CN105524839A - Trichoderma reesei of high-yield and high-temperature-resistant fungal alpha-amylase - Google Patents
Trichoderma reesei of high-yield and high-temperature-resistant fungal alpha-amylase Download PDFInfo
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- CN105524839A CN105524839A CN201410514080.2A CN201410514080A CN105524839A CN 105524839 A CN105524839 A CN 105524839A CN 201410514080 A CN201410514080 A CN 201410514080A CN 105524839 A CN105524839 A CN 105524839A
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- amylase
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Abstract
The invention discloses trichoderma reesei of high-yield and high-temperature-resistant fungal alpha-amylase. The trichoderma reesei is used as an original strain and is subjected to ultraviolet, lithium chloride and diethyl sulfate compound mutation to obtain a trichoderma reesei mutant strain of the high-temperature-resistant fungal alpha-amylase. The trichoderma reesei 901-18 is preserved in the China Center for Type Culture Collection on 24 November, 2013, and a preservation number of the trichoderma reesei 901-18 is CCTCC NO: M 2013602. The trichoderma reesei has the advantages that the enzyme activity of the high-temperature-resistant fungal alpha-amylase produced by the strain can reach 12000-15000 u/ml, the applicable temperature ranges are 50-75 DEG C, the pH (potential of hydrogen) is 4.5-7.0, and the trichoderma reesei is particularly applicable to industrial requirements on high reaction temperatures and coexistence of liquefaction processes and saccharification processes.
Description
Technical field:
The invention belongs to microbial technology field, be specifically related to the Trichodermareesei of a strain high-yield thermostable fungal alpha-amylase.
Background technology:
α-amylase full name is α-Isosorbide-5-Nitrae-glucan hydrolase (EC3.2.1.1), when acting on starch, α-1 can be cut from intramolecule, 4-glycosidic link and generate dextrin and reducing sugar, because the terminal glucose saccharide residue C1 carbon atom of product is α-configuration, therefore must be called α-amylase.
Common α-amylase has three kinds: bacterialα-amylase, fungal alpha-amylase and maltogenic alpha-amylases.Bacterialα-amylase is by bacterium as Bacillus subtilus, Bacillus licheniformis etc. produce, and the thermostability of bacterialα-amylase is stronger.
Fungal alpha-amylase allowed Ji extract from aspergillus oryzae early than 1896 by the peak of Japan, and used as digestive pharmaceutical.Since the fifties, the range of application of fungal alpha-amylase expands gradually, starts to be applied to starch refine dsugar, brewages, the field such as bread, beverage made of fruits or vegetables, medicine and feed.The novel syrup based on trisaccharide maltose can be produced for starch refine dsugar; Share with glycosyltransferase, can dextrinosan be prepared; In traditional maltose, rice wine production, use this enzyme, can production technique be simplified, improve raw material availability, save grain, reduce production cost; Can shorten fermentation period for bread manufacture, the bread even air hole distribution made, volume is large, long fresh-keeping period, also alternative in the past conventional bread additives potassium bromate; Can be used as digestive pharmaceutical for medicine, replace the enzyme extracted in animal; Can muddiness be eliminated for beverage made of fruits or vegetables, improve yield; Efficiency of feed utilization can be improved for feed.Therefore, the application of fungal alpha-amylase has wide market outlook.
But the optimum temperature of fungal alpha-amylase is at about 55 DEG C, when temperature just can inactivation gradually higher than 60 DEG C, and thermally-stabilised relatively low, be not suitable for the reaction process of comparatively high temps, therefore its application & development is by restriction to a certain extent.
Summary of the invention:
In order to solve the problems of the technologies described above, the invention provides the Trichodermareesei of a strain high-yield thermostable fungal alpha-amylase.
The Trichodermareesei of high-yield thermostable fungal alpha-amylase provided by the invention is specially Trichodermareesei 901-18Trichodermareesei901-18.This bacterial strain on November 24th, 2013 be preserved in China typical culture collection center (be called for short CCTCC, address is: China. Wuhan. Wuhan University), preserving number CCTCCNO:M2013602.
Described bacterial strain feature is as follows:
This bacterial strain is at PDA cultured on solid medium, and the colony characteristics of formation is bacterium colony is flocculence, and bacterium colony is light green, and bacterium colony is flat, high 0.2-0.8mm, colony edge white, neatly; Fast growth, 48h colony diameter reaches 1.5-8.5mm, and 72h reaches 30-55mm; White mycelium, has barrier film, and mycelia wall is smooth, and diameter is at 2-5 μm.Conidiophore occurs, to life on side shoot from the short lateral branch of mycelia.
Described Trichodermareesei 901-18Trichodermareesei901-18 is separated by a strain and obtains through UV-LiCl-ethyl sulfate Mutation screening from the Li's Trichoderma strains of Jinshi City river levee domestic fungus cultivating base soil sample, and concrete screening step is as follows:
(1) starting strain is inoculated on potato slant medium, continuous passage three times, activated spawn, add the spore that lower maturation is washed in sterilized water vibration, dispersed with stirring spore obtains spore suspension, under the spore suspension be stirred is placed in ultraviolet lamp, vertical range 30cm stirs and irradiates 200s, and above-mentioned uv irradiating all must carry out under the condition of lucifuge.
Suspension 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, in the bacterium colony grown, filter out hydrolysis circle 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 concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board after gradient dilution.
(2) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, put 30 DEG C and cultivate 48h, sift out hydrolysis circle the greater and choose to inclined-plane preservation, obtain three strain bacterium 901-11 after purifying, 901-15,901-18.
(3) shake flask fermentation sieves again
By the three strain bacterium 901-11 obtained, 901-15,901-18 carry out shake flask fermentation in the 250mL shaking flask containing 30mL potato liquid nutrient medium, seed inoculum size 10% (V/V), 30 DEG C, 150r/min cultivates 72h, centrifuging and taking fermented supernatant fluid obtains crude enzyme liquid.
(4) enzyme activity determination
The definition of Mei Huo unit: 1mL crude enzyme liquid, in 65 DEG C, under pH5.5 condition, 1min liquefies 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain 901-18 is stable most superior strain.
Cultural characteristic: this bacterial strain produces the optimal pH 3.0-6.5 of fungal alpha-amylase; Optimum temperuture is 27 ~ 30 DEG C.
The high fungal alpha-amylase that the 901-18 bacterial strain of gained can be obtained after fermentation condition optimization high temperature resistant, Heat stability is good and enzyme is lived, specific as follows:
The method adopting alternating temperature to regulate produces fungal alpha-amylase, and its fermentor cultivation stage comprises the steps:
By seed liquor with 6% inoculum size access fermentor tank, culture temperature 27 ~ 30 DEG C, stirring velocity 120-180r/m, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, first class seed pot fermented liquid is added access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 27 ~ 30 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/ml-8mg/ml, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/ml-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, soybean cake powder 15-25g, trehalose 30-40g, yeast powder 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium primary phosphate 1-2g, Trisodium Citrate 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 5-6.5,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, bean powder 15-25%, and insufficient section pure water is supplied, pH value 5-6.5,121-123 DEG C of sterilizing 30-40min.
Fermentation liquor is filtered, concentrated, allotment, essence filter, dry fungal alpha-amylase, its zymologic property is as follows:
(1) this enzyme temperature applicable range is wider, and between 50-75 DEG C, optimum temperature, at 65 DEG C, is preserved 3h and still had more than 80% enzyme work at 70 DEG C.
(2) this enzyme optimal reaction pH value is 5.5, and living at the glucose-6-phosphate dehydrogenase of pH value 4.5-7.0 remains on more than 70%.
(3) enzyme is lived: the Thermostable α-Amylase enzyme activity prepared by mutant strain 901-18 provided by the present invention can reach 12000-15000U/ml, compares the raising 3.6 times alive of starting strain enzyme.
Beneficial effect:
1, the present invention uses the method for UV-LiCl-ethyl sulfate complex mutation to obtain the Li's Trichoderma strains of a plant height product fungal alpha-amylase, this bacterial strain temperature-variable fermentation working condition lower produce enzyme and there is high temperature resistant, Heat stability is good, the feature that vigor is high.
2, having this bacterial strain to produce the high temperature resistant fungal alpha-amylase enzyme activity of gained up to 12000-15000U/ml, is 3.6 times of original strain; This enzyme thermal adaptation a wider range, between 50-75 DEG C, resistance to temperature is high, is 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 spore suspension
Add 10mL sterilized water by the starting strain inclined-plane after activation, fully spore suspension is made in concussion.
(2) UV-LiCl-ethyl sulfate complex mutation
3mL spore suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 200s under the ultraviolet lamp of power 15w.By the spore suspension through irradiating after gradient dilution (10
-1-10
-5) coat lithium chloride flat board, and contrast to be coated with flat board without the spore suspension 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 hydrolysis circle and chooses with colony diameter ratio the maximum and preserve to inclined-plane, after purifying, be mixed with bacteria suspension, after gradient dilution (10
-1-10
-5) get 20mL bacteria suspension and fully mix with 1mL ethyl sulfate stoste, and in 30 DEG C of concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 30 DEG C and cultivate 48h, on the flat board growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter ratio the greater, obtains three strain bacterium 901-11,901-15,901-18 after purifying;
(4) shake flask fermentation sieves again
By the three strain bacterium 901-11 obtained, 901-15,901-18 carry out shake flask fermentation in the 250mL shaking flask containing 30mL potato liquid nutrient medium, seed inoculum size 10% (V/V), 30 DEG C, 100r/min cultivates 72h, centrifuging and taking fermented supernatant fluid obtains crude enzyme liquid.
Described lithium chloride is dull and stereotyped: starch 1%, peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%.
Embodiment 2: the mensuration of enzymic activity
(1) definition of Mei Huo unit: 1mL crude enzyme liquid, in 65 DEG C, under pH5.5 condition, 1min liquefies 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with U/mL.
(2) adopt DNS method to measure the amylase activity of 901-11,901-15,901-18, learn, 901-18 has stable most enzymatic activity high.
Embodiment 3: the mensuration of zymologic property
(1) impact of temperature on enzymic activity and the thermostability of enzyme
Same reaction system, measures this amylase enzyme activity respectively at 40-80 DEG C, and result shows that this enzyme all shows high enzyme activity between 50-75 DEG C, and thermal adaptation a wider range of this enzyme is described.And thermally-stabilised experiment shows, this enzyme, after 70 DEG C of insulation 3h, in 65 DEG C, still has more than 80% enzyme alive fixed under pH5.5 condition, temperature is higher than 75 DEG C, and enzyme loss alive is serious.
(2) pH is on the impact of enzymic activity
Same reaction system, surveys the work of this enzyme enzyme respectively under pH3-8.5, and result shows that this enzyme all has the enzyme work of more than 70% when pH4.5-7, and reaches maximum value during pH5.5.
Embodiment 4: a kind of alternating temperature regulates the method for producing Thermostable α-Amylase, comprises the steps:
(1) actication of culture
The slant strains of intact Trichodermareesei 901-18 is inoculated in potato slant medium, cultivates 48h for 30 DEG C and carry out actication of culture, so activation 3 times;
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: the spore suspension that the slant strains after step (1) being activated is made accesses in 500 ml shake flasks, substratum loading amount 100 milliliters, and after inoculation, spore concentration reaches 10
5individual/mL, shaking table 120r/min, culture temperature 30 DEG C, incubation time 10h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100r/min, culture temperature 30 DEG C, incubation time 10-15h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, stirring velocity 150r/min, ventilation (V/V) 1:1.5, tank pressure 0.05Mpa, incubation time 15h;
Institute's one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.4%, glucose 1.2%, peptone 0.4%, extractum carnis 0.6%, dipotassium hydrogen phosphate 1.1%, trehalose 2%, calcium sulfate 1g, magnesium chloride 2g, Trisodium Citrate 2g, insufficient section pure water is supplied, pH value 5.5,123 DEG C of sterilizing 40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 10%, yeast powder 0.6%, trehalose 2%, peptone 0.3%, corn steep liquor 0.3%, dipotassium hydrogen phosphate 1.1%, magnesium sulfate 0.08%, Trisodium Citrate 0.3%, insufficient section pure water is supplied, pH value 5.5,123 DEG C of sterilizing 40min.
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 30 DEG C, stirring velocity 150r/min, ventilation (V/V) 1:2, incubation time 12h; Then with 2 DEG C/h rate of temperature fall slow cooling to 12 DEG C, constant temperature culture 18h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 4 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 25h; Finally slowly be warming up to 12 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 30 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 20%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 5.5;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/ml-8mg/ml, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/ml-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 70% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 100g, Semen Maydis powder 55g, soybean cake powder 20g, 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 5.5,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 25%, Semen Maydis powder 15%, bean powder 20%, and insufficient section pure water is supplied, pH value 5.5,123 DEG C of sterilizing 40min.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant fungal alpha-amylase.
The fungal alpha-amylase liquid enzymes vigor obtained through above-mentioned preparation method is 15000u/ml.
Claims (3)
1. the Trichodermareesei of a strain high-yield thermostable fungal alpha-amylase, it is characterized in that, described Trichodermareesei is specially Trichodermareesei 901-18Trichodermareesei901-18, and deposit number is CCTCCNO:M2013602.
2. the Trichodermareesei of a strain high-yield thermostable fungal alpha-amylase as claimed in claim 1, it is characterized in that, described Trichodermareesei can produce high temperature resistant fungal alpha-amylase.
3. the Trichodermareesei of a strain high-yield thermostable fungal alpha-amylase as claimed in claim 1, it is characterized in that, the method that described Trichodermareesei produces high temperature resistant fungal alpha-amylase is:
Bacterial strain 901-18 is entered the ferment tank stage after three grades of seed fermentations and first class seed pot fermentation;
By the first class seed pot fermented liquid of fermented bacterium with 6% inoculum size access fermentor tank, culture temperature 27-30 DEG C, stirring velocity 120-180r/m, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, continue first class seed pot fermented liquid to add access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 27-30 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/ml-8mg/ml, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/ml-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly;
Fermentation liquor is filtered, concentrated, allotment, essence filter, dry high temperature resistant fungal alpha-amylase.
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Cited By (2)
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| CN106434383A (en) * | 2016-11-01 | 2017-02-22 | 青岛蔚蓝生物集团有限公司 | Aspergillus oryza strain capable of highly producing fungal amylase |
| CN108823185A (en) * | 2018-06-25 | 2018-11-16 | 安徽新熙盟生物科技有限公司 | The cultural method of high enzyme activity fermentation liquid and the method for extracting acidproof alpha-amylase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434383A (en) * | 2016-11-01 | 2017-02-22 | 青岛蔚蓝生物集团有限公司 | Aspergillus oryza strain capable of highly producing fungal amylase |
| CN108823185A (en) * | 2018-06-25 | 2018-11-16 | 安徽新熙盟生物科技有限公司 | The cultural method of high enzyme activity fermentation liquid and the method for extracting acidproof alpha-amylase |
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Application publication date: 20160427 |