CN105524900A - High-temperature-resistant fungal alpha-amylase - Google Patents
High-temperature-resistant fungal alpha-amylase Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant fungal alpha-amylase and belongs to the field of enzymic preparations. The high-temperature-resistant fungal alpha-amylase is prepared from Trichoderma reesei 901-18 which is preserved with the preservation number of CCTCC No: M 2013602 in China Center for Type Culture Collection on November 24, 2013. The high-temperature-resistant fungal alpha-amylase has the advantages that the temperature application range is wide and is between 50 DEG C and 75 DEG C, an optimum temperature is 65 DEG C, and the high-temperature-resistant fungal alpha-amylase has more than 80% of enzyme activity when being stored at the temperature of 70 DEG C, thereby being high in heat stability; an optimum pH value of the high-temperature-resistant fungal alpha-amylase is 5.5, and the high-temperature-resistant fungal alpha-amylase has high enzyme activity when the pH value is between 4.5 and 7.0; the enzyme activity of the high-temperature-resistant fungal alpha-amylase is 12000-15000U/ml which is increased by 3.6 times as compared with that of an original strain.
Description
Technical field
The invention belongs to enzymic preparation field, particularly a kind of high temperature resistant 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
The object of this invention is to provide a kind of resistant to elevated temperatures fungal alpha-amylase.
Described resistant to elevated temperatures fungal alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, between 50-75 DEG C, optimum temperature, at 65 DEG C, is preserved 3h and is still had more than 80% enzyme work, have good thermostability at 70 DEG C.
(2) this enzyme optimal reaction pH value is 5.5, and living at pH value 4.5-7.0 enzyme remains on more than 70%.
(3) enzymic activity: by mutant strain 901-18 provided by the present invention, the high temperature resistant fungal alpha-amylase enzyme activity of preparation is 12000-15000U/ml, compares the raising 3.6 times alive of starting strain enzyme.
The preparation method of described high temperature resistant fungal alpha-amylase, comprises the steps:
(1) actication of culture
The PDA slant strains of intact Trichodermareesei is inoculated in slant medium, cultivates 36-48h for 27-30 DEG C and carry out actication of culture, so activation 2-3 time;
(2) liquid seeds enlarged culturing
By the bacterial classification after activation through three grades of shake flask fermentations and and seeding tank fermentation.
Institute's one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, extractum carnis 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, trehalose 1-3%, calcium sulfate 1g, magnesium chloride 2g, Trisodium Citrate 1-3g, insufficient section pure water is supplied, pH value 5-6.5,121-123 DEG C of sterilizing 30-40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 5-15%, yeast powder 0.4-0.8%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, Trisodium Citrate 0.1-0.5%, insufficient section pure water is supplied, pH value 5-6.5,121-123 DEG C of sterilizing 30-40min.
(3) ferment tank
By first class seed pot fermented liquid with 6% inoculum size access fermentor tank, culture temperature 27-30 DEG C, stirring velocity 120-180r/min, 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 high temperature resistant fungal alpha-amylase.
Described high temperature resistant fungal alpha-amylase is by Trichodermareesei 901-18Trichodermareesei901-18.This bacterial strain on November 24th, 2013 be preserved in middle China typical culture collection center (be called for short CCTCC, address is: China. Wuhan. Wuhan University), preserving number is CCTCCNO:M2013602.
Described Trichodermareesei 901-18 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, described bacterial strain feature is as follows: this bacterial strain is at PDA cultured on solid medium, the colony characteristics formed is bacterium colony is flocculence, bacterium colony is light green, 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.This bacterial strain produces the optimal pH 3.0-6.5 of fungal alpha-amylase; Optimum temperuture is 27 ~ 30 DEG C.
Beneficial effect:
1, the method for the high temperature resistant fungal alpha-amylase of production of the present invention is with Trichodermareesei 901-18 for bacterial classification, and this bacterial classification is first public.
2, the high temperature resistant fungal alpha-amylase adopting the method for the invention to produce has following features: enzyme activity is up to 12000-15000u/ml; Enzyme thermal adaptation a wider range, between 50-75 DEG C, optimum temperature, at 65 DEG C, is preserved 3h and is still had more than 80% enzyme work, have good thermostability and preserve active at 70 DEG C; 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%.Higher than existing high temperature resistant fungal alpha-amylase enzyme activity, enzyme effect optimum pH wide scope, 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.
3, the high temperature resistant fungal alpha-amylase in the present invention in preparation process due to substratum implement full optimization, the zymotechnique of gradient cooling and gradient increased temperature is adopted to significantly improve the anti-stress ability of Trichodermareesei 901-18, the enzymatic productivity of bacterial classification is caused to manifest to greatest extent, the present invention is produced, and high temperature resistant fungal alpha-amylase enzyme activity is high, action condition is wide in range, stability is strong, be suitable for suitability for industrialized production, has better stability and preserves active.
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: 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 2: 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 has the enzymic activity of more than 82% 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, under pH3-8.5, survey this enzyme enzyme respectively live, result shows that this enzyme enzymic activity when pH4.5-7 remains on more than 70%, and reaches maximum value during pH5.5.
Embodiment 3:
Alternating temperature regulates a method of producing high temperature resistant fungal alpha-amylase, comprises the steps:
(1) actication of culture
The PDA slant strains of intact Trichodermareesei 901-18 is inoculated in slant medium, cultivates 24h for 27 DEG C and carry out actication of culture, so activation 2 times;
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: step (1) is activated the spore suspension made and accesses in 500 ml shake flasks, make spore concentration reach 10
5individual/mL, substratum loading amount 100 milliliters, rotary shaker 120r/min, culture temperature 27 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 27 DEG C, incubation time 10h;
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 27 DEG C, stirring velocity 200rpm, ventilation (V/V) 1:1, tank pressure 0.05Mpa, incubation time 10h;
Institute's one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.3%, glucose 1%, peptone 0.3%, extractum carnis 0.5%, dipotassium hydrogen phosphate 0.8%, trehalose 1%, calcium sulfate 1g, magnesium chloride 2g, Trisodium Citrate 1g, insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 5%, yeast powder 0.4%, trehalose 1%, peptone 0.1%, corn steep liquor 0.1%, dipotassium hydrogen phosphate 0.8%, magnesium sulfate 0.05%, Trisodium Citrate 0.1%, insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 27 DEG C, stirring velocity 120r/m, ventilation (V/V) 1:1, incubation time 10h; Then with 1 DEG C/h rate of temperature fall slow cooling to 10 DEG C, constant temperature culture 15h; Continue with 1 DEG C/h rate of temperature fall slow cooling to 2 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 20h; Finally slowly be warming up to 10 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate; Continue slowly to be warming up to 27 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 15%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 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 60% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 50g, Semen Maydis powder 50g, soybean cake powder 15g, trehalose 30g, yeast powder 4g, corn steep liquor 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium primary phosphate 1g, Trisodium Citrate 1g, defoamer 0.1g, pure water l000mL, pH value 5,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20%, Semen Maydis powder 10%, bean powder 15%, and insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant fungal alpha-amylase.
The high temperature resistant fungal alpha-amylase liquid enzymes vigor obtained through above-mentioned preparation method is 12000u/ml.
Embodiment 4:
Alternating temperature regulates a method of producing high temperature resistant fungal alpha-amylase, comprises the steps:
(1) actication of culture
The PDA slant strains of intact Trichodermareesei 901-18 is inoculated in slant medium, cultivates 30h 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: step (1) is activated the spore suspension made and accesses in 500 ml shake flasks, make spore concentration reach 10
5individual/mL, substratum loading amount 100 milliliters, rotary shaker 150r/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 150r/min, culture temperature 30 DEG C, incubation time 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 high temperature resistant fungal alpha-amylase liquid enzymes vigor obtained through above-mentioned preparation method is 15000u/ml.
Embodiment 5:
Alternating temperature regulates a method of producing high temperature resistant fungal alpha-amylase, comprises the steps:
(1) actication of culture
The slant strains of intact Trichodermareesei 901-18 is inoculated in PDA slant medium, cultivates 36h for 28 DEG C and carry out actication of culture, so activation 3 times;
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: step (1) is activated the spore suspension made and accesses in 500 ml shake flasks, make spore concentration reach 10
5individual/mL, substratum loading amount 100 milliliters, rotary shaker 180r/min, culture temperature 28 DEG C, incubation time 15h;
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 180r/min, culture temperature 28 DEG C, incubation time 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 28 DEG C, stirring velocity 180r/min, ventilation (V/V) 1:2, tank pressure 0.05Mpa, incubation time 20h;
Institute's one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.5%, glucose 1.5%, peptone 0.5%, extractum carnis 0.8%, dipotassium hydrogen phosphate 1.5%, trehalose 3%, calcium sulfate 1g, magnesium chloride 2g, Trisodium Citrate 3g, insufficient section pure water is supplied, pH value 6.5,123 DEG C of sterilizing 40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 15%, yeast powder 0.8%, trehalose 3%, peptone 0.5%, corn steep liquor 0.5%, dipotassium hydrogen phosphate 1.5%, magnesium sulfate 0.1%, Trisodium Citrate 0.5%, insufficient section pure water is supplied, pH value 6.5,123 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 28 DEG C, stirring velocity 180r/min, ventilation (V/V) 1:3, incubation time 15h; Then with 2 DEG C/h rate of temperature fall slow cooling to 15 DEG C, constant temperature culture 20h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 5 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 30h; Finally slowly be warming up to 15 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 28 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 6.5;
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 80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 150g, Semen Maydis powder 60g, soybean cake powder 25g, trehalose 40g, yeast powder 8g, corn steep liquor 5g, ammonium sulfate 3g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 5g, defoamer 1g, pure water l000mL, pH value 6.5,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 30%, Semen Maydis powder 20%, bean powder 25%, and insufficient section pure water is supplied, pH value 6.5,123 DEG C of sterilizing 40min.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant fungal alpha-amylase.
The high temperature resistant fungal alpha-amylase liquid enzymes vigor obtained through above-mentioned preparation method is 13000u/ml.
Embodiment 6: temperature-variable fermentation preserves active impact to enzyme
The high temperature resistant fungal alpha-amylase obtained with the preparation method described in embodiment 4 is for sample 1, the high temperature resistant fungal alpha-amylase obtained with the preparation method being changed into 27 DEG C of ferment at constant temperature the ferment tank stage in embodiment 4 is for sample 2, and the preservation of comparing two kinds of samples is active.At pH5.5, preserve above-mentioned sample 18h under 35 DEG C of conditions, measure enzyme according to the method described in embodiment 1 afterwards and live, result shows: the remnant enzyme activity of sample 1 is more than 75%, and the remnant enzyme activity of sample 2 remains on about 60%.
Claims (6)
1. a high temperature resistant fungal alpha-amylase, is characterized in that, described high temperature resistant fungal alpha-amylase has following characteristic:
(1) this enzyme thermal adaptation a wider range, between 50-75 DEG C, optimum temperature, at 65 DEG C, is preserved 3h and is 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) enzymic activity: enzyme activity is 12000-15000U/ml, compares the raising 3.6 times alive of starting strain enzyme.
2. a kind of high temperature resistant fungal alpha-amylase as claimed in claim 1, is characterized in that, the production bacterial strain of described high temperature resistant fungal alpha-amylase is specially Trichodermareesei 901-18Trichodermareesei901-18, and preserving number is CCTCCNO:M2013602.
3. a kind of high temperature resistant fungal alpha-amylase as claimed in claim 1 or 2, is characterized in that, the preparation method of described high temperature resistant fungal alpha-amylase, comprises the steps:
Production bacterial strain 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.
4. the preparation method of a kind of high temperature resistant fungal alpha-amylase as claimed in claim 3, is characterized in that, described one-level, secondary, three grades of seed culture medium weight consist of: yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, extractum carnis 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, trehalose 1-3%, calcium sulfate 1g, magnesium chloride 2g, Trisodium Citrate 1-3g, insufficient section pure water is supplied, pH value 5-6.5,121-123 DEG C of sterilizing 30-40min.
5. the preparation method of a kind of high temperature resistant fungal alpha-amylase as claimed in claim 3, is characterized in that, described seed tank culture basic weight amount consists of: maltodextrin 5-15%, yeast powder 0.4-0.8%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, Trisodium Citrate 0.1-0.5%, insufficient section pure water is supplied, pH value 5-6.5,121-123 DEG C of sterilizing 30-40min.
6. the application of a kind of high temperature resistant fungal alpha-amylase as claimed in claim 1 in amylorrhexis industry.
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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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