CN102888415A - Improved method for producing beta-mannase - Google Patents
Improved method for producing beta-mannase Download PDFInfo
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- CN102888415A CN102888415A CN2011102063800A CN201110206380A CN102888415A CN 102888415 A CN102888415 A CN 102888415A CN 2011102063800 A CN2011102063800 A CN 2011102063800A CN 201110206380 A CN201110206380 A CN 201110206380A CN 102888415 A CN102888415 A CN 102888415A
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Abstract
The invention relates to a high-yield beta-mannase producing method. The method comprises the following steps of: chemically synthesizing a polynucleotide coded as endo-beta-1,4-mannan mannanohydrolase, coloning the polynucleotide; allowing the polynucleotide to be bonded with an alpha-factor signal peptide; allowing the polynucleotide bonded with peptide to be expressed outside cells (under the control of an alcohol oxidase AOX1 promoter); and the like. In a 500-L bioreactor, the selected engineering strain can secrete about 4g of protein in per liter of culture solution, and produces about 4305U of active beta-mannase per ml. Co2<+> and Mn2<+> can improve the hydrolytic activity of manna. The produced beta-mannase can hydrolyze mannan, glucomannan and galactomannan efficiently.
Description
Invention field
Present technique belongs to biological technical field, and a kind of method of improved production 'beta '-mannase is provided, and the application of beta-mannase enzymic hydrolysis mannosans, glucomannan and polygalactomannan.
Background technology
Plant cell wall comprises 2 kinds of main polysaccharide: Mierocrystalline cellulose and hemicellulose.The hemicellulose that is connected by β-Isosorbide-5-Nitrae-D-glycosidic link mainly is comprised of mannosans.The mannosans major part is present in the elementary plant cell wall, and content is less in the secondary cell wall.The annual agricultural wastes that contain in a large number mannosans that produce in the whole world.In South East Asia, annual generation millions of tons contains the palm kernel cake of mannosans.In South America, coffee industry and Oleum Cocois industry produce coffee refuse and the coconut cake of millions of tons.Palm kernel cake, coconut cake can only be as rudimentary ruminant feeds owing to being rich in the antinutritional factor such as mannosans.'beta '-mannase is that a class can be hydrolyzed the mannooligo saccharide that contains β-Isosorbide-5-Nitrae-sweet key of seminose and the inscribe lytic enzyme of mannocarolose (comprising mannosans, polygalactomannan, glucomannan etc.).The palm kernel cake that mannase was processed and coconut cake have demonstrated good feed value, can use in animal and fowl fodder.In addition, mannase has had good effect in paper-making industry and stain remover industry.After becoming a reality with the biomaterial production biofuel that comprises mannosans, mannase can be more and more important.
Pichia spp is a kind of methylotrophic yeasts, can produce than high-cell density in ordinary culture medium.Its good expression system can produce a large amount of enzymes and protein, such as, dextranase, zytase, tannase, phytase and mannase.
In the prior art, the technology of producing mannase is as follows:
PCT WO/1999/024588 has announced a cDNA clone body with coding beta-mannase restriction endonuclease that derives from tomato, can accelerate the maturation of fruit with this gene of promotor overexpression.
PCT WO/1993/024622 has announced the wooden mould dna sequence dna of a coding endomannanase, and it is imported to yeast or fungi, induces the generation endomannanase, also is a kind of method of separating coding endomannanase gene.
PCT WO/2006/126589 has announced snail, Pacific Ocean abalone especially, the characteristic of a kind of mannase of generation, and the technology that is applicable to a large amount of production zymins in aquatic products field.
PCT WO/2008/062317 has announced 'beta '-mannase, polynucleotide, the expression system of this albumen of encoding and the host cell that contains these polynucleotide that coffee berry produces.
PCT WO/2001/098462 and United States Patent (USP) 6,984,406 have been announced a strain can be created in the Bacillus strain that shows high enzyme mannase alive in neutral and the acidic culture.Mannase is used as additive in feed, good effect is arranged in the decomposition of hemicellulose.
PCT WO/2004/113538 has announced the new gene of the coding mannase of a Bacillus licheniformis WL-12, recombinant plasmid and an intestinal bacteria transformant that comprises recombinant plasmid of carrying this gene.Mannase is degraded to seminose, mannobiose and mannotriose to galactomannan sugar, and enzyme has higher activity under pH neutral and proper temperature condition, and this condition is that the animal physiological condition is close.
PCT WO/2003/012110 and United States Patent (USP) 7,112,429 have been announced the heat-resisting mannase that Acidothermus cellulolyticus produces.The present invention has further been announced mode and the application of zymin in food-processing and foodstuff additive of making the ManA recombinant type.
PCT WO/1994/025576 has announced the enzyme that can show mannase work that microorganism Aspergillus aculeatus CBS.101.43. produces, and thinks that this kind of enzyme is used for degraded or improvement plant or alga cells wall material.
PCT WO/2008/009673 has disclosed mutant and nucleic acid and the aminoacid sequence of the mannase of a wild-type Trichodermareesei generation, this mutant inserts, deletes and/or substituted one section amino acid, has the stability under higher thermostability, proteolysis stability, specific activity, the low pH condition.
PCT WO/2000/077155, WO/1999/009126, WO/2000/042146, and WO/1999/009128 have announced a prescription that comprises mannase, improve for food, makeup are dirty and whitening agent can clean-up performance.
PCT WO/1995/014809 has announced the technique for preparing bleached cellulose paper pulp with mannase.
PCT WO/2001/062951 and WO/2001/064932 has announced the technique with emulsifying agent and the mannase Combined Processing coconut dregs of rice production modification coconut dregs of rice.By this method, release mannosans that can be effective, economic.
PCT WO/2001/064830 has announced the technique that a compound enzymic preparation that contains Mierocrystalline cellulose restriction endonuclease, zytase, 'beta '-mannase and/or α-amylase is processed wine brewing Weibull in the lumps of wood.
PCT WO/2003/062409 has announced a prescription that contains at least 2 kinds of thermostable enzymes for animal-feed, and these 2 kinds of enzymes are from endoglucanase, zytase, phytase, proteolytic enzyme, Galactanase, mannase, glycanase screens in the alpha-galactosidase.First-selected zytase is from aspergillus, bacillus, and Humicola, the tea toadstool belongs to and separates acquisition with Trichoderma.
PCT WO/1996/036569 has announced 1 prescription that stops or remove the surface biological film, wherein contains at least mannase, optionally with carbohydrase, proteolytic enzyme, lipase and glucoproteinase at least a combination.
PCT WO/2001/075084 has announced the dna fragmentation of a kind of enzyme of can encoding at least that obtains from coffee, this enzyme can be hydrolyzed the polysaccharide that is connected to form by Isosorbide-5-Nitrae-glycosidic link by the straight or branched mannosans.
PCT WO/1994/020667 has announced a kind of with the raw-material technique of enzyme pre-treatment timber, and this processing can reduce mechanical energy consumption of pulling an oar, and improves the performance of fiber, and used enzyme is cellobiohydrolase and mannase.
PCT WO1998/020750 has announced mannase preparation in the application that improves forage plant protein raw materials value.
PCT WO/1997/041739 has announced and added the method that hemicellulase (such as mannase) improves the monogastric animal capacity usage ratio in the low energy feed.
PCT WO/2005/003319 and PCT WO/2007/095398 has announced the polypeptide of mannosans enzymic activity.
United States Patent (USP) 7,148,399 have announced the dna fragmentation of a kind of enzyme of can encoding at least that obtains from coffee, and this enzyme can be hydrolyzed the polysaccharide that is connected to form by Isosorbide-5-Nitrae-glycosidic link by the straight or branched mannosans.
United States Patent (USP) 6,566,114 have announced the new mannase by genus bacillus I633 genes encoding.This mannase is alkalescence, can be used as the clean-out system composition, and is used in the fracturing liquid and is used for fracturing stratum, is used for the improvement vegetable material, and for the treatment of fabric.
United States Patent (USP) 6,060,299 have announced a new mannase that subtilis 168 polynucleotide molecules coding produces.This mannase is alkalescence, can be used as the clean-out system composition, is used for the improvement vegetable material, and for the treatment of fabric.
United States Patent (USP) 4,895,800 have described cloning vector; The expression of the use of methanol induction promotor and metaprotein in the pichia spp.
Relevant in pichia spp the information of protein expression platform, such as, promotor, method for transformation etc., at United States Patent (USP) 4,683,293; 4,808,537; 4,812,405; 4,818,700; 4,837,148; 4,855,231; 4,857,467; 4,879,231; 4,882,279; 4,885,242; 4,929,555; 5,002,876 5,004,688 5,032,516; 5,122,465; 5,135,868; Can see in 5,166,329.
Summary of the invention
At first, the invention provides a kind of high-yield method of 'beta '-mannase.May further comprise the steps:
(1) a kind of synthetic promotor of beta-mannase gene in dna vector is combined, and produces an expression cassette; (2) dna vector is inoculated in the yeast cell, the yeast of inoculation is pichia pastoris phaff, belongs to Pichia; (3) under the small-scale culture condition, go out the high expression level bacterial strain by measuring the beta-mannase activity screen; (4) in the bio-reactor of 500L, the bacterial strain of screening high expression level 'beta '-mannase, the bacterial strain of high expression level 'beta '-mannase produces the 'beta '-mannase enzyme of 4305U/mL and lives.
Expression cassette is comprised of the signal peptide that an indication 'beta '-mannase is secreted in the nutrient solution of extracellular.First-selected signal peptide is α-factor, the 'beta '-mannase mature peptide is inserted into the below of complete α-factor encoding sequence, and expression cassette and outer membrane protein have carried out combination, thereby can make the 'beta '-mannase of expression be secreted into the extracellular, and this is conducive to acquisition and the active raising of enzyme.Wherein beta-mannase gene contains the major portion of Seq ID1 sequence, and the polynucleotide sequence of Seq ID1 number is seen shown in the sequence table.Beta-mannase gene is a kind of polynucleotide that at least 85% homology fragment is arranged with SeqID1 that contain.Promotor is an inducible promoter, is subject to alcohol and induces, and is subject to methanol induction most as the AOX1 promotor of pichia pastoris phaff.
Secondly, the invention provides the method for improved hydrolysis mannosans, glucomannan and a polygalactomannan, the product after the hydrolysis is for being fit to the free sugar of liquid biofuel production and animal feeding.May further comprise the steps with the method for hydrolysis of 'beta '-mannase to mannosans, glucomannan and polygalactomannan: (1) carries out pre-treatment to the substrate that contains mannosans, glucomannan and polygalactomannan etc.Damping fluid and the pretreated substrate that (2) will contain the enzyme mixture of 'beta '-mannase are cultivated.Pre-treatment can increase the touch opportunity of substrate and enzyme; Need during reaction in system, to add a certain amount of damping fluid, and regulate pH between 4.5-6.5, be preferably 5.5, and then add the Mn of lower concentration in the pre-treatment substrate
2+, Co
2+And Fe
2+Salts solution, ionic concn is greatly between 0.1-10mmol/L.The mixture of enzyme contains 'beta '-mannase.The substrate of hydrolysis is to be rich in the palm kernel meal of mannosans and the coconut dregs of rice etc.
Description of drawings:
Fig. 1: different metal ion and chemical reagent are on the impact of mannase.Significant difference (P＜0.05) is compared in a representative with control group.
Fig. 2: the accumulation of mannase in the bioreactor culture base.The 2-9 swimming lane represents respectively the sample collected after 24,36,48,50,64,76,96 and 108 hours behind the methanol induction.Each swimming lane adds the medium supernatant of 4 μ l.The M swimming lane is Bio-Rad protein labeling (Catalog#161-0373).No. 1 swimming lane is to induce medium supernatant before.
Fig. 3 A: the optimum pH of mannase.The variation range of tested pH value is 1.5-10.0, determines therein optimal pH.
Fig. 3 B: mannosans Enzymic stability under the different pH condition.In the damping fluid of pH1.0-10.0, cultivate the remnant enzyme activity after 1 hour.
Fig. 4 A: the optimum temperuture of mannase.In 20-80 ℃ temperature range, measure the activity of enzyme, determine optimum temperuture.
Fig. 4 B: mannosans Thermostability.Remnant enzyme activity after under 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ of temperature, cultivating 150 minutes.
Embodiment
Embodiment 1: the chemosynthesis of beta-mannase gene
Gene order is by DNA Star programanalysis, the prediction chemosynthesis coding molecule amount be that 35.8kDa contains 355 amino acid whose mannase precursor protein genes, this precursor protein can be cut open between Gly18 and Ala19, produces a molecular weight and be the maturation protein that is comprised of 317 amino acid of 35.8kDa.In ncbi database, mannase is carried out the blast sequence alignment, the putative protein An15g07760 that the result is presented on the amino acid levels with aspergillus niger C513.88 has 98% similarity, and only has respectively 6.6%, 9.3%, 8.7% identical with homologous protein in sulfuraspergillus, microorganism Aspergillus aculeatus and the Trichodermareesei.
Embodiment 2: being configured to of pichia pastoris phaff transformant of expressing mannase gene makes up the pichia pastoris phaff expression vector, utilizes primer 5 ' CCGGAATTCGCTTCCAACCAGACTCTGTCCTAT and 5 ' ATATGCGGCCGCTTAAGCCCCCTCCCAGTTCAGAGC amplification coding mannase mature peptide (according to predicting from Gly
18To Ala
335) dna sequence dna.With PCR product enzymolysis, and enzymolysis product is inserted into the corresponding site of pPIC9 with restriction enzyme EcoRI and NotI, forms the p-mannase.
Prepare the pichia pastoris phaff electricity according to the specification sheets of Pichia anomala expression test kit and turn competent cell, and change plasmid DNA over to prepared competent cell (Invitrogen, USA) by electrophoretic.Utilize BglII that 10 μ g p-mannase enzymes are cut and make it linearizing (New England Biolabs, USA).The mannosans enzymic activity of bacterium colony in 1mL BMMY (from 3mL BMGY subculturing cell) substratum that relatively forms on the MD plate.Have 54 to show the beta-mannase enzymic activity in more than 1000 transformant, screening mannase enzyme the highest bacterial strain alive carries out large-scale enzyme production in the bio-reactor of 500L from this 54 strain again.
Embodiment 3: the beta-mannase enzyme assay
The beta-mannase enzymic activity is measured by the DNS method.At first, enzyme liquid is diluted between 1000 to 5000 times with 0.1mol/L sodium-acetate buffer (pH 5.5), then gets 500 μ L enzyme liquid and mixes with the pretreated mannosans substrate solution (with identical damping fluid dilution) that 500 μ L contain 1% Viscogum BE.Reaction is 5 minutes under 50 ℃ of temperature, then adds 3mL DNS termination reaction and boils 5 minutes.Calculate the quantity of the reducing sugar of release in the absorbancy at 540nm place with the seminose standard substance.Under temperature is 50 ℃, pH5.5 condition, generates the needed enzyme amount of 1 μ mol seminose with per minute and be defined as a mannosans unit of enzyme activity.Obtain specific activity with the total protein content that the BCA protein determination kit is measured in the substratum.
Embodiment 4: the high-density culture of the Pichia Pastoris body of restructuring
In the 500L bio-reactor, adopt the batch feeding mode to cultivate pichia pastoris phaff and produce mannase.Per hour injecting about 3ml/L methanol solution comes inducible protein to express.Every 12 hours nutrient solution is taken a sample, measure the work of 'beta '-mannase enzyme, protein level and cell quantity in the supernatant liquor.The accumulation volume of 'beta '-mannase is measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and method for detecting enzymatic activity.Mannase is expressed as the albumen of one group of molecular weight between about 70 to 100kDa.(Fig. 2) appearred in the peak value of protein expression at 8-9 days.Inducing when finishing, is under 50 ℃ in temperature, is containing 1mmol/L Mn
2+Sodium acetate buffer solution in measure the beta-mannase enzymic activity, activity is 4305U/mL.Be about 4g/L by protein content in the BCA albuminometry detection culture filtrate, converting to than vigor is 1075U/mg.
Embodiment 5: the character of the 'beta '-mannase after the restructuring
Determine the optimum pH of 'beta '-mannase by in a series of buffered soln, carrying out the LBG hydrolysis reaction, damping fluid for example comprises: 0.1mol/L glycine-hydrochloride (pH 1.5-3.0), 0.1mol/L sodium-acetate (pH 3.5-6.0), 0.1mol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (pH 6.0-8.5), 0.1mol/L glycine-sodium hydroxide (pH 8.5-10), all mensuration are all 50 ℃ of reactions 5 minutes.The results are shown in Figure 3A.
Be determined at Enzymic stability under the different pH of buffer conditions with aforesaid method in 1 hour 50 ℃ of lower cultivations.Under standard conditions, detect residual enzymic activity.The results are shown in Figure 3B.
In 20-100 ℃ of scope, detect the activity of enzyme, determine the optimal reactive temperature of enzyme.Enzyme is 30,40, and 50,60 or 70 ℃ of lower cultivations after 1-150 minute (in 0.1mol/L sodium acetate buffer solution) are measured remaining enzymic activity and come the comparison Thermostability under standard conditions.The results are shown in Figure 4A and 4B.
Embodiment 6: the impact that different metal ions is lived on the mannase enzyme
By in enzyme liquid, adding vitriolate of tartar, sodium-chlor, rose vitriol, cesium chloride, copper sulfate, sal epsom, ferric sulfate, Manganous chloride tetrahydrate, zinc sulfate and the Silver Nitrate of 1mmol/L, measure different metal ions to the impact of enzymic activity.Add the Mn of 1mmol/L
2+And Co
2+Make the mannosans enzymic activity increase respectively 75% and 44%.The results are shown in Table 1.
Embodiment 7: the specificity of mannase
Mannase liquid dilutes 2000 times with 0.1mol/L sodium acetate buffer solution (pH5.5), get 500 μ l enzymes and mix with the pretreated substrate solution of 500 μ l, pretreated substrate solution (comprising 1%LGB, starch, filter paper and CMC) is 100 ℃ of lower heating 1 hour in damping fluid.Hydrolysis reaction is to react 5 minutes under 50 ℃ of conditions in temperature, then adds 3mL DNS reagent and boils 5 minutes termination reactions.In coming, the absorbancy at 540nm place calculates the amount of the reducing sugar of release with the seminose standard substance, when starch, filter paper or CMC do not detect activity during as substrate.This proof mannase is the enzyme of a specificity hydrolysis mannosans.
Embodiment 8: the pre-treatment of mannosans substrate
1gLGB is slowly added in the 100mL0.1mol/L sodium acetate buffer solution (pH5.5), and continuous heating is 60 minutes under 100 ℃ of continuous agitation conditions.Substrate was deposited for 2 weeks at 4 ℃ and could be used.
Embodiment 9: the zymologic property of 'beta '-mannase
Live by the enzyme that detects under different pH, temperature of reaction and the Metal Ions Conditions, analyzed the suitableeest hydrolysising condition of mannase.Can find out from Fig. 3 A, the pH scope that enzyme is lived is between 4.5-6.0, and optimal pH is 5.5.PH when 2.5-4.5 and 6.0-7.5, the enzyme fast reducing of living.PH between 4.0-8.5 the time enzyme also highly stable, cultivate to have greater than 65% enzyme behind the 1h and live.PH is below 4 and 9 when above, Enzymic stability significantly descend (Fig. 3 B).
The optimum temperuture that enzyme is lived is about 50 ℃ (Fig. 4 A).When temperature was elevated to 55 ℃, enzyme was extremely unstable, inactivation almost completely when reaching 80 ℃.The detailed variation of enzyme stability such as Fig. 4 B under the differing temps.When temperature was 50 ℃, the transformation period of enzyme was for being 65 minutes, and when temperature was elevated to 60 ℃ and 70 ℃, the transformation period that enzyme is lived only had respectively 20 and 15 minutes.Enzyme can keep higher activity under the temperature close with room temperature.When the activity of enzyme is approximately 50 ℃ in the time of 40 ℃ 70%.
Embodiment 10: the present invention produces the superiority of mannase
Up to the present, the present invention at first adopts the genetic expression of chemosynthesis to produce mannase.The expression level of this gene (3.6g/L) is 14 times (Chen, et al, 2007) of sulfuraspergillus (262mg/L), and is all higher than the result who reported in the past.Gained enzyme of the present invention is lived and is 4305U/ml, than the 350U/ml high 12 times (Chen, et al, 2007) that reported in the past.The optimal pH of recombinase is approximately 5.5, by the optimum pH 3 of aspergillus niger mannase that solid fermentation produces, although optimum temperuture is similar, is approximately 50 ℃ before being significantly higher than.The restructuring 'beta '-mannase that the present invention produces under 37-40 ℃ of condition, have higher activity, be the enzyme that is adapted at using under the cold condition.Yet, can be as before the detergent additives at it, it is to be further improved in addition to the resistibility of washing composition.
Claims (13)
1. method of producing 'beta '-mannase may further comprise the steps:
(1) a kind of synthetic promotor of beta-mannase gene in dna vector is combined, and produces an expression cassette,
(2) dna vector is inoculated in the yeast cell,
(3) under the small-scale culture condition, go out the high expression level bacterial strain by measuring the beta-mannase activity screen,
(4) in the bio-reactor of 500L, the bacterial strain of screening high expression level 'beta '-mannase
2. according to claim 1 method, the bacterial strain of high expression level 'beta '-mannase produces the 'beta '-mannase enzyme of 4305U/mL and lives.
3. according to claim 1 method, beta-mannase gene contains the major portion of Seq ID1 sequence, and the polynucleotide sequence of Seq ID 1 number is seen Fig. 1.
4. according to claim 1 method, beta-mannase gene is a kind of polynucleotide that at least 85% homology fragment is arranged with Seq ID1 that contain.
5. according to claim 1 method, methyl alcohol is the best inductor of promotor.
6. according to claim 1 method, the yeast of inoculation belongs to Pichia.
7. according to claim 1 method, the yeast of inoculation is pichia pastoris phaff.
8. according to claim 1 method, 'beta '-mannase is secreted in the outer nutrient solution of born of the same parents.
9. a yeast cell comprises the dna vector in the claim 1.
10. may further comprise the steps with the method for hydrolysis of 'beta '-mannase to mannosans, glucomannan and polygalactomannan:
(1) substrate that contains mannosans, glucomannan and polygalactomannan etc. is carried out pre-treatment.
Damping fluid and the pretreated substrate that (2) will contain the enzyme mixture of 'beta '-mannase are cultivated.
11. method for hydrolysis according to claim 9 contains Mn in the damping fluid
2+, Fe
2+And Co
2+A kind of in the metal ion.
12. method for hydrolysis according to claim 9, the pH of damping fluid is between 4.5-6.5.
13. method for hydrolysis according to claim 9, enzyme mixture contains 'beta '-mannase.
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| CN106387923A (en) * | 2016-09-07 | 2017-02-15 | 中国农业大学 | Soluble dietary fibers rich in galactomannan and preparation method of soluble dietary fibers |
| CN111040966A (en) * | 2019-12-23 | 2020-04-21 | 河北科技大学 | Bacillus licheniformis KD-1, β -mannase produced by same and application thereof |
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Cited By (3)
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| CN111040966A (en) * | 2019-12-23 | 2020-04-21 | 河北科技大学 | Bacillus licheniformis KD-1, β -mannase produced by same and application thereof |
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Application publication date: 20130123 |