CN103820356B - The Bacillus licheniformis of one plant height heat production stability beta-glucanase - Google Patents
The Bacillus licheniformis of one plant height heat production stability beta-glucanase Download PDFInfo
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- CN103820356B CN103820356B CN201310716849.4A CN201310716849A CN103820356B CN 103820356 B CN103820356 B CN 103820356B CN 201310716849 A CN201310716849 A CN 201310716849A CN 103820356 B CN103820356 B CN 103820356B
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Abstract
The invention discloses the B. licheniformis strain of a plant height heat production stability beta-glucanase.This bacterial strain is by original strain, obtains through ultraviolet-lithium chloride-ethyl sulfate complex mutation.Described Bacillus licheniformis (Β acillus? licheniformis) β-10-25 is preserved in China typical culture collection center, does is preserving number CCTCC? NO:M2013538.The beta-glucanase enzyme activity that this bacterial strain produces, up to 8000-10000u/ml, is 2.5 times of original strain.Higher than existing beta-glucanase enzyme activity, enzyme effect optimum pH wide scope, thermostability and stability in storage are all higher, are particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to the Bacillus licheniformis of a plant height heat production stability beta-glucanase.
Background technology
Beta-glucan is a kind of natural polysaccharide, is usually present in the cell walls of the bacterium of particular variety, yeast and fungi, or the bag of higher plant seed by.Beta-glucan is the structural non-starch polysaccharide of one forming gramineae plant cell walls, and in the Formation of Endosperm Cell Walls of barley, rye, jowar, rice and millet, content is especially high.With general carbohydrate mainly with α-1,4-glycosidic link be combined into into linear molecule unlike, beta-glucan in plant cell wall is the β-(1 mixed, 3), (1,4) the D type glucose polymer that is connected to form of glycosidic link, therefore beta-glucan can be dissolved in the water with very large molecular weight, thus have that viscosity is large, wetting ability is high, water-swelling power and the high character of retentiveness.When using barley as beer production raw material, above-mentioned character just can affect the filtration velocity of wort, reduces the leaching yield of solid substance, also can reduce the quality of beer because of gel precipitation effect; Simultaneously when the crop containing beta-glucan is used as feed, non-intestinal juice viscosity of ruminating poultry can be increased, one of antinutritional factor becoming feed, reduce the utilization ratio of feed.
Beta-glucanase is the Major Enzymes of efficient, single-minded decomposition beta-glucan, and in the production process of beer and feed, add this enzyme can effectively solve the problem.In fodder industry, it can be hydrolyzed 1-3 or the 1-4 glycosidic link in beta-glucan, make it to be degraded to low molecular sugar, beta-glucan after degraded loses wetting ability and viscosity, adhesion of can not expanding in animal and bird intestines, and the digestibility of chyme and the capacity usage ratio of feed are all improved.Beta-glucanase is applied in beer production, can shift and decompose the very high various barley beta-glucan of viscosity, loose barley endosperm cell walls, promote the excessive of entocyte, improve raw material availability, reduce Wort viscosity, thus beer production is increased, quality is promoted.
At present, the states such as the U.S., Japan, Denmark, Germany, Australia, Canada have adopted beta-glucanase as the Major Enzymes preparation of beer and fodder industry all, and it also displays gradually in medicine, weaving, wastewater treatment and the otherwise using value such as daily-use chemical industry, biological control, prospect is very wide.But poor heat stability and enzyme activity is low has become the restraining factors affecting its effect.
Chinese patent CN103013873A discloses the bacterial strain of a strain heat production stability beta-glucanase, what this invention provided that a strain derives from hot spring first can produce β-1,3-1, the high ground bacillus of 4-dextranase, and clone obtains the gene of this enzyme, this enzyme still can keep the enzyme activity of 90% preserve 1h under 60 DEG C of conditions after.
Chinese patent CN102719416A provides a kind of method improving β-1,3-1,4-dextranase thermostability, passes through HNO
2chemically modified is carried out to β-1,3-1,4-dextranase, β-1,3-1, the 4-dextranase T of gained
50value and t
(1/2,60 DEG C)improve 4.76% and 62.1% respectively.
Before, on market the beta-glucanase of high-quality almost monopolize by external zymin production company, although the enzyme activity of production by biological beta-glucanase that China reported in recent years has met or exceeded world level, the beta-glucanase that has high reactivity and thermostability concurrently can be obtained and produced bacterial strain and remained a large problem urgently to be resolved hurrily in current industrial application.
Summary of the invention
The object of this invention is to provide the Bacillus licheniformis of a plant height heat production stability beta-glucanase.
The bacterial strain of high yield thermostability beta-glucanase provided by the invention is specially Bacillus licheniformis (Β acilluslicheniformis) β-10-25.This bacterial strain is preserved in China typical culture collection center (be called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College) on November 3rd, 2013, and preserving number is CCTCCNO:M2013538.
Described bacterial strain feature is as follows:
On solid plate, bacterium colony is white, and edge is irregular, and surface drying is opaque, and microscopy is gram positive bacterium, and cellular form is rod-short, raw in gemma, oval, does not expand.
Described bacterial strain physiological and biochemical property: V-P tests (+), Starch Hydrolysis (+), casein hydrolysis (+), gelatin hydrolysis (+), nitrate reduction (+), indole test (-), utilizes Citrate trianion (+), nitrate reductase (+), N.F,USP MANNITOL (+), wood sugar (+).
Described Bacillus licheniformis (Β acilluslicheniformis) β-10-25 is separated the Bacillus licheniformis warp of the product beta-glucanase obtained by strain our company laboratory: original, bacterial classification β-10 → test tube activation → ultraviolet (UV)-lithium chloride (LiCl)-ethyl sulfate (DES) complex mutation → dull and stereotyped primary dcreening operation (high yield) → shaking flask is sieved the steps such as (high yield) → thermal stability determination → mitotic stability test again and screened acquisition.
Concrete screening step is as follows:
1. starting strain activation
The activation of starting strain: get Bacillus licheniformis starting strain β-10 one ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL seed culture medium, cultivates about 24h in 37 DEG C, continuously activation three times.
Described seed culture medium is: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%.
2. UV-LiCl-ethyl sulfate complex mutation breeding
1) preparation of bacteria suspension
The bacterial strain list bacterium colony grown after plate streaking is separated is accessed in L Β substratum, after 37 DEG C of cultivation 24h, use brine twice after getting 1mL medium centrifugal, and be resuspended in 9mL physiological saline.
2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 60s under the ultraviolet lamp of power 15w.Bacterium liquid through irradiating is coated lithium chloride flat board after gradient dilution, and contrasts to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 30 DEG C and cultivate 48h, the flat board growing bacterium colony filters out transparent circle and colony diameter ratio the maximum choose and preserve to inclined-plane, bacteria suspension is mixed with after purifying, fully mix with ethyl sulfate stoste after gradient dilution, and in 30 DEG C of static process 40min, the bacterium liquid processed is coated on lithium chloride flat board after gradient dilution.
Described lithium chloride is dull and stereotyped: lichenstarch 0.3%, Congo red 0.1%, peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%.
3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, put 30 DEG C and cultivate 48h, the separating plate growing bacterium colony just sifts out transparent circle and colony diameter odds ratio the greater and chooses and preserve to inclined-plane, ten strain bacterium β-10-21 are obtained after purifying, β-10-22, β-10-23, β-10-24, β-10-25, β-10-26, β-10-27, β-10-28, β-10-29, β-10-30.
4) shake flask fermentation sieves again
The ten strain bacterium obtained are carried out shake flask fermentation in the 250mL shaking flask that 50mL fermention medium is housed, seed inoculum size 5% (V/V), rear employing DNS method of having fermented measures enzyme and lives, result shows β-10-23, the enzymatic productivity comparatively original strain raising 55%, 62%, 47% respectively of β-10-25, β-10-26.
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 50mL fermention medium, inoculum size 5% (V/V), 100r/min, 37 DEG C of fermentation culture 72h.
3, thermostability screening
By the bacterial strain β-10-23 of three high producing beta-glucanases that shake flask fermentation obtains, β-10-25, β-10-26 carries out shake flask fermentation, 200r/min.37 DEG C ferment 2 days, fermented liquid is centrifugal, get supernatant liquor and be beta-glucanase crude enzyme liquid, be placed in 4 DEG C of preservations, under 50 DEG C of conditions, measure enzyme after above-mentioned three strain beta-glucanases that bacterial strain produces are preserved 10min, 30min, 1h under 50,60,70,80,90 DEG C of conditions live, result represents that the beta-glucanase that β-10-25 produces has best thermostability, and it still can keep the enzyme of more than 80% to live after preserving 1h at 60 DEG C, therefore determine that β-10-25 is experimental strain.
The production method of described beta-glucanase is as follows:
(1) seed culture
By β-10-25 slant strains through three grades of shake flask fermentations and first class seed pot fermentation;
The substratum of described seed culture is: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%, pH4.5-7.0,121-123 DEG C of sterilizing 30-40min.
(2) ferment tank
First class seed pot fermented liquid is contained in the fermentor tank of 3L fermention medium with 3% inoculum size access, culture temperature 35-45 DEG C, stirring velocity 200-300r/min, ventilation (V/V) 1:1-3, incubation time 10-15h; Then by 1L through 121 DEG C of sterilizing 20min, temperature is that the fermention medium of 10 DEG C fills into fermentor tank, constant temperature culture 15-20h when temperature rises to 35-45 DEG C; Now, first class seed pot fermented liquid is added access fermentor tank with 2% inoculum size, constant temperature culture 10-15h; Again by 1L through 121 DEG C of sterilizing 20min, temperature is that the fermention medium of 10 DEG C fills into fermentor tank, constant temperature culture 10-15h when temperature rises to 35-45 DEG C;
Described fermention medium consists of: wheat bran 75g, Semen Maydis powder 55g, rice bran 25g, soybean cake powder 20g, beta-glucan 2g, herbal mediciment powder 40g, trehalose 35g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water l000mL, pH value 4.5-7.0,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
Take Radix Astragali 20-30 part; Radix Codonopsis 10-18 part; Radix bupleuri 10-15 part; The tuber of dwarf lilyturf 10-15 part; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then being cooled to 45-60 DEG C, adding mixed enzyme agent and carry out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, ultrasonic extraction 0.5 ~ 1.5h under 110W power, filter; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder;
Described mixed enzyme addition is the 5-10% of mixture gross weight;
The parts by weight of described mixed enzyme consist of: beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, polygalacturonase 10-15 part, aspartic protease 10-15 part, papoid 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part;
The mass ratio of described ethanol and propyl alcohol is 1:1-1.5.
(3) fermentation liquor filter, concentrated, essence filter, dry beta-glucanase.
The beta-glucanase liquid enzymes vigor obtained through above-mentioned preparation method is 8000-10000u/ml.
The beta-glucanase obtained by bacterial strain β-10-25 fermentation, zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature is 65 DEG C, has higher enzyme and live between 50-70 DEG C.
(2) this enzyme optimal reaction pH value is 5.0, the enzyme complete stability alive when pH value is 4.0.
(3) enzymic activity: by bacterial strain β-10-25 provided by the present invention, the beta-glucanase enzyme activity of preparation is 8000-10000u/ml, compares the raising 2.5 times alive of starting strain enzyme.
(4) thermostability: this enzyme still can keep more than 80% enzyme to live preserve 1h under 60 DEG C of conditions after, keeps more than 20% enzyme to live after preserving 10min under 70 DEG C of conditions; This enzyme is at room temperature preserved the loss alive of enzyme after 6 months and is less than 25%, preserves the loss alive of enzyme after 12 months for 4 DEG C and is less than 20%.
Beneficial effect:
1, the present invention uses the method for UV-LiCl-ethyl sulfate complex mutation to obtain the Bacillus licheniformis β-10-25 of a plant height heat production stability beta-glucanase, enzyme that this bacterial strain produces has Heat stability is good, it is active good to store, the feature that enzyme activity is high.
2, having this bacterial strain to produce the beta-glucanase enzyme activity of gained up to 8000-10000u/ml, is 2.5 times of original strain; Under 50-70 DEG C of condition, have high enzyme live, optimal reactive temperature is 65 DEG C; Optimal reaction pH value is 5.0; At room temperature preserve enzyme loss alive after 6 months and be less than 25%, preserve the loss alive of enzyme after 12 months for 4 DEG C and be less than 20%.Higher than existing beta-glucanase enzyme activity, enzyme effect optimum pH wide scope, thermostability and stability in storage are all higher, are particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1: UV-LiCl-ethyl sulfate Mutation screening
(1) preparation of bacteria suspension
The single bacterium colony of β-10 grown after plate streaking is separated is accessed in seed culture medium, 100r/min, after 37 DEG C of cultivation 48h, use brine twice after getting 1mL medium centrifugal, and be resuspended in 9mL physiological saline.
Described seed culture medium is: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%.
(2) UV-LiCl-ethyl sulfate complex mutation
3mL bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.By the bacterium liquid through irradiating through 10
-1-10
-5coat lithium chloride flat board after gradient dilution, and contrast to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 37 DEG C and cultivate 48h, the flat board growing bacterium colony filters out hydrolysis circle and chooses with colony diameter ratio the maximum and preserve to inclined-plane, be mixed with bacteria suspension after purifying, through 10
-1-10
-5get 20mL bacteria suspension after gradient dilution fully to mix with 1mL ethyl sulfate stoste, and in 37 DEG C of concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board.
Described lithium chloride is dull and stereotyped: lichenstarch 0.3%, Congo red 0.1%, peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 37 DEG C and cultivate 48h, on the separating plate growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter odds ratio the greater, ten strain bacterium β-10-21 are obtained after purifying, β-10-22, β-10-23, β-10-24, β-10-25, β-10-26, β-10-27, β-10-28, β-10-29, β-10-30.
(4) shake flask fermentation sieves again
The ten strain bacterium obtained are carried out shake flask fermentation in the 250mL shaking flask containing 50mL fermention medium, get the centrifugal 10min of fermented liquid 5000r/min after having fermented and remove thalline, supernatant liquor carries out ultrafiltration and concentration through 10000MWCO, obtain crude enzyme liquid, the enzyme adopting DNS method to measure the crude enzyme liquid of above-mentioned ten strain bacterial strains is respectively lived.
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 50mL fermention medium, inoculum size 5% (V/V), 100r/min, 37 DEG C of fermentation culture 72h.
The mensuration of embodiment 2: β-glucanase activity
(1) definition of Mei Huo unit: 1mL crude enzyme liquid, in 50 DEG C, under pH6.0 condition, 1min is that the final decline of 4mg/ml beta-glucan solution solves 1 μm of ol reducing sugar from concentration, is 1 Ge Meihuo unit (1U/ml)
(2) enzyme activity determination method: adopt DNS method to measure
1. get 5 test tubes, numbering 1,2,3,4,5, adds 0.5ml dextran solution respectively;
2. in 1,2, No. 3 test tube, add 0.5ml crude enzyme liquid to be measured, in 50 DEG C of water-baths, react 15min;
3. in 4, No. 5 test tubes, 0.5ml crude enzyme liquid to be measured is added;
4. in 5 test tubes, respectively add 3mlDNS reagent, shake up rear boiling water bath 5min;
5., after water-bath cooling, with 4, No. 5 test tubes for contrast, 1,2, No. 3 absorbancy under 540nm is measured;
6. contrast glucose standard curve calculating enzyme to live.
Embodiment 3: the mensuration of zymologic property
(1) temperature is on the impact of beta-glucanase enzymic activity
At 40,50,60,70,80,90 DEG C, measure the vigor of gained beta-glucanase crude enzyme liquid respectively, result shows that this enzyme all has higher enzymic activity between 50-70 DEG C, and thermal adaptation a wider range of this enzyme is described, its optimum temperature is 65 DEG C.(2) thermostability of beta-glucanase
Under 50 DEG C of conditions, measure enzyme after the crude enzyme liquid of gained is preserved 10min, 30min, 1h at 40,50,60,70,80,90 DEG C live, result shows still to keep more than 80% enzyme to live after this enzyme preserves 1h under 60 DEG C of conditions, keeps more than 20% enzyme to live after preserving 10min under 70 DEG C of conditions.
(3) storage stability of beta-glucanase
Under 50 DEG C of conditions, measure enzyme after the crude enzyme liquid of gained is preserved 1 day, 15 days, 1 month, 3 months, 6 months and 12 months under 4 DEG C and room temperature condition to live.Result shows that this enzyme is at room temperature preserved the loss alive of enzyme after 6 months and is less than 25%, preserves the loss alive of enzyme after 12 months for 4 DEG C and is less than 20%.
(4) pH is on the impact of enzymic activity
At 50 DEG C, survey this enzyme enzyme under pH3-9 respectively and live, result shows that this enzyme enzymic activity when pH4.5-7.0 is higher, and optimal pH is 6.5.
Claims (3)
1. Bacillus licheniformis (Β acilluslicheniformis) β-10-25 of a plant height heat production stability beta-glucanase, deposit number is CCTCCNO:M2013538.
2. Bacillus licheniformis (Β acilluslicheniformis) β-10-25 of high yield thermostability beta-glucanase as claimed in claim 1, it is characterized in that, the beta-glucanase enzyme activity that described Bacillus licheniformis β-10-25 produces is 8000-10000u/ml, more than 80% enzyme still can be kept preserve 1h under 60 DEG C of conditions after to live, after preserving 10min under 70 DEG C of conditions, keep more than 20% enzyme to live; This enzyme is at room temperature preserved the loss alive of enzyme after 6 months and is less than 25%, preserves the loss alive of enzyme after 12 months for 4 DEG C and is less than 20%.
3. Bacillus licheniformis (Β acilluslicheniformis) β-10-25 is producing the application in beta-glucanase according to claim 1.
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| CN114214247A (en) * | 2021-12-27 | 2022-03-22 | 威海市世代海洋生物科技股份有限公司 | Bacillus licheniformis and application thereof |
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| CN103013873A (en) * | 2012-12-10 | 2013-04-03 | 南京农业大学 | Strain generating heat-stable Beta-glucanase and application of strain |
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