CN102093992B - Method for producing low-temperature protease by microbial fermentation - Google Patents
Method for producing low-temperature protease by microbial fermentation Download PDFInfo
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- CN102093992B CN102093992B CN 201010162644 CN201010162644A CN102093992B CN 102093992 B CN102093992 B CN 102093992B CN 201010162644 CN201010162644 CN 201010162644 CN 201010162644 A CN201010162644 A CN 201010162644A CN 102093992 B CN102093992 B CN 102093992B
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Abstract
The invention relates to a method for producing low-temperature protease by microbial fermentation, which comprises the following steps: carrying out low-temperature domestication on protease-producing microbes step by step, so that the microbes can well grow in a low temperature environment; carrying out amplification culture on the protease-producing microbes, which are subjected to low-temperature domestication, at 14-20 DEG C step by step; inoculating the protease-producing microbes, which account for 3-9% of a fermentation liquid by volume, into a protease-producing culture medium; culturing at 14-20 DEG C for 48-120 hours, and finishing the production of the low-temperature protease by microbial fermentation; centrifugating the fermentation liquid at 4000-8000 rpm, and collecting the liquid which is a crude enzyme liquid; and according to different demands and different use objects, further concentrating the crude enzyme liquid, and purifying by separation to obtain enzyme preparations with different activities, purities and forms.
Description
Technical field
The present invention relates to the fields such as microbiology, enzyme engineering, fermentation engineering, biological chemistry, be specifically related to a kind of low-temperature protease microbial fermentation production method.The low-temperature protease that this invention is produced is mainly used in other proteolytic enzyme application industries such as feedstuff industry, food-processing, brewage, milk preparation production, cosmetic industry, printing and dyeing, washing, leather manufacture and environment protection.Can improve utilization ratio, transformation efficiency and the productivity of raw material, reduce production costs, improve and improve the quality of products.
Background technology
Proteolytic enzyme is the general name of the class of enzymes of protein hydrolysate peptide bond.Proteolytic enzyme has been widely used in other proteolytic enzyme application industries such as feedstuff industry, food-processing, brewage, milk preparation production, cosmetic industry, printing and dyeing, washing, leather manufacture and environment protection as the important industrial enzymes of a class.The proteolytic enzyme that uses at present generally all is middle gentle high temperature modification proteolytic enzyme (Chen Xiulan etc., industrial microorganism, 2001).Research about low-temperature protease still is in the starting stage, mainly concentrates on aspect (Wang Quanfu etc., Chinese aquatic science, 2005 such as strain improvement, fermentation condition optimization, zymologic property and Cloning of Genes Related; Chi Naiyu etc., microbiology circular, 2006; Zhang Xiu etc., Chinese aquatic science, 2006; Feng Xiuping etc., Chinese biological engineering magazine, 2009); And the industrialization of low-temperature protease, large-scale production and application yet there are no report (Wang Haiting etc., marine fishery research, 2002; Zhao Yuanyuan etc., household chemicals science, 2005).Low-temperature protease is compared with middle gentle high temperature proteolytic enzyme has very large application advantage, lives and high catalytic efficiency because low-temperature protease has high enzyme at low temperatures, can greatly shorten the time for the treatment of processes and save expensive heating or refrigeration costs; Aspect energy-conservation, sizable advantage is arranged; Can make the forfeiture of cold induced proteins enzyme activity through gentle thermal treatment, and low temperature or thermophilic can not affect the quality of product, this will help the promotion and application of low-temperature protease.Utilize the low-temperature protease of psychrophile fermentative production, in application, can save heating and cooling apparatus and technique, improved production efficiency, reduced production cost; Can improve and improve the quality of product and local flavor (grandson is quiet etc., marine fishery research, 2002) in Applications in Food Industry.The cold induced proteins application of enzymes will be to traditional grain processing, food, brewage, fermentation, textiles, medicine and other fields produce far-reaching influence, also will provide actual opportunity for the mankind break away from the predicaments such as resource, energy dilemma and environmental pollution that faces in the survival and development.Low-temperature protease has boundless potentiality to be exploited and application prospect.
Summary of the invention
The method that the purpose of this invention is to provide a kind of producing low-temperature protease by microbial fermentation, the method mainly is that microorganism is through after the domestication by low temperature, produce the method for low-temperature protease at 14～20 ℃ of liquid fermentings, the low-temperature protease crude enzyme liquid activity that this production method obtains can reach 240U/ml, through separating and purifying, can obtain the zymin of different concns and purity as again.This zymin application operating is simple, convenient, fast, cost is low.Can fundamentally avoid heating, cooling apparatus and the technique of middle temperature, high temperature enzyme.
The method of a kind of producing low-temperature protease by microbial fermentation of the present invention specifically may further comprise the steps:
The microorganism that (1) will produce proteolytic enzyme is domestication by low temperature step by step, and acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃, makes its well-grown in low temperature environment from high to low;
(2) according to a conventional method with the protease-producing bacterium after the domestication by low temperature 14～20 ℃ of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed;
(3) with liquid first order seed or secondary seed, press in 3～9% inoculum sizes access culture medium of fermentating liquid volume, when cultivating 48～120h for 14～20 ℃, namely producing low-temperature protease by microbial fermentation finishes;
(4) with the fermented liquid of (3) 4,000～8, the centrifugal collection liquid of 000rpm; The liquid of collecting is crude enzyme liquid;
(5) different with the use object according to different needs, with further concentrated, the separation and purification of crude enzyme liquid that (4) obtain, be prepared into the zymin of different activities, purity and formulation.
The bacterial classification that uses among the present invention derives from China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and initial stage activation and growth conditions are undertaken by the explanation that culture presevation unit provides.Protease-producing bacterium (CGMCC bacterium numbering: 1.504 or 1.775 or 1.2172 etc.), bacterial strain activates first, again through domestication by low temperature step by step, cultivate by the inventive method during the fermentative production low-temperature protease, bacterial strain after the domestication by low temperature can be preserved 2 months in 4 ℃ of environment, bacteria suspension for a long time preservation under subzero 80 ℃ of conditions made from 25～30% glycerine.
Embodiment:
Embodiment one:
(1), substratum preparation
1. strain activation and culture base: extractum carnis 0.3%, peptone 1.0%, NaCl 0.5%, casein food grade 2%, agar 2%, tap water 1000ml, pH7.4,0.1Mpa, 121 ℃ of sterilization 20min.
2. bacterial classification domestication by low temperature substratum: extractum carnis 0.1%～0.5%, peptone 0.7%～1.2%, NaCl 0.4%～0.6%, agar 2%, casein food grade 1%～4%, tap water 1000ml, pH 7.4,0.1Mpa, 121 ℃ of sterilization 20min.
3. liquid seed culture medium: glucose 0.8%～1.2%, peptone 0.3%～0.7%, yeast extract paste 0.3%～0.8%, K
2HPO
40.08%～0.12%, MgSO
40.01%～0.04%, Na
2CO
30.8%～1.2%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min.
4. culture medium: glucose 0.9%～1.3%, peptone 0.2%～0.6%, yeast extract paste 0.3%～0.7%, K
2HPO
40.07%～0.13%, MgSO
40.01%～0.03%, Na
2CO
30.8%～1.3%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min.
(2) will produce the bacterial classification of proteolytic enzyme, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) (2) are activated step by step domestication by low temperature of good bacterial classification, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃, makes its well-grown in low temperature environment from high to low;
(4) according to a conventional method the protease-producing bacterium after the domestication by low temperature is cultivated 24～48h at 14～20 ℃, the inoculum size by 4% is carried out step by step enlarged culturing, is prepared into liquid first order seed and secondary seed;
The inoculum size of (5) secondary seed of (4) preparation being pressed fermentating liquid volume 3～5% accesses in the culture medium of 10L, cultivates 96～120h at 14～16 ℃, and namely producing low-temperature protease by microbial fermentation finishes;
(6) with the fermented liquid of (5) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(7) different with the use object according to different needs, with further concentrated, the separation and purification of crude enzyme liquid that (6) obtain, be prepared into the cold induced proteins zymin of different activities, purity and formulation.
Embodiment two:
(1), substratum preparation
1. strain activation and culture base: extractum carnis 0.3%, peptone 1.0%, NaCl 0.5%, casein food grade 2%, agar 2%, tap water 1000ml, pH7.4,0.1Mpa, 121 ℃ of sterilization 20min.
2. bacterial classification domestication by low temperature substratum: extractum carnis 0.1%～0.5%, peptone 0.7%～1.2%, NaCl 0.4%～0.6%, agar 2%, casein food grade 1%～4%, tap water 1000ml, pH 7.4,0.1Mpa, 121 ℃ of sterilization 20min.
3. liquid seed culture medium: glucose 0.8%～1.2%, peptone 0.3%～0.7%, yeast extract paste 0.3%～0.8%, K
2HPO
40.08%～0.12%, MgSO
40.01%～0.04%, Na
2CO
30.8%～1.2%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min.
4. culture medium: glucose 0.9%～1.3%, peptone 0.2%～0.6%, yeast extract paste 0.3%～0.7%, K
2HPO
40.07%～0.13%, MgSO
40.01%～0.03%, Na
2CO
30.8%～1.3%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min.
(2) will produce the bacterial classification of proteolytic enzyme, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) (2) are activated step by step domestication by low temperature of good bacterial classification, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃, makes its well-grown in low temperature environment from high to low;
(4) according to a conventional method the protease-producing bacterium after the domestication by low temperature is cultivated 24～48h at 14～20 ℃, the inoculum size by 5% is carried out step by step enlarged culturing, is prepared into liquid first order seed and secondary seed;
The inoculum size of (5) secondary seed of (4) preparation being pressed fermentating liquid volume 5～7% accesses in the culture medium of 50L, cultivates 72～96h at 16～18 ℃, and namely producing low-temperature protease by microbial fermentation finishes;
(6) with the fermented liquid of (5) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(7) different with the use object according to different needs, with further concentrated, the separation and purification of crude enzyme liquid that (6) obtain, be prepared into the cold induced proteins zymin of different activities, purity and formulation.
Embodiment three:
(1), substratum preparation
1. strain activation and culture base: extractum carnis 0.3%, peptone 1.0%, NaCl 0.5%, casein food grade 2%, agar 2%, tap water 1000ml, pH7.4,0.1Mpa, 121 ℃ of sterilization 20min.
2. bacterial classification domestication by low temperature substratum: extractum carnis 0.1%～0.5%, peptone 0.7%～1.2%, NaCl 0.4%～0.6%, agar 2%, casein food grade 1%～4%, tap water 1000ml, pH 7.4,0.1Mpa, 121 ℃ of sterilization 20min.
3. liquid seed culture medium: glucose 0.8%～1.2%, peptone 0.3%～0.7%, yeast extract paste 0.3%～0.8%, K
2HPO
40.08%～0.12%, MgSO
40.01%～0.04%, Na
2CO
30.8%～1.2%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min.
4. culture medium: glucose 0.9%～1.3%, peptone 0.2%～0.6%, yeast extract paste 0.3%～0.7%, K
2HPO
40.07%～0.13%, MgSO
40.01%～0.03%, Na
2CO
30.8%～1.3%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min.
(2) will produce the bacterial classification of proteolytic enzyme, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) (2) are activated step by step domestication by low temperature of good bacterial classification, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃, makes its well-grown in low temperature environment from high to low;
(4) according to a conventional method the protease-producing bacterium after the domestication by low temperature is cultivated 24～48h at 14～20 ℃, the inoculum size by 5% is carried out step by step enlarged culturing, is prepared into liquid first order seed and secondary seed;
The inoculum size of (5) secondary seed of (4) preparation being pressed fermentating liquid volume 7～9% accesses in the culture medium of 100L, cultivates 48～72h at 18～20 ℃, and namely producing low-temperature protease by microbial fermentation finishes;
(6) with the fermented liquid of (5) 4,000～8, the centrifugal collection liquid of 000rpm, the liquid of collecting is crude enzyme liquid;
(7) different with the use object according to different needs, with further concentrated, the separation and purification of crude enzyme liquid that (6) obtain, be prepared into the cold induced proteins zymin of different activities, purity and formulation.
Claims (1)
1. the method for a producing low-temperature protease by microbial fermentation, it may further comprise the steps:
(1) bacterial classification derives from China Committee for Culture Collection of Microorganisms common micro-organisms center, bacterium numbering: 1.504 or 1.775 or 1.2172, and bacterial strain activates first; With the microorganism of above-mentioned generation proteolytic enzyme domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃, makes its well-grown in low temperature environment from high to low; Bacterial classification domestication by low temperature substratum: extractum carnis 0.1%～0.5%, peptone 0.7%～1.2%, NaCl 0.4%～0.6%, agar 2%, casein food grade 1%～4%, tap water 1000ml, pH 7.4,0.1Mpa, 121 ℃ of sterilization 20min;
(2) according to a conventional method with the protease-producing bacterium after the above-mentioned domestication by low temperature 14～20 ℃ of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Liquid seed culture medium: glucose 0.8%～1.2%, peptone 0.3%～0.7%, yeast extract paste 0.3%～0.8%, K
2HPO
40.08%～0.12%, MgSO
40.01%～0.04%, Na
2CO
30.8%～1.2%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min;
(3) with liquid first order seed or secondary seed, press in 3～9% inoculum sizes access culture medium of fermentating liquid volume, cultivate 48～120 h at 14～20 ℃, namely producing low-temperature protease by microbial fermentation finishes; Culture medium: glucose 0.9%～1.3%, peptone 0.2%～0.6%, yeast extract paste 0.3%～0.7%, K
2HPO
40.07%～0.13%, MgSO
40.01%～0.03%, Na
2CO
30.8%～1.3%, pH 8.5,0.1Mpa, 121 ℃ of sterilization 20min;
(4) with the fermented liquid of (3) at the centrifugal collection liquid of 4,000～8,000 rpm, the liquid of collecting is crude enzyme liquid;
(5) different with the use object according to different needs, with further concentrated, the separation and purification of crude enzyme liquid that (4) obtain, be prepared into the zymin of different activities, purity and formulation.
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| CN102653750A (en) * | 2012-04-12 | 2012-09-05 | 大连大学 | Method for producing douchi fibrinolytic enzyme through microbial fermentation |
| CN102653748A (en) * | 2012-04-12 | 2012-09-05 | 大连大学 | Fermenting production method of low-temperature xylanase by microorganisms |
| CN102776167A (en) * | 2012-04-12 | 2012-11-14 | 大连大学 | Method for producing plasmin through microbial fermentation |
| CN102653749A (en) * | 2012-04-13 | 2012-09-05 | 大连大学 | Method for producing low-temperature chitosanase through microbial fermentation |
| CN104630187A (en) * | 2015-02-16 | 2015-05-20 | 大连大学 | Method for producing low-temperature glucomannanase by microbial fermentation |
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| 王晓辉等.海洋低温BS070623菌株选育及其发酵培养基优化（I）.《渤海大学学报（自然科学版）》.2009,第30卷(第2期),97-100. * |
| 迟乃玉等.海洋低温蛋白酶菌株发酵条件的研究(Ⅱ).《微生物学通报》.2006,第33卷(第2期),106-108. * |
| 陈秀兰等.低温蛋白酶的研究进展与应用前景.《工业微生物》.2001,第31卷(第1期),52-53，57. * |
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