CN102653747A - Fermenting production method of low-temperature beta-galactosidase by marine microorganisms - Google Patents
Fermenting production method of low-temperature beta-galactosidase by marine microorganisms Download PDFInfo
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- CN102653747A CN102653747A CN2012101096021A CN201210109602A CN102653747A CN 102653747 A CN102653747 A CN 102653747A CN 2012101096021 A CN2012101096021 A CN 2012101096021A CN 201210109602 A CN201210109602 A CN 201210109602A CN 102653747 A CN102653747 A CN 102653747A
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Abstract
The invention provides a fermenting production method of low-temperature beta-galactosidase by marine microorganisms. The method comprises the following steps: gradually directionally domesticating microorganisms which produce beta-galactosidase to lead the beta-galactosidase to grow excellently in a natural condition; gradually propagating the beta-galactosidase producing strain at a temperature between 10 and 16 DEG C after directional domestication; inoculating into a liquid ferment culture medium in an inoculum size of 3 to 9 percent of the volume of a fermentation broth, and culturing for 48 to 84 hours at a temperature between 10 and 16 DEG C to complete fermenting production of the low-temperature beta-galactosidase by the marine microorganisms; centrifuging the fermentation broth in 4000 to 8000rpm to collect a strain; collecting precipitate after multiple cleaning times, suspending in a buffer solution, and grinding with quartz sands; centrifugally collecting supernatant in 10000 to 14000rpm to obtain a crude enzyme; and further concentrating, separating and purifying the crude enzyme according to different requirements and different use objects to prepare the enzyme preparations with different activities, purities and preparation 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 beta-galactosidase enzymes microbial fermentation production method.The low temperature beta-galactosidase enzymes that this invention is produced is mainly used in foodstuffs industry, environmental protection, medicine and other fields.This cold-adapted enzyme preparation can reduce production costs, and reduces technical process, enhances productivity, and increases economic benefit, improves quality product.
Background technology
(β-Galactosidase EC3.2.1.23) claims Sumylact L, β-D-galactoside galactohydrolase again to beta-galactosidase enzymes, is a kind of biological enzyme formulation that has no side effect (Zhang Li, Northeast Agricultural University's journal, 2009).It is hydrolysis β-D-semi-lactosi glycosidic bond (β-1,4-glycosidic link) under given conditions, hydrolyzes lactose into α D-glucose and β D-semi-lactosi; Sumylact L has the effect of shifting galactoside simultaneously, can be connected to semi-lactosi on the lactose, generates oligomeric galactose (Lu Lili etc., mikrobe journal, 2008).Early stage beta-galactosidase enzymes working method mainly is from mammalian tissues and some plants, to extract, and has high, the problems (Zhang Li, Northeast Agricultural University's journal, 2009) such as purity is low, complex manufacturing of production cost; Utilize microbial fermentation to produce that beta-galactosidase enzymes technology is fairly simple, production cost is lower, purity high (Zhou Chunlei etc., polar research, 2010).Warm enzyme during at present domestic research to microbial fermentation production beta-galactosidase enzymes mainly concentrates on; Research to cold-adapted enzyme is less; Mainly be research (Liu Wenyu etc., Xinjiang agricultural sciences, 2007 of the screening of low temperature beta-galactosidase bacteria, physico-chemical property and enzyme molecular characterization; Li Yu is strong etc., foodstuff additive, 2001).To the research of marine microorganism fermentative prodn low temperature beta-galactosidase enzymes still in the starting stage, industrialization, large-scale production and use and also do not appear in the newspapers.Compare with the medium and high temperature beta-galactosidase enzymes; The marine low temperature beta-galactosidase enzymes has loose, submissive molecular structure; Make it have lower activation energy, Km value and catalyzed reaction temperature under field conditions (factors), these characteristics can be strengthened its avidity to substrate, improve substrate utilization ratio; Thereby cut down the consumption of energy, shorten the time of mechanism; In addition, the low temperature beta-galactosidase enzymes is very sensitive to heat, and above-mentioned these characteristics of marine low temperature beta galactose enzyme help the popularization and the use (Li Xingfeng etc., Chinese food journal, 2003) of this zymin, and these advantages are that the medium and high temperature enzyme is incomparable.The low temperature beta-galactosidase enzymes that utilizes the marine microorganism fermentative prodn reduce production costs and energy consumption aspect huge advantage; Can fundamentally break away from heating, cooling apparatus and technology that the medium and high temperature enzyme is used, thereby reduce technical process, reduce production costs, enhance productivity, improve and improve the quality of products.The exploitation of this zymin is to utilizing oceanic resources, such as predicaments such as disease, energy dilemma and environmental pollutions actual opportunity is provided for what the mankind broke away from the survival and development to be faced.Therefore, the low temperature beta-galactosidase enzymes of marine microorganism fermentative prodn has broad prospect of application and huge potentiality to be exploited.
Summary of the invention
The method that the purpose of this invention is to provide a kind of marine microorganism fermentative prodn low temperature beta-galactosidase enzymes; This method mainly is after marine microorganism is tamed through orientation; Produce the low temperature beta-galactosidase enzymes by the ordinary method liquid fermenting; The marine low temperature beta-galactosidase enzymes vigor that this working method obtains can reach 120U/ml, as passing through the zymin that separation and purifying can obtain different concns and purity again.This zymin manufacturing cost is low, purity is high, and 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 of a kind of marine microorganism fermentative prodn low temperature beta-galactosidase enzymes of the present invention specifically may further comprise the steps:
The marine microorganism that (1) will produce beta-galactosidase enzymes carries out the orientation domestication, makes its well-grown under field conditions (factors);
(2) by ordinary method with the beta-galactosidase bacteria after the orientation domestication 10～16 ℃ 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 in the liquid fermentation medium, and when cultivating 48～84h for 10～16 ℃, promptly marine microorganism fermentative prodn low temperature beta-galactosidase enzymes finishes;
(4) with the fermented liquid 4,000～8 of (3), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(5) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the beta-galactosidase enzymes crude enzyme liquid;
(6) according to different needs with to use object different, can also the crude enzyme liquid that (5) obtain is further concentrated, separation and purification, be prepared into the zymin of different activities, purity and formulation.
The bacterial classification that uses in this project derives from Chinese common micro-organisms culture presevation administrative center (CGMCC), and initial stage activation and growth conditions are undertaken by the explanation that culture presevation unit provides.Beta-galactosidase bacteria (for example; CGMCC bacterium numbering: 1.3 or 1.11); After the activation of bacterial strain elder generation, the directed domestication; By condition of enzyme production fermentative prodn low temperature beta-galactosidase enzymes of the present invention, the bacterial strain after the domestication can be preserved 2 months in 4 ℃ of environment, bacteria suspension, the preservation for a long time under-80 ℃ of conditions of processing with 10～25% glycerine.
Embodiment
Embodiment one:
(1) medium preparation
1. strain activation and culture base: peptone 10.0g, yeast extract paste 15.0g, NaCl 5.0g, K
2HPO
42.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: lactose 5.0～15.0g, peptone 8.0～12.0g, yeast extract paste 5.0～15.0g, NaCl 4.0～8.0g, K
2HPO
42.0～4.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
3. liquid seed culture medium: peptone 8.0～12.0g, yeast extract paste 12.0～16.0g, NaCl 4.0～7.0g, K
2HPO
41.5～3.5g, agar 15.0～25.0g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
4. enzymatic production substratum: lactose 15.0～25.0g, peptone 5.0～15.0g, NaCl 4.0～7.0g, K
2HPO
41.0～3.5g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
(2) will produce the bacterial classification of beta-galactosidase enzymes, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) (2) activation is good bacterial classification carries out the orientation domestication, makes its well-grown under field conditions (factors);
(4) beta-galactosidase bacteria of pressing after ordinary method is tamed orientation is cultivated 24～36h for 10～16 ℃, and the inoculum size by 5～8% is carried out enlarged culturing step by step, is prepared into liquid first order seed and secondary seed;
(5) with liquid first order seed or secondary seed, 3～5% inoculum sizes of pressing fermentating liquid volume insert in the 10L liquid fermentation medium, and when cultivating 72～84h for 10～12 ℃, promptly marine microorganism fermentative prodn low temperature beta-galactosidase enzymes finishes;
(6) with the fermented liquid 4,000～8 of (5), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(7) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the beta-galactosidase enzymes crude enzyme liquid;
(8) different with the use object according to different needs, the crude enzyme liquid that (7) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Embodiment two:
(1) medium preparation
1. strain activation and culture base: peptone 10.0g, yeast extract paste 15.0g, NaCl 5.0g, K
2HPO
42.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: lactose 5.0～15.0g, peptone 8.0～12.0g, yeast extract paste 5.0～15.0g, NaCl 4.0～8.0g, K
2HPO
42.0～4.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
3. liquid seed culture medium: peptone 8.0～12.0g, yeast extract paste 12.0～16.0g, NaCl 4.0～7.0g, K
2HPO
41.5～3.5g, agar 15.0～25.0g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
4. enzymatic production substratum: lactose 15.0～25.0g, peptone 5.0～15.0g, NaCl 4.0～7.0g, K
2HPO
41.0～3.5g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
(2) will produce the bacterial classification of beta-galactosidase enzymes, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) (2) activation is good bacterial classification carries out the orientation domestication, makes its well-grown under conventional pressure, salt concentration conditions;
(4) beta-galactosidase bacteria of pressing after ordinary method is tamed orientation is cultivated 24～36h for 10～16 ℃, carries out enlarged culturing step by step by 5～8% inoculum sizes, is prepared into liquid first order seed and secondary seed;
(5) with liquid first order seed or secondary seed, 5～7% inoculum sizes of pressing fermentating liquid volume insert in the 50L liquid fermentation medium, and when cultivating 60～72h for 12～14 ℃, promptly marine microorganism fermentative prodn low temperature beta-galactosidase enzymes finishes;
(6) with the fermented liquid 4,000～8 of (5), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(7) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the beta-galactosidase enzymes crude enzyme liquid;
(8) different with the use object according to different needs, the crude enzyme liquid that (7) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Embodiment three:
(1) medium preparation
1. strain activation and culture base: peptone 10.0g, yeast extract paste 15.0g, NaCl 5.0g, K
2HPO
42.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: lactose 5.0～15.0g, peptone 8.0～12.0g, yeast extract paste 5.0～15.0g, NaCl 4.0～8.0g, K
2HPO
42.0～4.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
3. liquid seed culture medium: peptone 8.0～12.0g, yeast extract paste 12.0～16.0g, NaCl 4.0～7.0g, K
2HPO
41.5～3.5g, agar 15.0～25.0g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
4. enzymatic production substratum: lactose 15.0～25.0g, peptone 5.0～15.0g, NaCl 4.0～7.0g, K
2HPO
41.0～3.5g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
(2) will produce the bacterial classification of beta-galactosidase enzymes, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) (2) activation is good bacterial classification carries out the orientation domestication, makes its well-grown under conventional pressure, salt concentration conditions;
(4) beta-galactosidase bacteria of pressing after ordinary method is tamed orientation is cultivated 24～36h for 10～16 ℃, carries out enlarged culturing step by step by 5～8% inoculum sizes, is prepared into liquid first order seed and secondary seed;
(5) with liquid first order seed or secondary seed, 7～9% inoculum sizes of pressing fermentating liquid volume insert in the 200L liquid fermentation medium, and when cultivating 48～60h for 14～16 ℃, promptly marine microorganism fermentative prodn low temperature beta-galactosidase enzymes finishes;
(6) with the fermented liquid 4,000～8 of (5), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(7) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the beta-galactosidase enzymes crude enzyme liquid;
(8) different with the use object according to different needs, the crude enzyme liquid that (7) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Claims (3)
1. a microbial fermentation is produced the method for low temperature beta-galactosidase enzymes, may further comprise the steps:
The marine microorganism that (1) will produce beta-galactosidase enzymes carries out the orientation domestication, makes its well-grown under field conditions (factors);
(2) by ordinary method with the beta-galactosidase bacteria after the orientation domestication 10～16 ℃ 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 in the liquid fermentation medium, and when cultivating 48～84h for 10～16 ℃, promptly marine microorganism fermentative prodn low temperature beta-galactosidase enzymes finishes;
(4) with the fermented liquid 4,000～8 of (3), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(5) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the beta-galactosidase enzymes crude enzyme liquid;
(6) according to different needs with to use object different, can also the crude enzyme liquid that (5) obtain is further concentrated, separation and purification, be prepared into the zymin of different activities, purity and formulation.
2. method according to claim 1 further comprises in step (6) afterwards: the crude enzyme liquid that step (6) is obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
3. method according to claim 1 and 2, wherein bacterial classification domestication by low temperature substratum, liquid seed culture medium, enzymatic production substratum are respectively:
(1) bacterial classification domestication by low temperature substratum: lactose 5.0～15.0g, peptone 8.0～12.0g, yeast extract paste 5.0～15.0g, NaCl 4.0～8.0g, K
2HPO
42.0～4.5g, zero(ppm) water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
(2) liquid seed culture medium: peptone 8.0～12.0g, yeast extract paste 12.0～16.0g, NaCl 4.0～7.0g, K
2HPO
41.5～3.5g, agar 15.0～25.0g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
(3) enzymatic production substratum: lactose 15.0～25.0g, peptone 5.0～15.0g, NaCl 4.0～7.0g, K
2HPO
41.0～3.5g, tap water 1.0L, 7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630188A (en) * | 2015-02-16 | 2015-05-20 | 大连大学 | Method for producing low-temperature glucomannanase by fermentation of marine microorganisms |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN1932004A (en) * | 2005-09-16 | 2007-03-21 | 新疆农业科学院微生物应用研究所 | Low temperature beta-galactosidase strain, low temperature bata-galactosidase and its production process |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
| CN102174439A (en) * | 2011-01-27 | 2011-09-07 | 中国人民解放军第二军医大学 | Marine halomonas for producing low-temperature beta-galactosidase and application thereof |
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2012
- 2012-04-13 CN CN2012101096021A patent/CN102653747A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932004A (en) * | 2005-09-16 | 2007-03-21 | 新疆农业科学院微生物应用研究所 | Low temperature beta-galactosidase strain, low temperature bata-galactosidase and its production process |
| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
| CN102174439A (en) * | 2011-01-27 | 2011-09-07 | 中国人民解放军第二军医大学 | Marine halomonas for producing low-temperature beta-galactosidase and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630188A (en) * | 2015-02-16 | 2015-05-20 | 大连大学 | Method for producing low-temperature glucomannanase by fermentation of marine microorganisms |
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Application publication date: 20120905 |