CN101805727A - Preparation method of protease with wide adaptability - Google Patents
Preparation method of protease with wide adaptability Download PDFInfo
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- CN101805727A CN101805727A CN 201010180442 CN201010180442A CN101805727A CN 101805727 A CN101805727 A CN 101805727A CN 201010180442 CN201010180442 CN 201010180442 CN 201010180442 A CN201010180442 A CN 201010180442A CN 101805727 A CN101805727 A CN 101805727A
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Abstract
The invention relates to a preparation method of protease with wide adaptability, which comprises the following steps that: (1) Bacillus subtilis strain for producing the protease is rejuvenated and cultured to obtain the Bacillus subtilis rejuvenation strain; (2) Bacillus subtilis seed is cultured to obtain Bacillus subtilis seed culture liquid; (3) the Bacillus subtilis seed culture liquid is undertaken the liquid submerged fermentation culture to obtain the Bacillus subtilis liquid submerged culture liquid; (4) diatomaceous earth framework is used for compressing and filtering the Bacillus subtilis liquid submerged culture liquid to obtain prefiltration liquid; (5) the prefiltration liquid is concentrated to obtain concentration liquid; (6) the concentration liquid is frozen and dried to obtain the protease. The enzyme activity of the protease which is produced with the method reaches 250000 to 280000u/g. The protease is suitable for being used under the condition of pH 6 to pH 10.
Description
Technical field
The present invention relates to a kind of preparation method of proteolytic enzyme, especially relate to a kind of preparation method of protease with wide adaptability.
Background technology
Proteolytic enzyme is the general name of the various enzymes of catalysis peptide bond hydrolysis, extensively derives from microorganism, plant stem-leaf fruit, pluck etc.Typical case's representative of studying proteolytic enzyme the earliest is people such as German R hm in 1908, U.S. Hua Lesitan company used as the clarify beer agent with papoid first in 1911, since the thirties in 20th century, extract the main source that proteolytic enzyme becomes protease preparation by microbial engineering, at present, whole world zymin ultimate production is more than 2,000 ten thousand tons, and wherein about 20% is proteolytic enzyme.Made crystallization or highly purified proteolytic enzyme has kind more than 200, its primary structure even three-dimensional tertiary structure are also illustrated.
The proteolytic enzyme purposes is very extensive, and the application in fields such as fodder industry, foodstuffs industry, textile industry, paper industries has produced very remarkable economic efficiency and social ecological benefits.China's zymin output only accounts for about 2% of Gross World Product at present, mainly is enzymes such as saccharifying enzyme, amylase, Sumizyme MP, and domestic Sumizyme MP has reached the annual production about 1.4 ten thousand tons now.According to proteinase activity pH value optimum range, albumen divides and can be divided three classes: aspartic protease, and optimum pH is generally 4-5; Neutral protease, optimum pH is generally 6-7; Sumizyme MP, optimum pH is generally 8-10.Every proteinoid enzyme is the effect of its catalysis peptide bond hydrolysis of competence exertion in its suitable potential of hydrogen (i.e. Shi Yi pH scope) environment only, and in the production application, often can not have only a kind of potential of hydrogen environment.
Therefore, regardless of the proteolytic enzyme of producing in which kind of mode, its major defect is at present: it is narrow 1, to use the pH optimum range, can only use in certain series products; 2, production cost is higher relatively.
Summary of the invention
The objective of the invention is to overcome the above-mentioned defective that existing proteolytic enzyme preparation method exists, a kind of preparation method of protease with wide adaptability is provided.
Purpose of the present invention is achieved by the following technical programs: it comprises following operation steps:
(1) the Bacillus subtilus rejuvenation of spawn is cultivated: 1) rejuvenation substratum preparation: rejuvenation culture medium prescription: peptone 0.9-1.1wt%, and extractum carnis 0.5-0.7wt%, NaCl0.3-0.5wt%, agar 2wt%, pH8.5-10.0, all the other are water; With described batching mixing, divide to be put in the culture dish, 0.1Mpa120-122 ℃ of sterilization 30-35min, cool to room temperature is made the rejuvenation substratum, and is standby; 2) inoculation with cultivate: the Bacillus subtilus ring of the product proteolytic enzyme that the laboratory is preserved pipettes the 1-2 ring, is inoculated into described rejuvenation substratum, places 36 ℃ ± 1 ℃ constant temperature culture 60-70h, the rejuvenation bacterial classification, take out cryopreservation;
(2) Bacillus subtilus seed culture: 1) seed culture medium preparation: seed culture based formulas: glucose 0.4-0.6wt%, peptone 0.4-0.6wt%, yeast extract paste 0.4-0.6wt%, potassium primary phosphate 0.05-0.08wt%, sal epsom 0.01-0.02wt%, sodium-chlor 0.3-0.5wt%, all the other are water, pH8.5-10.0, with described batching mixing, 0.1MPa120-122 ℃ of sterilization 30-35min is cooled to 39-41 ℃, make seed culture medium, standby; 2) seed culture method: the Bacillus subtilus bacterial classification inoculation of getting rejuvenation, inoculum size is by the 1-2wt% of seed culture basic weight, be inoculated into the seed culture medium that is cooled to 39-41 ℃, 36 ℃ ± 1 ℃ constant temperature culture 60-70 hour, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300-320 rev/min, ventilation be 1.3-1.4 cube of gas/rise substratum/minute, make the Bacillus subtilus seed culture fluid;
(3) liquid submerged fermentation is cultivated: 1) liquid submerged fermentation culture medium prescription: glucose 4.0-6.0wt%, MgS039-41.01-0.02wt%, Na2HPO40.20-0.30wt%, peptone 0.15-0.25wt%, analysis for soybean powder 2.0-3.0wt%, pH8.5-10.0, all the other are water; To prepare burden behind the mixing, 0.1MPa120-122 ℃ of sterilization 30-35min is cooled to 39-41 ℃, makes the liquid submerged fermentation substratum, and be standby; 2) liquid submerged fermentation is cultivated: get the inoculation of Bacillus subtilus seed culture fluid, the inoculum size of seed culture fluid is cultivated the 2-3wt% of basic weight by liquid submerged fermentation, be inoculated into the liquid deep layer substratum that is cooled to 39-41 ℃, 36 ℃ ± 1 ℃ constant temperature culture 60-70 hour, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300-320 rev/min, ventilation be 1.3-1.4 cube of gas/rise substratum/minute, make Bacillus subtilus liquid submerged fermentation nutrient solution;
(4) coarse filtration: add the diatomite that is equivalent to the heavy 3-5% of fermentation culture in Bacillus subtilus submerged fermentation nutrient solution, mix, filter press gets coarse filtration liquid;
(5) the coarse filtration liquid film concentrates: with the 1/4-1/5 of coarse filtration liquid filtering and concentrating to its former weight, make concentrated solution with microfiltration membrane;
(6) lyophilize: concentrated solution is freezing under-30 ℃, vacuumize and be dried to powder.
Described Bacillus subtilus is known common microorganism (referring to " zymin industry " volume two, Zhang Shuzheng chief editor, Science Press's the third printing in 1998,395 pages).
Proteolytic enzyme enzyme activity detection method adopts ultraviolet spectrophotometry (referring to " zymin industry " volume two, Zhang Shuzheng chief editor, Science Press's the third printing in 1998,447 pages).。
By the proteolytic enzyme that the inventive method is produced, enzyme activity can reach 250000-280000u/g.Suit to use under the condition of pH6-10, pH is applied widely.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
(1) the Bacillus subtilus rejuvenation of spawn is cultivated: 1) take by weighing peptone 1.kg, extractum carnis 0.6kg, NaCl0.4kg, agar 2kg, water 96kg, mixing is transferred pH9.0, divides to be put in the culture dish, 0.1MPa121 ℃ sterilization 33min, cool to room temperature is made the rejuvenation substratum, and is standby; 2) the Bacillus subtilus ring of the product proteolytic enzyme that the laboratory is preserved pipettes 2 rings, is inoculated into described rejuvenation substratum, places 36 ℃ ± 1 ℃ constant temperature culture 65h, gets the rejuvenation bacterial classification, takes out cryopreservation;
(2) Bacillus subtilus seed culture: 1) take by weighing glucose 0.5kg, peptone 0.5kg, yeast extract paste 0.5kg, potassium primary phosphate 0.06kg, sal epsom 0.02kg, sodium-chlor 0.4kg, water 98.02kg, mixing, transfer pH9.0,0.1MPa121 ℃ of sterilization 33min is cooled to 40 ℃, make seed culture medium, standby; 2) get the Bacillus subtilus bacterial classification inoculation of rejuvenation, inoculum size is by the 1-2wt% of seed culture basic weight, be inoculated into the seed culture medium that is cooled to 40 ℃, 36 ℃ ± 1 ℃ constant temperature culture 65 hours, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300 rev/mins, ventilation be 1.3 cubes of gases/rise substratum/minute, make the Bacillus subtilus seed culture fluid;
(3) liquid submerged fermentation is cultivated: 1) take by weighing glucose 5kg, MgS039-41.02kg, Na2HPO40.30kg, peptone 0.2kg, analysis for soybean powder 3.0kg, water 91.48kg, mixing is transferred pH9.0,0.1MPa121 ℃ sterilization 35min is cooled to 40 ℃, makes the liquid submerged fermentation substratum, and is standby; 2) get the inoculation of Bacillus subtilus seed culture fluid, the inoculum size of seed culture fluid is cultivated the 2-3wt% of basic weight by liquid submerged fermentation, be inoculated into the liquid deep layer substratum that is cooled to 40 ℃, 36 ℃ ± 1 ℃ constant temperature culture 65 hours, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300 rev/mins, ventilation be 1.3 cubes of gases/rise substratum/minute, make Bacillus subtilus liquid submerged fermentation nutrient solution;
(4) coarse filtration: add the diatomite that is equivalent to the heavy 3-5% of nutrient solution in Bacillus subtilus submerged fermentation nutrient solution, mix, filter press gets coarse filtration liquid;
(5) the coarse filtration liquid film concentrates: with microfiltration membrane with coarse filtration liquid filtering and concentrating to 1/4 of the former weight of coarse filtration liquid, make concentrated solution;
(6) lyophilize: step (5) gained concentrated solution is freezing under-30 ℃, vacuumize and be dried to powder.
The proteolytic enzyme that present embodiment is produced records enzyme activity and reaches 265000u/g under the pH6 condition; Under the pH10 condition, record enzyme activity and reach 276000u/g.
Embodiment 2
(1) the Bacillus subtilus rejuvenation of spawn is cultivated: 1) take by weighing peptone 1.1kg, extractum carnis 0.5kg, NaCl0.5kg, agar 2kg, water 95.9kg, mixing, transferring pH is 10.0, divides to be put in the culture dish, 0.1MPa121 ℃ sterilization 35min, cool to room temperature is made the rejuvenation substratum, and is standby; 2) the Bacillus subtilus ring of the product proteolytic enzyme that the laboratory is preserved pipettes 1 ring, is inoculated into described rejuvenation substratum, places 36 ℃ ± 1 ℃ constant temperature culture 70h, gets the rejuvenation bacterial classification, takes out cryopreservation, and is standby;
(2) Bacillus subtilus seed culture: 1) take by weighing glucose 0.4kg, peptone 0.6kg, yeast extract paste 0.4kg, potassium primary phosphate 0.06kg, sal epsom 0.01kg, sodium-chlor 0.5kg, water 98.03kg, mixing, transferring pH is 10.0, and 0.1MPa121 ℃ of sterilization 30-35min is cooled to 40 ℃, make seed culture medium, standby; 2) seed culture method: the Bacillus subtilus bacterial classification inoculation of getting rejuvenation, inoculum size is by the 1-2wt% of seed culture basic weight, be inoculated into the seed culture medium that is cooled to 40 ℃, 36 ℃ ± 1 ℃ constant temperature culture 60-70 hour, during constant temperature culture, constantly stir and ventilate, stirring velocity is 320 rev/mins, ventilation be 1.4 cubes of gases/rise substratum/minute, make the Bacillus subtilus seed culture fluid, standby;
(3) liquid submerged fermentation is cultivated: 1) take by weighing glucose 6.0kg, MgS040.02kg, Na2HPO40.30kg, peptone 0.25kg, analysis for soybean powder 3.0kg, water 90.43kg, mixing, transferring pH is 10.0,0.1MPa121 ℃ sterilization 35min, be cooled to 40 ℃, make the liquid submerged fermentation substratum, standby; 2) get the inoculation of Bacillus subtilus seed culture fluid, the inoculum size of seed culture fluid is cultivated the 3wt% of basic weight by liquid submerged fermentation, be inoculated into the liquid submerged fermentation substratum that is cooled to 40 ℃, 36 ℃ ± 1 ℃ constant temperature culture 70 hours, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300 rev/mins, ventilation be 1.4 cubes of gases/rise substratum/minute, make Bacillus subtilus liquid submerged fermentation nutrient solution;
(4) coarse filtration: add in the Bacillus subtilus submerged fermentation nutrient solution and be equivalent to nutrient solution and weigh 5% diatomite, mix, filter press, coarse filtration liquid;
(5) the coarse filtration liquid film concentrates: with 1/5 of its former weight of coarse filtration liquid filtering and concentrating, make concentrated solution with microfiltration membrane;
(6) lyophilize: concentrated solution is freezing under-30 ℃, vacuumize and be dried to powder.
The proteolytic enzyme that present embodiment is produced records enzyme activity and reaches 263000u/g under the pH6 condition; Under the pH10 condition, record enzyme activity and reach 280000u/g.
Claims (1)
1. a preparation method of protease with wide adaptability is characterized in that, comprises the steps:
(1) the Bacillus subtilus rejuvenation of spawn is cultivated: 1) rejuvenation substratum preparation: rejuvenation culture medium prescription: peptone 0.9-1.1wt%, and extractum carnis 0.5-0.7wt%, NaCl0.3-0.5wt%, agar 2wt%, pH8.5-10.0, all the other are water; With described batching mixing, divide to be put in the culture dish, 0.1Mpa120-122 ℃ of sterilization 30-35min, cool to room temperature is made the rejuvenation substratum, and is standby; 2) inoculation with cultivate: the Bacillus subtilus ring of the product proteolytic enzyme that the laboratory is preserved pipettes the 1-2 ring, is inoculated into described rejuvenation substratum, places 36 ℃ ± 1 ℃ constant temperature culture 60-70h, the rejuvenation bacterial classification, take out cryopreservation;
(2) Bacillus subtilus seed culture: 1) seed culture medium preparation: seed culture based formulas: glucose 0.4-0.6wt%, peptone 0.4-0.6wt%, yeast extract paste 0.4-0.6wt%, potassium primary phosphate 0.05-0.08wt%, sal epsom 0.01-0.02wt%, sodium-chlor 0.3-0.5wt%, all the other are water, pH8.5-10.0, with described batching mixing, 0.1MPa120-122 ℃ of sterilization 30-35min is cooled to 39-41 ℃, make seed culture medium, standby; 2) seed culture method: the Bacillus subtilus bacterial classification inoculation of getting rejuvenation, inoculum size is by the 1-2wt% of seed culture basic weight, be inoculated into the seed culture medium that is cooled to 39-41 ℃, 36 ℃ ± 1 ℃ constant temperature culture 60-70 hour, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300-320 rev/min, ventilation be 1.3-1.4 cube of gas/rise substratum/minute, make the Bacillus subtilus seed culture fluid;
(3) liquid submerged fermentation is cultivated: 1) liquid submerged fermentation culture medium prescription: glucose 4.0-6.0wt%, MgS040.01-0.02wt%, Na2HPO40.20-0.30wt%, peptone 0.15-0.25wt%, analysis for soybean powder 2.0-3.0wt%, pH8.5-10.0, all the other are water; With described batching mixing, 0.1MPa120-122 ℃ of sterilization 30-35min is cooled to 39-41 ℃, makes the liquid submerged fermentation substratum, and be standby; (2) liquid submerged fermentation is cultivated: get the inoculation of Bacillus subtilus seed culture fluid, the inoculum size of seed culture fluid is cultivated the 2-3wt% of basic weight by liquid submerged fermentation, be inoculated into the liquid deep layer substratum that is cooled to 39-41 ℃, 36 ℃ ± 1 ℃ constant temperature culture 60-70 hour, during constant temperature culture, constantly stir and ventilate, stirring velocity is 300-320 rev/min, ventilation be 1.3-1.4 cube of gas/rise substratum/minute, make Bacillus subtilus liquid submerged fermentation nutrient solution;
(4) coarse filtration: add the diatomite that is equivalent to the heavy 3-5% of fermentation culture in Bacillus subtilus submerged fermentation nutrient solution, mix, filter press gets coarse filtration liquid;
(5) the coarse filtration liquid film concentrates: with the 1/4-1/5 of coarse filtration liquid filtering and concentrating to its former weight, make concentrated solution with microfiltration membrane;
(6) lyophilize: concentrated solution is freezing under-30 ℃, vacuumize and be dried to powder.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
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| CN1202245C (en) * | 2003-08-29 | 2005-05-18 | 天津科技大学 | Method for producing highly basic alkali protease |
| CN101368177A (en) * | 2008-10-13 | 2009-02-18 | 西北农林科技大学 | Process for preparing bacillus subtilis alkali proteinase with microorganism zymotechnics |
| WO2009132575A1 (en) * | 2008-04-28 | 2009-11-05 | 赖文育 | Nattokinase powders with super high activity and producing method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202245C (en) * | 2003-08-29 | 2005-05-18 | 天津科技大学 | Method for producing highly basic alkali protease |
| WO2009132575A1 (en) * | 2008-04-28 | 2009-11-05 | 赖文育 | Nattokinase powders with super high activity and producing method thereof |
| CN101368177A (en) * | 2008-10-13 | 2009-02-18 | 西北农林科技大学 | Process for preparing bacillus subtilis alkali proteinase with microorganism zymotechnics |
Non-Patent Citations (3)
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| 《中国调味品》 20081031 李秀凉等 枯草芽孢杆菌HD132产生的alpha－淀粉酶性质的初步研究 第45-55页 1 , 第10期 2 * |
| 《现代食品科技》 20081231 吴晖等 发酵条件对枯草芽孢杆菌发酵豆粕中的蛋白酶活力的影响 第973-976页 1 第24卷, 第10期 2 * |
| 《辽宁化工》 20041231 丁琳 枯草杆菌碱性磷酸酶制备的最佳工艺条件及酶促反应动力学性质的研究 第691-710页 1 第33卷, 第12期 2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
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