with the pH patience of research enzyme.Result (Fig. 2) shows that mannase is all very stable between pH3.0-10.0, and the residual enzyme activity is more than 80% behind the processing 60min in this pH scope, and this illustrates that this enzyme has satisfactory stability in very wide in range pH scope.
2, the optimum temperuture of mannase and thermal stability determination method are as follows:
Enzymatic reaction is carried out in being determined as under citric acid-Sodium phosphate dibasic damping fluid (pH5.0) buffer solution system and differing temps of the optimum temperuture of mannase.Temperature tolerance is determined as mannase and handles different time under differing temps, carries out enzyme assay again under 75 ℃.Enzyme reaction optimum temperuture measurement result (Fig. 3) shows that its optimum temperuture is 75 ℃, and lives at 85 ℃ of enzymes that still have about 60%.The thermostability test of enzyme shows (Fig. 4), and Man5XZ7 has good thermostability, at 50 ℃ of following incubation 1h, can keep original enzymic activity.
3, the K of mannase
mValues determination method is as follows:
Carob bean gum with different concns is substrate respectively, in citric acid-Sodium phosphate dibasic damping fluid (pH5.0) buffer solution system, measures enzymic activity down, calculates its K under 75 ℃ for 75 ℃
mValue.K when after measured, being substrate with the carob bean gum
mValue is respectively 6.6mg/mL, maximum reaction velocity V
MaxBe 886 μ mol/minmg.
4, different metal ion chemistry reagent is as follows to Man5XZ7 enzyme influence mensuration alive:
Add different metal ions and the chemical reagent of different concns in enzymatic reaction system, study it to the influence of enzymic activity, various material final concentrations are 1 and 5mmol/L.Under 75 ℃, pH5.0 condition, measure enzymic activity.The result shows, the active not influence of most ions enzymes, and beta-mercaptoethanol can improve its enzyme activity of 1.35 times.Ag
+, its activity of SDS (6.3%) strongly inhibited.Work as Cu
2+Can partly suppress the Man5XZ7 enzyme activity, and opposite beta-mercaptoethanol can improve its enzyme activity of 1.4 times.
5, mannase antitrypsin ability is measured as follows:
With pH7.0Tris-HCl damping fluid preparation 0.1mg/mL trypsinase.The enzyme liquid of getting the 0.5mL purifying after the pH7.0Tris-HCl damping fluid dilutes adds 0.5mL trypsinase and mixes, and 0.1,37 ℃ of insulation of proteolytic enzyme/mannase (w/w) ≈ 60min sampling is measured enzymic activity under pH5.0 and 75 ℃ of conditions.After experimental result showed that mannase Man5XZ7 is with trypsin treatment 60min, the enzyme of the Man5XZ7 after the trypsin treatment was lived and is not changed.
6, the substrate specificity of reorganization mannase
This enzyme is except acting on the carob bean gum, and for guar gum, Rhizoma amorphophalli powder also has certain Degradation (table 2).
Table 2. mannase Man5XZ7 substrate specificity is analyzed
Claims (9)
1. a high temperature alkali resistant mannase Man5XZ7 is characterized in that, its aminoacid sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.
2. a high temperature alkali resistant mannase gene man5XZ7 is characterized in that, the described high temperature alkali resistant mannase of coding claim 1 Man5XZ7.
3. high temperature alkali resistant mannase gene man5XZ7 as claimed in claim 2 is characterized in that, its base sequence is shown in SEQ ID NO.4 or SEQ ID NO.5.
4. the recombinant vectors that comprises the described high temperature alkali resistant mannase gene of claim 2 man5XZ7.
5. the recombinant vectors pPIC-Man5XZ7 that comprises the described high temperature alkali resistant mannase gene of claim 2 man5XZ7.
6. the recombinant bacterial strain that comprises the described high temperature alkali resistant mannase gene of claim 2 man5XZ7.
7. a method for preparing high temperature alkali resistant mannase gene man5XZ7 is characterized in that, may further comprise the steps:
1) with the recombinant vectors transformed host cell of claim 4, gets recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce reorganization mannosans expression of enzymes;
3) reclaim the also expressed mannase Man5XZ7 of purifying.
8. the application of the described high temperature alkali resistant mannase of claim 1 Man5XZ7.
9. the mould XZ7 of Tai Ruisisuo spore shell (Thielavia terrestris) is characterized in that, deposit number is CGMCC6544.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310062872.6A CN103275954B (en) | 2012-11-08 | 2013-02-28 | High temperature and alkali resisting mannanase Man5XZ7, gene and application thereof |
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| CN103642774A (en) * | 2013-11-13 | 2014-03-19 | 宁夏夏盛实业集团有限公司 | Mixed neutral cellulase, preparation method thereof and application thereof to papermaking beating |
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| CN105567541A (en) * | 2016-01-26 | 2016-05-11 | 湖北工业大学 | Mannan-oligosaccharide Chinese yam vinegar and preparing method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103525792A (en) * | 2013-10-29 | 2014-01-22 | 中国农业科学院饲料研究所 | High-temperature high-specific activity acidic beta-mannanase, and coding gene and application thereof |
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| CN103642774A (en) * | 2013-11-13 | 2014-03-19 | 宁夏夏盛实业集团有限公司 | Mixed neutral cellulase, preparation method thereof and application thereof to papermaking beating |
| CN103642774B (en) * | 2013-11-13 | 2016-08-17 | 宁夏夏盛实业集团有限公司 | Mixing neutral cellulase and preparation method thereof and the application in papermaking is pulled an oar |
| CN104004725A (en) * | 2014-06-11 | 2014-08-27 | 中国农业科学院饲料研究所 | Medium and low temperature neutral tannase TanXZ7 and gene and application thereof |
| CN104004725B (en) * | 2014-06-11 | 2016-05-11 | 中国农业科学院饲料研究所 | The neutral tannase TanXZ7 of low temperature and gene and application in one |
| CN105567541A (en) * | 2016-01-26 | 2016-05-11 | 湖北工业大学 | Mannan-oligosaccharide Chinese yam vinegar and preparing method thereof |
| CN105567541B (en) * | 2016-01-26 | 2018-04-17 | 湖北工业大学 | A kind of Oligomeric manna sugar yam vinegar and preparation method thereof |
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| CN103275954B (en) | 2014-07-30 |
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