CN102093992A - 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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- CN102093992A CN102093992A CN 201010162644 CN201010162644A CN102093992A CN 102093992 A CN102093992 A CN 102093992A CN 201010162644 CN201010162644 CN 201010162644 CN 201010162644 A CN201010162644 A CN 201010162644A CN 102093992 A CN102093992 A CN 102093992A
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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 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 manufacturing and environment protection.Can improve utilization ratio of raw materials, transformation efficiency and productivity, 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 manufacturing and environment protection as a class important industrial enzymes.The proteolytic enzyme that uses generally all is middle gentle high temperature modification proteolytic enzyme (Chen Xiulan etc., industrial microorganism, 2001) at present.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 genes involved clone; 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 big application advantage, lives and high catalytic efficiency because low-temperature protease has high enzyme at low temperatures, can shorten the time for the treatment of processes greatly and save expensive heating or refrigeration costs; Aspect energy-conservation, sizable advantage is arranged; Through gentle thermal treatment the vigor of low-temperature protease is lost, and low temperature or thermophilic can not influence 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 technology, 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 application of low-temperature protease will be to traditional grain processing, food, brewage, fermentation, textiles, medicine and other fields produce far-reaching influence, also will provide actual opportunity such as predicaments such as resource, energy dilemma and environmental pollutions for what the mankind broke away from the survival and development to be faced.Low-temperature protease has boundless potentiality to be exploited and application prospect.
Summary of the invention
The purpose of this invention is to provide a kind of microbial fermentation and produce the method for low-temperature protease, this 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 technology of middle temperature, high temperature enzyme.
The method that a kind of microbial fermentation of the present invention is produced low-temperature protease specifically may further comprise the steps:
(1) microorganism that will produce 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;
(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, 3～9% inoculum sizes of pressing fermentating liquid volume insert produces in the enzyme substratum, and when cultivating 48～120h for 14～20 ℃, promptly microbial fermentation is produced the low-temperature protease end;
(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, the crude enzyme liquid that (4) are obtained further concentrates, separation and purification, is 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 is activation earlier, 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 preservation for a long time under subzero 80 ℃ of conditions made from 25～30% glycerine.
Embodiment:
Embodiment one:
(1), medium 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. produce the enzyme substratum: 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 good bacterial classification 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;
(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 enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) inoculum size of the secondary seed of (4) preparation being pressed fermentating liquid volume 3～5% inserts in the product enzyme substratum of 10L, cultivates 96～120h at 14～16 ℃, and promptly microbial fermentation is produced the low-temperature protease end;
(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, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the cold induced proteins zymin of different activities, purity and formulation.
Embodiment two:
(1), medium 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. produce the enzyme substratum: 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 good bacterial classification 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;
(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 enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) inoculum size of the secondary seed of (4) preparation being pressed fermentating liquid volume 5～7% inserts in the product enzyme substratum of 50L, cultivates 72～96h at 16～18 ℃, and promptly microbial fermentation is produced the low-temperature protease end;
(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, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the cold induced proteins zymin of different activities, purity and formulation.
Embodiment three:
(1), medium 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. produce the enzyme substratum: 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 good bacterial classification 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;
(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 enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) inoculum size of the secondary seed of (4) preparation being pressed fermentating liquid volume 7～9% inserts in the product enzyme substratum of 100L, cultivates 48～72h at 18～20 ℃, and promptly microbial fermentation is produced the low-temperature protease end;
(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, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the cold induced proteins zymin of different activities, purity and formulation.
Claims (2)
1. a microbial fermentation is produced the method for low-temperature protease, and it may further comprise the steps:
(1) microorganism that will produce 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;
(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, 3～9% inoculum sizes of pressing fermentating liquid volume insert produces in the enzyme substratum, cultivates 48～120h at 14～20 ℃, and promptly microbial fermentation is produced the low-temperature protease end;
(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, the crude enzyme liquid that (4) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
2. method according to claim 1, wherein bacterial classification domestication by low temperature substratum, liquid seed culture medium, product enzyme substratum are respectively:
(1) 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) 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) produce the enzyme substratum: 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.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653750A (en) * | 2012-04-12 | 2012-09-05 | 大连大学 | Method for producing douchi fibrinolytic enzyme through microbial fermentation |
| CN102653749A (en) * | 2012-04-13 | 2012-09-05 | 大连大学 | Method for producing low-temperature chitosanase 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 |
| CN104630187A (en) * | 2015-02-16 | 2015-05-20 | 大连大学 | Method for producing low-temperature glucomannanase by microbial fermentation |
-
2010
- 2010-04-09 CN CN 201010162644 patent/CN102093992B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《工业微生物》 20010331 陈秀兰等 低温蛋白酶的研究进展与应用前景 52-53，57 1-2 第31卷, 第1期 * |
| 《微生物学通报》 20060430 迟乃玉等 海洋低温蛋白酶菌株发酵条件的研究(Ⅱ) 106-108 1-2 第33卷, 第2期 * |
| 《渤海大学学报（自然科学版）》 20090630 王晓辉等 海洋低温BS070623菌株选育及其发酵培养基优化（I） 97-100 1-2 第30卷, 第2期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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