CN103540580A - Mannanase and production method thereof - Google Patents
Mannanase and production method thereof Download PDFInfo
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01101—Mannan endo-1,6-alpha-mannosidase (3.2.1.101), i.e. endo-1,6-beta-mannanase
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Abstract
The invention discloses a production method of mannanase which is suitable for an alkaline environment and has good thermal stability. The production method is characterized by comprising the steps of culturing a strain, performing shaking flask fermentation and preparing an enzyme liquid, wherein the strain is Bacillussubtilis, the laboratory number is PARK9639, the accession number is CGMCCNO: 6226, the mannanase is an alkaline enzyme, does not produce cellulase and has no activity against crystalline cellulose and carboxymethyl cellulose, and the activity of the mannanase in the pH range of 5.5-10.0 and the temperature range of 40 DEG C-60 DEG C is stable. The mannanase disclosed by the invention has high enzyme activity, the standard mannanase solution is not less than 4500u/ml, the unit production cost is low, the fermentation period is short, the degree of industrialization is high, and the technological process is simple and easy to control; and the product has no activity of the cellulase, is suitable for applications in a paper-making industry, and can reduce paper-making production cost and environmental pollution and simultaneously facilitate the improvement of product quality of paper.
Description
Technical field
The present invention relates to a kind of suitability for industrialized production of biological enzyme formulation, specifically a kind of mannase and production method.
Background technology
Mannase comprises following four kinds: (1), mannosans inscribe-Isosorbide-5-Nitrae-beta-Mannosidase (Mannan endo-1,4-β-mannosidase), numbering EC 3.2.1.78; (2) mannosans-Isosorbide-5-Nitrae-β-mannobiose lytic enzyme (Mannan endo-1,4-β-mannobiohydrolase), numbering EC 3.2.1.100[claim again circumscribed-Isosorbide-5-Nitrae-β-mannobiose lytic enzyme (Exo-1,4-β-mannobiohydrolase)]; (3) beta-Mannosidase (β-Mannosidase), numbering EC 3.2.1.25; (4) mannosans inscribe-1,6-beta-Mannosidase (Mannan endo-1,6-β-mannosidase), numbering EC 3.2.1.101.
At the beginning of last century, just relevant for the report that decomposes plant mannosans, in recent years along with the continuous discovery of people to mannase purposes, promoted the development of mannase research, within 1958, Courtios has reported the fungi that produces mannase for the first time; Nineteen sixty William and Doetsch road produce the bacterium of mannase; Nineteen sixty-five, Reese and Shibata have proposed concept and the definition of mannase; After the seventies, people have obtained large development to the research of the essential property of mannase, the nineties people are applied to feed cultivation industry by mannase, obtained good development, along with people are to the exploitation of nature hemicellulose resource and to the deepening continuously of zytase research, mannase is widely used research in many-sides such as feed, food, papermaking, oil production and biological study technology.But, aspect papermaking, there is no all the time industrial applications, major cause is that the required enzyme requirement of papermaking is used under higher pH condition, and require the Heat stability is good of enzyme, low price, and mannase majority is on the market acidicenzym, action pH, generally in pH3.5～6.0, is difficult to be applied to field of papermaking.
Summary of the invention
The object of this invention is to provide a kind of mannase and production method that is adapted to alkaline environment, Heat stability is good.
Enzyme activity determination method of the present invention is to utilize the disodium hydrogen phosphate dodecahydrate of 0.05mol/L pH6.0 and the locust bean gum solution of monohydrate potassium damping fluid preparation 0.5%.In this substrate solution of 0.9ml, add the suitably enzyme solution of dilution of 0.1ml, 50 ℃ of accurate clock reaction 10min, in 550nm photometry absorption value.Its enzyme activity is defined as, and under condition determination, hydrolysis locust bean gum per hour produces the required enzyme amount of 1mg reducing sugar (in seminose) and is defined as an enzyme activity unit (u).
The present invention adopts following technical scheme to realize its goal of the invention, a kind of production method of mannase, it comprises spawn culture, shake flask fermentation, enzyme liquid preparation process, described bacterial classification is subtilis (Bacillus subtilis), laboratory is numbered PARK9639, preserving number: CGMCC NO:6226, its biological characteristics is: G+ bacteria, produce gemma, it is shaft-like that thalline is, 0.7～0.8 * 2～3 microns of individual cells, peritrichous, can move, in binary fission mode, breed; 0.6～0.9 * 1.0～1.5 microns of ellipses of gemma are positioned at thalline central authorities or slightly inclined to one side, and after sporulation, thalline does not expand, and on substratum of the present invention, bacterium colony is rounded, dirty white, uniform coloring, and neat in edge, tarnish, sticky.
Bacterial classification of the present invention is in Ningxia, China collection, in strain improvement screening process, filter out the superior strain that a strain laboratory is numbered PARK9639, according to major physiological biochemical character and 16S rRNA gene order, the comprehensive analysis of the experimental datas such as gyrB gene order is accredited as subtilis (Bacillus subtilis), this bacterial classification has been submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on 06 15th, 2012, preserving number: CGMCC NO:6226, and in having submitted on the same day preservation survival proof to.
Spawn culture of the present invention is CGMCC NO:6226 bacterial classification well-grown on the inclined-plane of following preparation, slant culture based formulas is: in 1000mL distilled water, add extractum carnis 3g～8g, yeast extract paste 3g～8g, peptone 8g～12g, glucose 3g～5g, sodium-chlor 3g～5g, adjust pH 6.9～7.1, 0.1MPa sterilizing 28 minutes～32 minutes, make test tube slant, inoculate this bacterial classification, 34 ℃～36 ℃, cultivate after 20 hours～24 hours, put into 5 ℃ of refrigerators standby, or to injecting after sterilising liq paraffin preservation on bacterium inclined-plane or take skimmed milk as the preservation of carrier lyophilize vacuum seal.
Shake flask fermentation of the present invention is that CGMCC NO:6226 bacterial classification can produce the mannase of high vigor under the culture condition of regulation, this culture condition is: in mass, culture medium prescription is: Rhizoma amorphophalli powder 3 ﹪～4 ﹪, wheat bran 2 ﹪～4 ﹪, corn steep liquor 0.5 ﹪～1 ﹪ of nitrogen content 40%, yeast extract paste 0.5 ﹪～1 ﹪, Secondary ammonium phosphate 0.2 ﹪～0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪～0.05 ﹪, peptone 0.1 ﹪～0.2 ﹪, sal epsom 0.02 ﹪～0.05 ﹪, sodium carbonate 0.20 ﹪～0.25 ﹪, water 89.15 ﹪～93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is pH8.0～pH8.5, 0.1MPa sterilizing 30 minutes～35 minutes, culture condition: 32 ℃～37 ℃, shaking speed 260 r/min～280r/min, cultivate 34 hours～42 hours.
Enzyme liquid of the present invention is prepared as by eggplant bottle inclined-plane and makes cell liquid seeds to seeding tank, then to producing enzymic fermentation tank, second order fermentation, preparation standard enzyme liquid.
Seed culture based formulas of the present invention is: in mass, corn steep liquor 1 ﹪～2 ﹪ of wheat bran 5 ﹪～6 ﹪, Semen Maydis powder 1 ﹪～2 ﹪, nitrogen content 40 ﹪, sodium-chlor 0.4 ﹪～0.6 ﹪, sodium carbonate 1 ﹪～2 ﹪, ammonium sulfate 0.3 ﹪～0.5 ﹪, water 86.9 ﹪～91.3 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5～7.0,0.1MPa sterilizing 30 minutes～35 minutes; Seeding tank charging cumulative volume is no more than 70 ﹪, culture condition is: 33 ℃～38 ℃ of temperature, mixing speed 180 r/min～220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.25～0.27,4 hours～6 hours, 1:0.33～0.36, after 6 hours, 1:0.50～0.55, tank pressure 0.06 MPa～0.07MPa, culture cycle is 7 hours～9 hours.
The culture medium prescription that the present invention produces enzymic fermentation tank is: in mass, Rhizoma amorphophalli powder 3 ﹪～4 ﹪, wheat bran 2 ﹪～4 ﹪, corn steep liquor 0.5 ﹪～1 ﹪ of nitrogen content 40%, yeast extract paste 0.5 ﹪～1 ﹪, Secondary ammonium phosphate 0.2 ﹪～0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪～0.05 ﹪, peptone 0.1 ﹪～0.2 ﹪, sal epsom 0.02 ﹪～0.05 ﹪, sodium carbonate 0.20 ﹪～0.25 ﹪, water 89.15 ﹪～93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0～8.5, 0.1MPa sterilizing 30 minutes～35 minutes, charging cumulative volume is no more than 70 ﹪, culture condition: 33 ℃～38 ℃ of temperature, mixing speed 180 r/min～220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.30～0.32,4 hours～6 hours, 1:0.40～0.42,1:0.50～0.55 after 6 hours, tank pressure 0.06 MPa～0.07MPa, fermentation period is 34 hours～42 hours.
The present invention, in preparation standard enzyme liquid, through Plate Filtration, diatomite degerming, obtains through rotproofing the fermentation liquid that produces enzymic fermentation tank deposit under three months conditions 15 ℃ of sealings, and enzyme activity conservation rate is standard mannase liquid more than 95 ﹪.
The present invention is impurity elimination degerming, improve the quality of products, fermentation liquid, after Plate Filtration, adds disodium hydrogen phosphate dodecahydrate and CALCIUM CHLORIDE DIHYDRATE to get assorted degerming in ferment filtrate, and its consumption is respectively 2.8 ﹪～3.5 ﹪ and 1.0 ﹪～1.3 ﹪ of ferment filtrate quality.
A kind of mannase of producing as aforesaid method, described mannase is alkaline enzyme, cellulase-producing not, crystalline cellulose, carboxymethyl cellulose are not all had to activity, within the scope of 40 ℃～60 ℃ of pH5.5～10.0, temperature, mannase is activity stabilized, the suitableeest scope is pH6.0～8.0, and temperature is 45 ℃～55 ℃.
Owing to adopting technique scheme, the present invention has realized goal of the invention preferably, and its enzyme activity is high, standard mannase liquid >=4500u/ml, and the unit cost of production is low, and fermentation period is short, and industrialization degree is high, and technological process is simple and easy to control; Product is cellulose enzymic activity not, is adapted at paper industry application, can reduce paper cost and environmental pollution, helps the product quality that improves paper simultaneously.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
A kind of production method of mannase, it comprises spawn culture, shake flask fermentation, enzyme liquid preparation process, and described bacterial classification is subtilis (Bacillus subtilis), and laboratory is numbered PARK9639, preserving number: CGMCC NO:6226, its biological characteristics is: G+ bacteria, produce gemma, and it is shaft-like that thalline is, 0.7～0.8 * 2～3 microns of individual cells, peritrichous, can move, and in binary fission mode, breeds; 0.6～0.9 * 1.0～1.5 microns of ellipses of gemma are positioned at thalline central authorities or slightly inclined to one side, and after sporulation, thalline does not expand, and on substratum of the present invention, bacterium colony is rounded, dirty white, uniform coloring, and neat in edge, tarnish, sticky.
Bacterial classification of the present invention is in Ningxia, China collection, in strain improvement screening process, filter out the superior strain that a strain laboratory is numbered PARK9639, according to major physiological biochemical character and 16S rRNA gene order, the comprehensive analysis of the experimental datas such as gyrB gene order is accredited as subtilis (Bacillus subtilis), this bacterial classification has been submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on 06 15th, 2012, preserving number: CGMCC NO:6226, and in having submitted on the same day preservation survival proof to.
Spawn culture of the present invention is CGMCC NO:6226 bacterial classification well-grown on the inclined-plane of following preparation, slant culture based formulas is: in 1000mL distilled water, add extractum carnis 3g～8g, yeast extract paste 3g～8g, peptone 8g～12g, glucose 3g～5g, sodium-chlor 3g～5g, adjust pH 6.9～7.1, 0.1MPa sterilizing 28 minutes～32 minutes, make test tube slant, inoculate this bacterial classification, 34 ℃～36 ℃, cultivate after 20 hours～24 hours, put into 5 ℃ of refrigerators standby, or to injecting after sterilising liq paraffin preservation on bacterium inclined-plane or take skimmed milk as the preservation of carrier lyophilize vacuum seal.
The present embodiment slant culture based formulas is: in 1000mL distilled water, add extractum carnis 5g, yeast extract paste 5g, peptone 10g, glucose 5g, sodium-chlor 5g, adjusts adjust pH 7.0 with sodium hydroxide, 0.1MPa sterilizing 30 minutes, make test tube slant, inoculate this bacterial classification, 35 ℃, cultivate after 22 hours, put into 5 ℃ of refrigerators standby.
After this slant strains checks bacterial classification enzymatic productivity qualified by shake flask fermentation, can be used as seed and spread cultivation and use or enzymatic production is used.
Shake flask fermentation of the present invention is that CGMCC NO:6226 bacterial classification can produce the mannase of high vigor under the culture condition of regulation, this culture condition is: in mass, culture medium prescription is: Rhizoma amorphophalli powder 3 ﹪～4 ﹪, wheat bran 2 ﹪～4 ﹪, corn steep liquor 0.5 ﹪～1 ﹪ of nitrogen content 40%, yeast extract paste 0.5 ﹪～1 ﹪, Secondary ammonium phosphate 0.2 ﹪～0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪～0.05 ﹪, peptone 0.1 ﹪～0.2 ﹪, sal epsom 0.02 ﹪～0.05 ﹪, sodium carbonate 0.20 ﹪～0.25 ﹪, water 89.15 ﹪～93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is pH8.0～pH8.5, 0.1MPa sterilizing 30 minutes～35 minutes, culture condition: 32 ℃～37 ℃, shaking speed 260 r/min～280r/min, cultivate 34 hours～42 hours.
The present embodiment culture medium prescription is: corn steep liquor 1 ﹪ of Rhizoma amorphophalli powder 3.5 ﹪, wheat bran 2.5 ﹪, nitrogen content 40%, yeast extract paste 1 ﹪, Secondary ammonium phosphate 0.25 ﹪, dipotassium hydrogen phosphate 0.02 ﹪, peptone 0.2 ﹪, sal epsom 0.02 ﹪, sodium carbonate 0.20 ﹪, water 91.31 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is pH8.2,0.1MPa sterilizing 30 minutes, culture condition: 35 ℃, shaking speed 280r/min, cultivates 40 hours.Fermented liquid centrifuging and taking supernatant liquor is crude enzyme liquid, and enzyme activity is 4800u/ml.
Enzyme liquid of the present invention is prepared as by eggplant bottle inclined-plane and makes cell liquid seeds to seeding tank, then to producing enzymic fermentation tank, second order fermentation, preparation standard enzyme liquid.
Seed culture based formulas of the present invention is: in mass, corn steep liquor 1 ﹪～2 ﹪ of wheat bran 5 ﹪～6 ﹪, Semen Maydis powder 1 ﹪～2 ﹪, nitrogen content 40 ﹪, sodium-chlor 0.4 ﹪～0.6 ﹪, sodium carbonate 1 ﹪～2 ﹪, ammonium sulfate 0.3 ﹪～0.5 ﹪, water 86.9 ﹪～91.3 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5～7.0,0.1MPa sterilizing 30 minutes～35 minutes; Seeding tank charging cumulative volume is no more than 70 ﹪, culture condition is: 33 ℃～38 ℃ of temperature, mixing speed 180 r/min～220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.25～0.27,4 hours～6 hours, 1:0.33～0.36, after 6 hours, 1:0.50～0.55, tank pressure 0.06 MPa～0.07MPa, culture cycle is 7 hours～9 hours.
The present embodiment seed culture based formulas is: in mass, corn steep liquor 2.0 ﹪ of wheat bran 5 ﹪, Semen Maydis powder 1.5 ﹪, nitrogen content 40 ﹪, sodium-chlor 0.5 ﹪, sodium carbonate 1.5 ﹪, ammonium sulfate 0.3 ﹪, water 89.2 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5,0.1MPa sterilizing 30 minutes; Seeding tank charging cumulative volume is no more than 70 ﹪, and culture condition is: 37 ℃ of temperature, and mixing speed 220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.25,4 hours～6 hours, 1:0.35, after 6 hours, 1:0.55, tank pressure 0.07MPa, culture cycle is 8 hours.After cultivating 4h, sample microscopy, observe bacteria growing and grow, breeding situation.When cultivating, to somatic cells chain 70 ﹪, be fractured into individual cells, show that seed has approached ripe, do every preparation work of improvement tank inoculation.
The culture medium prescription that the present invention produces enzymic fermentation tank is: in mass, Rhizoma amorphophalli powder 3 ﹪～4 ﹪, wheat bran 2 ﹪～4 ﹪, corn steep liquor 0.5 ﹪～1 ﹪ of nitrogen content 40%, yeast extract paste 0.5 ﹪～1 ﹪, Secondary ammonium phosphate 0.2 ﹪～0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪～0.05 ﹪, peptone 0.1 ﹪～0.2 ﹪, sal epsom 0.02 ﹪～0.05 ﹪, sodium carbonate 0.20 ﹪～0.25 ﹪, water 89.15 ﹪～93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0～8.5, 0.1MPa sterilizing 30 minutes～35 minutes, charging cumulative volume is no more than 70 ﹪, culture condition: 33 ℃～38 ℃ of temperature, mixing speed 180 r/min～220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.30～0.32,4 hours～6 hours, 1:0.40～0.42,1:0.50～0.55 after 6 hours, tank pressure 0.06 MPa～0.07MPa, fermentation period is 34 hours～42 hours.
The culture medium prescription that the present embodiment produces enzymic fermentation tank is: in mass, corn steep liquor 1 ﹪ of Rhizoma amorphophalli powder 4 ﹪, wheat bran 2 ﹪, nitrogen content 40%, yeast extract paste 1 ﹪, Secondary ammonium phosphate 0.25 ﹪, dipotassium hydrogen phosphate 0.02 ﹪, peptone 0.2 ﹪, sal epsom 0.02 ﹪, sodium carbonate 0.23 ﹪, water 91.28 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0,0.1MPa sterilizing 33 minutes, and charging cumulative volume is no more than 70 ﹪; Culture condition: 35 ℃ of temperature, mixing speed 220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.30,4 hours～6 hours, 1:0.42,1:0.55 after 6 hours, tank pressure 0.06 MPa, fermentation period is 40 hours.
Its enzyme activity can reach 6000u/ml, more than mean level (ML) can reach 5200u/ml.
Put tank condition: tank temperature no longer rises; Enzyme activity no longer increases; PH7.0 left and right; More than 95% there is gemma in cell, and has part self-dissolving.Fermentation liquid pH is adjusted to 7.0 left and right.Filtrate enzyme activity 5500u/ml.
The present invention, in preparation standard enzyme liquid, through Plate Filtration, diatomite degerming, obtains through rotproofing the fermentation liquid that produces enzymic fermentation tank deposit under three months conditions 15 ℃ of sealings, and enzyme activity conservation rate is standard mannase liquid more than 95 ﹪.
The present invention is impurity elimination degerming, improve the quality of products, fermentation liquid is after Plate Filtration, in ferment filtrate, add disodium hydrogen phosphate dodecahydrate and CALCIUM CHLORIDE DIHYDRATE to get assorted degerming, its consumption is respectively 2.8 ﹪～3.5 ﹪ (the present embodiment is 3.2 ﹪) and 1.0 ﹪～1.3 ﹪ (the present embodiment is 1.2 ﹪) of ferment filtrate quality.
The present invention also can use other inorganic salt combination replacement, for example sodium sulfate and CALCIUM CHLORIDE DIHYDRATE combination.
CGMCC NO:6226 bacterial classification of the present invention can utilize multiple carbon and nitrogen sources to produce enzyme.Described carbon source, as bagasse, wheat bran, maize spike stalk powder, corn stalk powder, rice straw powder, the corn dregs of fat, cellulosic powder, peanut shell powder etc., can separately or be mixed and use.Described nitrogenous source is as the physiologically acid salts such as the ammonium sulfate in inorganic nitrogen, Secondary ammonium phosphate, ammonium chloride, SODIUMNITRATE physiological alkalinity salt, ammonium nitrate physiology neutral salt; The organonitrogens such as urea, corn steep liquor, corn starch, Zein powder, soybean protein powder, soy peptone, fish peptone, yeast extract paste, yeast powder, extractum carnis, can separately or mix and use.
A kind of mannase of producing as aforesaid method, described mannase is alkaline enzyme, cellulase-producing not, crystalline cellulose, carboxymethyl cellulose are not all had to activity, within the scope of 40 ℃～60 ℃ of pH5.5～10.0, temperature, mannase is activity stabilized, the suitableeest scope is pH6.0～8.0, and temperature is 45 ℃～55 ℃.
Mannase of the present invention is cellulose enzymic activity not, and enzyme activity is high, and its operative temperature and pH are adapted at paper industry application, can reduce paper cost and environmental pollution, help the product quality that improves paper.
Claims (9)
1. the production method of a mannase, it is characterized in that it comprises spawn culture, shake flask fermentation, enzyme liquid preparation process, described bacterial classification is subtilis (Bacillus subtilis), and laboratory is numbered PARK9639, preserving number: CGMCC NO:6226, its biological characteristics is: G+ bacteria, produce gemma, and it is shaft-like that thalline is, 0.7～0.8 * 2～3 microns of individual cells, peritrichous, can move, and in binary fission mode, breeds; 0.6～0.9 * 1.0～1.5 microns of ellipses of gemma are positioned at thalline central authorities or slightly inclined to one side, and after sporulation, thalline does not expand, and on substratum of the present invention, bacterium colony is rounded, dirty white, uniform coloring, and neat in edge, tarnish, sticky.
2. the production method of a kind of mannase according to claim 1, it is characterized in that described spawn culture is CGMCC NO:6226 bacterial classification well-grown on the inclined-plane of following preparation, slant culture based formulas is: in 1000mL distilled water, add extractum carnis 3g～8g, yeast extract paste 3g～8g, peptone 8g～12g, glucose 3g～5g, sodium-chlor 3g～5g, adjust pH 6.9～7.1, 0.1MPa sterilizing 28 minutes～32 minutes, make test tube slant, inoculate this bacterial classification, 34 ℃～36 ℃, cultivate after 20 hours～24 hours, put into 5 ℃ of refrigerators standby, or to injecting after sterilising liq paraffin preservation on bacterium inclined-plane or take skimmed milk as the preservation of carrier lyophilize vacuum seal.
3. the production method of a kind of mannase according to claim 1, it is characterized in that described shake flask fermentation is that CGMCC NO:6226 bacterial classification can produce the mannase of high vigor under the culture condition of regulation, this culture condition is: in mass, culture medium prescription is: Rhizoma amorphophalli powder 3 ﹪～4 ﹪, wheat bran 2 ﹪～4 ﹪, corn steep liquor 0.5 ﹪～1 ﹪ of nitrogen content 40%, yeast extract paste 0.5 ﹪～1 ﹪, Secondary ammonium phosphate 0.2 ﹪～0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪～0.05 ﹪, peptone 0.1 ﹪～0.2 ﹪, sal epsom 0.02 ﹪～0.05 ﹪, sodium carbonate 0.20 ﹪～0.25 ﹪, water 89.15 ﹪～93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is pH8.0～pH8.5, 0.1MPa sterilizing 30 minutes～35 minutes, culture condition: 32 ℃～37 ℃, shaking speed 260 r/min～280r/min, cultivate 34 hours～42 hours.
4. the production method of a kind of mannase according to claim 1, is characterized in that described enzyme liquid is prepared as by eggplant bottle inclined-plane to make cell liquid seeds to seeding tank, then to product enzymic fermentation tank, second order fermentation, preparation standard enzyme liquid.
5. the production method of a kind of mannase according to claim 4, it is characterized in that seed culture based formulas is: in mass, corn steep liquor 1 ﹪～2 ﹪ of wheat bran 5 ﹪～6 ﹪, Semen Maydis powder 1 ﹪～2 ﹪, nitrogen content 40 ﹪, sodium-chlor 0.4 ﹪～0.6 ﹪, sodium carbonate 1 ﹪～2 ﹪, ammonium sulfate 0.3 ﹪～0.5 ﹪, water 86.9 ﹪～91.3 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5～7.0,0.1MPa sterilizing 30 minutes～35 minutes; Seeding tank charging cumulative volume is no more than 70 ﹪, culture condition is: 33 ℃～38 ℃ of temperature, mixing speed 180 r/min～220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.25～0.27,4 hours～6 hours, 1:0.33～0.36, after 6 hours, 1:0.50～0.55, tank pressure 0.06 MPa～0.07MPa, culture cycle is 7 hours～9 hours.
6. the production method of a kind of mannase according to claim 4, it is characterized in that the culture medium prescription that produces enzymic fermentation tank is: in mass, Rhizoma amorphophalli powder 3 ﹪～4 ﹪, wheat bran 2 ﹪～4 ﹪, corn steep liquor 0.5 ﹪～1 ﹪ of nitrogen content 40%, yeast extract paste 0.5 ﹪～1 ﹪, Secondary ammonium phosphate 0.2 ﹪～0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪～0.05 ﹪, peptone 0.1 ﹪～0.2 ﹪, sal epsom 0.02 ﹪～0.05 ﹪, sodium carbonate 0.20 ﹪～0.25 ﹪, water 89.15 ﹪～93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0～8.5, 0.1MPa sterilizing 30 minutes～35 minutes, charging cumulative volume is no more than 70 ﹪, culture condition: 33 ℃～38 ℃ of temperature, mixing speed 180 r/min～220 r/min, air flow is in v/v: 0 hour～4 hours, 1:0.30～0.32,4 hours～6 hours, 1:0.40～0.42,1:0.50～0.55 after 6 hours, tank pressure 0.06 MPa～0.07MPa, fermentation period is 34 hours～42 hours.
7. the production method of a kind of mannase according to claim 4, it is characterized in that in preparation standard enzyme liquid, fermentation liquid process Plate Filtration, the diatomite degerming of enzymic fermentation tank will be produced, through rotproofing, obtain depositing under three months conditions 15 ℃ of sealings, enzyme activity conservation rate is standard mannase liquid more than 95 ﹪.
8. the production method of a kind of mannase according to claim 7, it is characterized in that fermentation liquid is after Plate Filtration, in ferment filtrate, add disodium hydrogen phosphate dodecahydrate and CALCIUM CHLORIDE DIHYDRATE to get assorted degerming, its consumption is respectively 2.8 ﹪～3.5 ﹪ and 1.0 ﹪～1.3 ﹪ of ferment filtrate quality.
9. the mannase that method is produced according to claim 1, it is characterized in that described mannase is alkaline enzyme, cellulase-producing not, crystalline cellulose, carboxymethyl cellulose are not all had to activity, within the scope of 40 ℃～60 ℃ of pH5.5～10.0, temperature, mannase is activity stabilized, the suitableeest scope is pH6.0～8.0, and temperature is 45 ℃～55 ℃.
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| CN110004166A (en) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | The recombined bacillus subtilis bacterial strain and its preparation method of high efficient expression secretion 'beta '-mannase |
| CN114045241A (en) * | 2021-11-18 | 2022-02-15 | 河南省科学院生物研究所有限责任公司 | Bacillus subtilis HKS018 and application thereof in production of beta-mannase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110004166A (en) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | The recombined bacillus subtilis bacterial strain and its preparation method of high efficient expression secretion 'beta '-mannase |
| CN114045241A (en) * | 2021-11-18 | 2022-02-15 | 河南省科学院生物研究所有限责任公司 | Bacillus subtilis HKS018 and application thereof in production of beta-mannase |
| CN114045241B (en) * | 2021-11-18 | 2023-05-12 | 河南省科学院生物研究所有限责任公司 | Bacillus subtilis HKS018 and application thereof in production of beta-mannase |
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