CN103540580B - A kind of mannase and production method - Google Patents
A kind of mannase and production method Download PDFInfo
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- CN103540580B CN103540580B CN201310490356.3A CN201310490356A CN103540580B CN 103540580 B CN103540580 B CN 103540580B CN 201310490356 A CN201310490356 A CN 201310490356A CN 103540580 B CN103540580 B CN 103540580B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/011—Mannan 1,4-mannobiosidase (3.2.1.100)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- 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
Abstract
The invention discloses a kind of mannase production method being adapted to alkaline environment, Heat stability is good, it is characterized in that it comprises spawn culture, shake flask fermentation, enzyme liquid preparation process, described bacterial classification and subtilis (Bacillus? subtilis), laboratory is numbered PARK9639, preserving number: CGMCC? NO:6226, described mannase is alkaline enzyme, not cellulase-producing, all do not have activity to crystalline cellulose, carboxymethyl cellulose, within the scope of pH5.5 ~ 10.0, temperature 40 DEG C ~ 60 DEG C, Mannanase Activity is stablized; Enzyme activity of the present invention 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 reduces paper cost and environmental pollution, help the product quality improving paper simultaneously.
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), mannan endo-Isosorbide-5-Nitrae-beta-Mannosidase (Mannanendo-1,4-β-mannosidase), numbering EC3.2.1.78; (2) mannosans-Isosorbide-5-Nitrae-β-mannobiose lytic enzyme (Mannanendo-1,4-β-mannobiohydrolase), numbering EC3.2.1.100[is also known as circumscribed-Isosorbide-5-Nitrae-β-mannobiose lytic enzyme (Exo-1,4-β-mannobiohydrolase)]; (3) beta-Mannosidase (β-Mannosidase), numbering EC3.2.1.25; (4) mannan endo-1,6-beta-Mannosidase (Mannanendo-1,6-β-mannosidase), numbering EC3.2.1.101.
At the beginning of last century, just having the report about decomposing plant mannosans, in recent years along with people are to the continuous discovery of mannase purposes, having promoted the development of mannase research, Courtios first time in 1958 reports the fungi producing mannase; Nineteen sixty William and Doetsch road to produce the bacterium of mannase; Nineteen sixty-five, Reese and Shibata proposes concept and the definition of mannase; After the seventies, the basic nature Quality Research of people to mannase achieves large development, mannase is applied to feed cultivation industry by the nineties people, achieve good development, along with people are to the exploitation of nature hemicellulose resource and deepening continuously to zytase research, mannase is widely used research in many-sides such as feed, food, papermaking, oil production and biological study technology.But, all the time industrial applications is not had in papermaking, major cause is that needed for papermaking, enzyme requirement uses 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 and be a kind ofly adapted to alkaline environment, the mannase of Heat stability is good and production method.
Enzyme activity determination method of the present invention is, utilizes the disodium hydrogen phosphate dodecahydrate of 0.05mol/LpH6.0 and the locust bean gum solution of monohydrate potassium buffer 0.5%.In this substrate solution of 0.9ml, add the enzyme solution that 0.1ml suitably dilutes, 50 DEG C of accurate clock reaction 10min, in 550nm light-metering 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 and subtilis (Bacillussubtilis), laboratory is numbered PARK9639, preserving number: CGMCCNO:6226, its biological characteristics is: G+ bacteria, produce gemma, thalline is shaft-like, individual cells 0.7 ~ 0.8 × 2 ~ 3 microns, peritrichous, can move, breed in binary fission mode; Gemma 0.6 ~ 0.9 × 1.0 ~ 1.5 microns of ellipses are positioned at thalline central authorities or slightly inclined, 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 is matt, 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 16SrRNA gene order, the experimental datas such as gyrB gene order are comprehensively analyzed and are accredited as subtilis (Bacillussubtilis), this bacterial classification 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: CGMCCNO:6226, and prove in have submitted preservation survival on the same day.
Spawn culture of the present invention is CGMCCNO: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 DEG C ~ 36 DEG C, cultivate after 20 hours ~ 24 hours, put into 5 DEG C of refrigerators for subsequent use, or on bacterium inclined-plane, inject preservation or be the preservation of carrier lyophilize vacuum seal with skimmed milk after sterilising liq paraffin.
Shake flask fermentation of the present invention is the mannase that CGMCCNO:6226 bacterial classification can produce 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 ﹪, magnesium sulfate 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 DEG C ~ 37 DEG C, shaking speed 260r/min ~ 280r/min, cultivate 34 hours ~ 42 hours.
Enzyme liquid of the present invention is prepared as and obtains cellular liquid seed to seeding tank by eggplant bottle inclined-plane, 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: temperature 33 DEG C ~ 38 DEG C, mixing speed 180r/min ~ 220r/min, air flow 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 0.06MPa ~ 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 ﹪, magnesium sulfate 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: temperature 33 DEG C ~ 38 DEG C, mixing speed 180r/min ~ 220r/min, air flow was 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 0.06MPa ~ 0.07MPa, fermentation period is 34 hours ~ 42 hours.
The present invention is in preparation standard enzyme liquid, and by degerming through Plate Filtration, diatomite for the fermentation liquid producing enzymic fermentation tank, obtain under three months conditions are deposited in 15 DEG C of sealings through rotproofing, enzyme activity conservation rate is the standard mannase liquid of 95 more than ﹪.
The present invention is that impurity elimination is degerming, improve the quality of products, fermentation liquid, after Plate Filtration, adds disodium hydrogen phosphate dodecahydrate and CALCIUM CHLORIDE DIHYDRATE is got 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 produced as aforesaid method, described mannase is alkaline enzyme, not cellulase-producing, all activity is not had to crystalline cellulose, carboxymethyl cellulose, within the scope of pH5.5 ~ 10.0, temperature 40 DEG C ~ 60 DEG C, Mannanase Activity is stablized, the suitableeest scope is pH6.0 ~ 8.0, and temperature is 45 DEG C ~ 55 DEG C.
Owing to adopting technique scheme, the present invention achieves 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 reduces paper cost and environmental pollution, help the product quality improving 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, described bacterial classification and subtilis (Bacillussubtilis), and laboratory is numbered PARK9639, preserving number: CGMCCNO:6226, its biological characteristics is: G+ bacteria, and produce gemma, thalline is shaft-like, individual cells 0.7 ~ 0.8 × 2 ~ 3 microns, peritrichous, can move, breed in binary fission mode; Gemma 0.6 ~ 0.9 × 1.0 ~ 1.5 microns of ellipses are positioned at thalline central authorities or slightly inclined, 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 is matt, 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 16SrRNA gene order, the experimental datas such as gyrB gene order are comprehensively analyzed and are accredited as subtilis (Bacillussubtilis), this bacterial classification 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: CGMCCNO:6226, and prove in have submitted preservation survival on the same day.
Spawn culture of the present invention is CGMCCNO: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 DEG C ~ 36 DEG C, cultivate after 20 hours ~ 24 hours, put into 5 DEG C of refrigerators for subsequent use, or on bacterium inclined-plane, inject preservation or be the preservation of carrier lyophilize vacuum seal with skimmed milk after sterilising liq paraffin.
The present embodiment slant culture based formulas is: in 1000mL distilled water, adds extractum carnis 5g, yeast extract paste 5g, peptone 10g, glucose 5g, sodium-chlor 5g, by sodium hydroxide key pH value 7.0,0.1MPa sterilizing 30 minutes, make test tube slant, inoculate this bacterial classification, 35 DEG C, cultivate after 22 hours, put into 5 DEG C of refrigerators for subsequent use.
After this slant strains is qualified by shake flask fermentation inspection bacterial classification enzymatic productivity, can spreads cultivation as seed and use or enzymatic production use.
Shake flask fermentation of the present invention is the mannase that CGMCCNO:6226 bacterial classification can produce 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 ﹪, magnesium sulfate 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 DEG C ~ 37 DEG C, shaking speed 260r/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 ﹪, magnesium sulfate 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 DEG C, 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 and obtains cellular liquid seed to seeding tank by eggplant bottle inclined-plane, 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: temperature 33 DEG C ~ 38 DEG C, mixing speed 180r/min ~ 220r/min, air flow 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 0.06MPa ~ 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: temperature 37 DEG C, mixing speed 220r/min, and air flow was 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.Sample microscopy after cultivating 4h, observe bacteria growing and grow, breeding situation.When cultivation is fractured into individual cells to somatic cells chain 70 ﹪, show seed close to 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 ﹪, magnesium sulfate 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: temperature 33 DEG C ~ 38 DEG C, mixing speed 180r/min ~ 220r/min, air flow was 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 0.06MPa ~ 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 ﹪, magnesium sulfate 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: temperature 35 DEG C, mixing speed 220r/min, air flow was in v/v: 0 hour ~ 4 hours, and 1:0.30,4 hours ~ 6 hours, 1:0.42,1:0.55, tank pressure 0.06MPa after 6 hours, fermentation period was 40 hours.
Its enzyme activity can reach 6000u/ml, and mean level (ML) can reach more than 5200u/ml.
Put tank condition: tank temperature no longer rises; Enzyme activity no longer increases; About pH7.0; There is gemma in cell more than 95%, and has partial autolysis.Fermentation liquid pH is adjusted to about 7.0.Filtrate enzyme activity 5500u/ml.
The present invention is in preparation standard enzyme liquid, and by degerming through Plate Filtration, diatomite for the fermentation liquid producing enzymic fermentation tank, obtain under three months conditions are deposited in 15 DEG C of sealings through rotproofing, enzyme activity conservation rate is the standard mannase liquid of 95 more than ﹪.
The present invention is that impurity elimination is degerming, improve the quality of products, fermentation liquid is after Plate Filtration, in ferment filtrate, add disodium hydrogen phosphate dodecahydrate and CALCIUM CHLORIDE DIHYDRATE is got 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, and such as sodium sulfate and CALCIUM CHLORIDE DIHYDRATE combine.
CGMCCNO: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., separately or can mix and use.Described nitrogenous source as physiologically acid salts such as the ammonium sulfate in inorganic nitrogen, Secondary ammonium phosphate, ammonium chlorides, SODIUMNITRATE physiological alkalinity salt, ammonium nitrate physiological 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, separately or can mix and use.
A kind of mannase produced as aforesaid method, described mannase is alkaline enzyme, not cellulase-producing, all activity is not had to crystalline cellulose, carboxymethyl cellulose, within the scope of pH5.5 ~ 10.0, temperature 40 DEG C ~ 60 DEG C, Mannanase Activity is stablized, the suitableeest scope is pH6.0 ~ 8.0, and temperature is 45 DEG C ~ 55 DEG C.
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 improving paper.
Claims (5)
1. a production method for mannase, is characterized in that it comprises spawn culture, shake flask fermentation, enzyme liquid preparation process, described bacterial classification and subtilis (
bacillussubtilis), laboratory is numbered PARK9639, preserving number: CGMCCNO:6226, and its biological characteristics is: G+ bacteria, and produce gemma, thalline is shaft-like, individual cells 0.7 ~ 0.8 × 2 ~ 3 microns, and peritrichous can move, breed in binary fission mode; Gemma 0.6 ~ 0.9 × 1.0 ~ 1.5 microns of ellipses are positioned at thalline central authorities or slightly inclined, and after sporulation, thalline does not expand, and on slant medium, bacterium colony is rounded, dirty white, uniform coloring, and neat in edge is matt, sticky;
Described spawn culture is CGMCCNO:6226 bacterial classification well-grown on the inclined-plane of 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 DEG C ~ 36 DEG C, cultivate after 20 hours ~ 24 hours, put into 5 DEG C of refrigerators for subsequent use, or on bacterium inclined-plane, inject preservation or be the preservation of carrier lyophilize vacuum seal with skimmed milk after sterilising liq paraffin,
Described shake flask fermentation is the mannase that CGMCCNO:6226 bacterial classification can produce 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 ﹪, magnesium sulfate 0.02 ﹪ ~ 0.05 ﹪, sodium carbonate 0.20 ﹪ ~ 0.25 ﹪, water 89.15 ﹪ ~ 93.46 ﹪, each component sum is 100 ﹪, before sterilizing, adjust pH is pH8.0 ~ pH8.5, 0.1MPa sterilizing 30 minutes ~ 35 minutes, culture condition: 32 DEG C ~ 37 DEG C, shaking speed 260r/min ~ 280r/min, cultivate 34 hours ~ 42 hours,
Described enzyme liquid is prepared as and obtains cellular liquid seed to seeding tank by eggplant bottle inclined-plane, then to producing enzymic fermentation tank, second order fermentation, preparation standard enzyme liquid.
2. the production method of a kind of mannase according to claim 1, 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 ﹪, 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: temperature 33 DEG C ~ 38 DEG C, mixing speed 180r/min ~ 220r/min, air flow 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 0.06MPa ~ 0.07MPa, culture cycle is 7 hours ~ 9 hours.
3. the production method of a kind of mannase according to claim 1, it is characterized in that the culture medium prescription producing 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 ﹪, magnesium sulfate 0.02 ﹪ ~ 0.05 ﹪, sodium carbonate 0.20 ﹪ ~ 0.25 ﹪, water 89.15 ﹪ ~ 93.46 ﹪, 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: temperature 33 DEG C ~ 38 DEG C, mixing speed 180r/min ~ 220r/min, air flow was 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 0.06MPa ~ 0.07MPa, fermentation period is 34 hours ~ 42 hours.
4. the production method of a kind of mannase according to claim 1, it is characterized in that in preparation standard enzyme liquid, by degerming through Plate Filtration, diatomite for the fermentation liquid producing enzymic fermentation tank, through rotproofing, obtain under three months conditions are deposited in 15 DEG C of sealings, standard mannase liquid that enzyme activity conservation rate is 95 more than ﹪.
5. the production method of a kind of mannase according to claim 4, it is characterized in that fermentation liquid is after Plate Filtration, in ferment filtrate, add disodium hydrogen phosphate dodecahydrate and CALCIUM CHLORIDE DIHYDRATE impurity elimination is degerming, its consumption is respectively 2.8 ﹪ ~ 3.5 ﹪ and 1.0 ﹪ ~ 1.3 ﹪ of ferment filtrate quality.
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| Purification, characterization, and production of β-mannanase from Bacillus subtilis TJ-102 and its application in gluco-mannooligosaccharides preparation.;Mengfan Wang et al.;《Eur Food Res Technol》;20130507;399-408 * |
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