CN104630167A - Method for producing low-temperature glucose oxidase by fermentation of marine microorganisms - Google Patents
Method for producing low-temperature glucose oxidase by fermentation of marine microorganisms Download PDFInfo
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- CN104630167A CN104630167A CN201510084670.0A CN201510084670A CN104630167A CN 104630167 A CN104630167 A CN 104630167A CN 201510084670 A CN201510084670 A CN 201510084670A CN 104630167 A CN104630167 A CN 104630167A
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- glucose oxidase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Abstract
The invention relates to a method for producing low-temperature glucose oxidase by fermentation of marine microorganisms. The method specifically comprises the following steps: activating a glucose oxidase strain; carrying out step-by-step low-temperature domestication, wherein the strain grows well in a low-temperature environment; carrying out step-by-step enlarge cultivation on a glucose oxidase producing strain after low-temperature domestication at 10-16 DEG C; inoculating the strain to a liquid fermentation culture medium according to an inoculation amount which is 3-9% by volume of a fermentation liquid; culturing for 72-144 hours at 10-16 DEG C to obtain the low-temperature glucose oxidase; and further concentrating, separating and purifying the collected coarse enzyme according to different demands and using objects to prepare enzyme preparations with different activities, purities and dosage forms. The low-temperature glucose oxidase produced by the invention has high enzyme activity and high catalytic efficiency at the low temperature. The processing process can be simplified, the processing time is shortened, the production cost is lowered, the production efficiency is improved, and the product quality is improved and enhanced. The enzyme preparation is simple, convenient and rapid to apply and operate and has relatively broad industrial development value and market application advantages.
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 glucose oxidase marine microorganism fermentation method for producing.
Background technology
Glucose oxidase (Glucose oxidase, E.C 1.1.3.4, is abbreviated as GOD) specificity catalysis β-D-Glucose under aerobic conditions generates gluconic acid and hydrogen peroxide (Ahmed H etc., Eur Biophys J, 1998).GOD is mainly distributed in animal, plant and microbe, wherein in animal and plant tissue, GOD content is limited, and microorganism is owing to himself having the advantages such as wide material sources, growth cycle be short, it is the main source producing GOD, at present, the main production bacterial strain of GOD is aspergillus niger and mould (Wang Zhixin etc., Chinese food journal, 2007).The production mainly interior extraction of driven, plant materials of early stage GOD, but the method is difficult to obtain the high GOD of purity, and microbe fermentation method to have fermentation manufacturing technique simple, extraction step is relatively simple and easy, the purifying process of enzyme is stablized, produce not by features such as raw material sources affect, and the GOD produced has greater advantages in security, be applicable to batch production, easily realize industrialization, can also be united with foodstuff production, fodder production and medical test material, there are broad mass market application prospect (Xing Liangying etc., food science and technology, 2007; Zhang Yuexun etc., fodder industry, 2011).Abroad to the research relatively morning of GOD, mainly in the cloning and expression etc. of the separation and purification of the optimization of bacterial screening, condition of enzyme production, enzyme, enzymatic property and GOD gene, there are further investigation (Sisak C etc., Enzyme and Microbial Technology, 2006; Mislovicova D etc., Process Biochemistry, 2007; ).Compare external, China to start late (Zhu Yun equality about the research work of GOD, Food Additives Used in China, 2013) though at present China has GOD product to sell, zymin purity and active generally lower, poor stability, production cost is higher, main dependence on import (Han Jianchun etc., Food science, 2011).
Cold-adapted enzyme has high enzymatic activity and high catalytic efficiency at low temperatures, greatly can shorten the time for the treatment of processes and save expensive heating or refrigeration costs; Sizable advantage is had in energy-conservation; The vigor of cold-adapted enzyme can be made to lose through gentle thermal treatment, and low temperature or thermophilic process can not affect quality (Lu little Bo etc., biotechnology circular, 2006 of product; Zeng Yinxin etc., Chinese biological engineering magazine, 2003) low temperature GOD utilizes the above-mentioned advantage of cold-adapted enzyme to make it have boundless potentiality to be exploited and application prospect.Low temperature GOD provides actual opportunity by breaking away from the such as disease, resource, the predicament such as energy dilemma and environmental pollution that face in survival and development for the mankind, has boundless potentiality to be exploited and application prospect.Have no the relevant report of low temperature GOD at present both at home and abroad.
Summary of the invention
The object of this invention is to provide a kind of method of marine microorganism fermentative production low temperature glucose oxidase, the method mainly microorganism after domestication by low temperature, the method of low temperature glucose oxidase is produced at 10 ~ 16 DEG C of liquid fermentings, the low temperature glucose oxidase crude enzyme liquid activity that this production method obtains can reach 227.1U/ml, as again through abstraction and purification, the zymin of different concns and purity can be obtained.The method that a kind of fermentable of the present invention produces low temperature glucose oxidase specifically comprises the following steps:
(1) bacterial classification that can produce glucose oxidase first activates, again through domestication by low temperature step by step, acclimation temperature from high to low, 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures; The domestication by low temperature substratum of this bacterial classification is: glucose 20.0 ~ 50.0g, peptone 1.0 ~ 5.0g, (NH
4)
2sO
42.0 ~ 3.0g, CaCO
31.0 ~ 5.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
The described bacterial classification that can produce glucose oxidase derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), bacterium numbering: 1.10601 or 1.6446 or 1.10904;
(2) according to a conventional method by the glucose oxidase producing strains after domestication by low temperature in step (1) in liquid seed culture medium in 10 ~ 16 DEG C of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Described liquid seed culture medium is: peptone 7.0 ~ 12.0g, extractum carnis 2.0 ~ 7.0g, NaCl 8.0 ~ 11.0g, and pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
(3) pressed by liquid two stage seed in the inoculum size access liquid culture medium of fermentating liquid volume 3 ~ 9%, when cultivating 72 ~ 144h for 10 ~ 16 DEG C, namely fermentable production low temperature glucose oxidase terminates; Described liquid culture medium is: glucose 75.0 ~ 85.0g, peptone 1.0 ~ 5.0g, KH
2pO
41.4 ~ 2.0g, MgSO
47H
2o 0.7 ~ 0.8g, KCl 0.2 ~ 0.6g, pH value is 5.6, in 103kpa, 121 DEG C of autoclaving 30min;
(4) by the fermented liquid of (3) at 4,000 ~ 8,000rpm collected by centrifugation liquid, the supernatant liquor collected is crude enzyme liquid;
(5) different with use object according to difference needs, the crude enzyme liquid that (4) can also be obtained is concentrated, separation and purification further, is prepared into the low temperature glucose oxidase preparation of different activities, purity and formulation.
Further, the strain activation and culture base producing glucose oxidase in step (1) is: peptone 10.0g, yeast extract paste 5.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min.
The explanation that the bacterial classification initial activation that can produce glucose oxidase used in the present invention and growth conditions provide by China Committee for Culture Collection of Microorganisms's common micro-organisms center is carried out.Glucose oxidase producing strains bacterial strain first activates, produce low temperature glucose oxidase through domestication by low temperature secondary fermentation step by step again cultivates by condition of enzyme production of the present invention, bacterial strain after domestication by low temperature can preserve 2 months in 4 DEG C of environment, the bacteria suspension made with 10 ~ 25vt% glycerine, can long term storage under-80 DEG C of conditions.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the present invention utilizes marine microorganism fermentative production low temperature glucose oxidase, the vigor of cold-adapted enzyme can be made to lose through gentle thermal treatment, and low temperature or thermophilic process all can not affect the quality of product, particularly low temperature lipoxygenase still has high enzymatic activity and high catalytic efficiency at low temperatures, effectively improves and improves the quality of products;
(2) the inventive method simplifies production technique, shortens the link that process period also fundamentally saves heating in the use of middle temperature enzyme, cooling apparatus, saves expensive heating or refrigeration costs, in energy-conservation, have sizable advantage;
(3) the glucose oxidase application operating produced of the inventive method is simple, convenient, fast, cost is low, the emerging field such as industry or biofuel, biosensor and biochip such as to bleaching and dyeing all be widely used in foodstuffs industry, feedstuff industry, medicine industry, weaving.
Embodiment
Below by embodiment, the present invention will be further described, but do not limit the present invention.If no special instructions, the present invention is raw materials used all commercially.
Embodiment 1
(1) medium preparing
1. strain activation and culture base: peptone 10.0g, yeast extract paste 5.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
2. bacterial classification domestication by low temperature substratum: glucose 20.0g, peptone 1.0g, (NH
4)
2sO
42.0g, CaCO
31.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
3. liquid seed culture medium: peptone 7.0g, extractum carnis 2.0g, NaCl 8.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
4. liquid culture medium: glucose 75.0g, peptone 1.0g, KH
2pO
41.4g, MgSO
47H
2o 0.7g, KCl 0.2g, pH value is 5.6, in 103kpa, 121 DEG C of autoclaving 30min;
(2) will produce the bacterial classification of glucose oxidase, the bacterial strain provided by China General Microbiological culture presevation administrative center (CGMCC) illustrates and carries out initial activation;
(3) by the bacterial classification that activated in (2) domestication by low temperature step by step in domestication by low temperature substratum, acclimation temperature from high to low, 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, tame about 8 days, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures;
(4), after according to a conventional method the glucose oxidase producing strains after domestication by low temperature being cultivated 48 ~ 72h in 10 ~ 12 DEG C in liquid seed culture medium, carry out enlarged culturing step by step by the inoculum size of 5vt%, be prepared into liquid first order seed and secondary seed;
(5), in the liquid culture medium of the secondary seed (4) prepared by the inoculum size access 10L of fermentating liquid volume 3 ~ 5%, when cultivating 120 ~ 144h for 10 ~ 12 DEG C, namely marine microorganism fermentative production low temperature glucose oxidase terminates;
(6) by the fermented liquid of (5) at 4,000 ~ 8,000rpm collected by centrifugation liquid, the supernatant liquor collected is crude enzyme liquid;
(7) different with use object according to difference needs, the crude enzyme liquid that (6) can also be obtained is concentrated, separation and purification further, is prepared into the low temperature glucose oxidase preparation of different activities, purity and formulation.
Embodiment 2
(1) medium preparing
1. strain activation and culture base: peptone 10.0g, yeast extract paste 5.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
2. bacterial classification domestication by low temperature substratum: glucose 25.0g, peptone 3.0g, (NH
4)
2sO
42.5g, CaCO
33.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
3. liquid seed culture medium: peptone 10.0g, extractum carnis 5.0g, NaCl 10.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
4. liquid culture medium: glucose 80.0g, peptone 3.0g, KH
2pO
41.6g, MgSO
47H
2o 0.7g, KCl 0.6g, pH value is 5.6, in 103kpa, 121 DEG C of autoclaving 30min;
(2) will produce the bacterial classification of glucose oxidase, the bacterial strain provided by China General Microbiological culture presevation administrative center (CGMCC) illustrates and carries out initial activation;
(3) bacterial classification (2) activated domestication by low temperature step by step in domestication by low temperature substratum, acclimation temperature from high to low, 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, tame about 8 days, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures;
(4) according to a conventional method the glucose oxidase producing strains after domestication by low temperature after 12 ~ 14 DEG C of cultivation 48 ~ 72h, is carried out enlarged culturing step by step by the inoculum size of 5vt%, is prepared into liquid first order seed and secondary seed in liquid seed culture medium;
(5), in the culture medium of the secondary seed (4) prepared by the inoculum size access 50L of fermentating liquid volume 5 ~ 7%, when cultivating 96 ~ 120h for 12 ~ 14 DEG C, namely fermentable production low temperature glucose oxidase terminates;
(6) by the fermented liquid of (5) at 4,000 ~ 8,000rpm collected by centrifugation liquid, the supernatant liquor collected is crude enzyme liquid;
(7) different with use object according to difference needs, the crude enzyme liquid that (6) can also be obtained is concentrated, separation and purification further, is prepared into the low temperature glucose oxidase preparation of different activities, purity and formulation.
Embodiment 3
(1), medium preparing
1. strain activation and culture base: peptone 10.0g, yeast extract paste 5.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
2. bacterial classification domestication by low temperature substratum: glucose 50.0g, peptone 5.0g, (NH
4)
2sO
43.0g, CaCO
35.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
3. liquid seed culture medium: peptone 12.0g, extractum carnis 7.0g, NaCl 11.0g, pH value nature, 121 DEG C of autoclaving 30min;
4. culture medium: glucose 85.0g, peptone 5.0g, KH
2pO
42.0g, MgSO
47H
2o 0.8g, KCl 0.6g, pH value is 5.6, in 103kpa, 121 DEG C of autoclaving 30min;
(2) will produce the bacterial classification of glucose oxidase, the bacterial strain provided by China General Microbiological culture presevation administrative center (CGMCC) illustrates and carries out initial activation;
(3) bacterial classification (2) activated domestication by low temperature step by step in domestication by low temperature substratum, acclimation temperature from high to low, 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, tame about 8 days, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures;
(4) according to a conventional method the glucose oxidase producing strains after domestication by low temperature is cultivated after 48 ~ 72h in 14 ~ 16 DEG C at liquid seed culture medium, carry out enlarged culturing step by step by the inoculum size of 5vt%, be prepared into liquid first order seed and secondary seed;
(5), in the culture medium of the secondary seed (4) prepared by the inoculum size access 100L of fermentating liquid volume 7 ~ 9%, when cultivating 72 ~ 96h for 14 ~ 16 DEG C, namely fermentable production low temperature glucose oxidase terminates;
(6) by the fermented liquid of (5) at 4,000 ~ 8,000rpm collected by centrifugation liquid, the supernatant liquor collected is crude enzyme liquid;
(7) different with use object according to difference needs, the crude enzyme liquid that (6) can also be obtained is concentrated, separation and purification further, is prepared into the low temperature glucose oxidase preparation of different activities, purity and formulation.
By the low temperature glucose oxidase that the method for embodiment 1,2 and 3 is obtained, average enzymic activity is 800U/ml, the active low temperature glucose oxidase for 1000U/ml or 1000U/g is obtained through concentrated, abstraction and purification, by the amount of 0.01 ‰ ~ 0.5 ‰, be applied in the fields such as animal-feed, food-processing and medicine detection, under 8 ~ 25 DEG C of conditions, process 0.5 ~ 2 hour, particular content please refer to table 1.
Table 1 low temperature glucose oxidase application example
Experimental result shows: low temperature glucose oxidase is applied in the fields such as animal-feed, food-processing and medicine detection, can improve the transformation efficiency of raw material, the quality guaranteed period extending food and product special flavour, reduce production cost, improve and improve the quality of products; Also illustrate that cold-adapted enzyme has high enzymatic activity and high catalytic efficiency at low temperatures, greatly can shorten the time for the treatment of processes and save expensive heating or refrigeration costs simultaneously; Sizable advantage is had in energy-conservation; This zymin application operating is simple, convenient, fast, cost is low; Fundamentally can avoid the heating of middle temperature, high temperature enzyme, cooling apparatus and technique, improve the utilization ratio of raw material, transformation efficiency and productivity, reduce production cost, improve and improve the quality of products.
Claims (2)
1. the method for marine microorganism fermentative production low temperature glucose oxidase, is characterized in that, comprise the following steps:
(1) bacterial classification that can produce glucose oxidase first activates, again through domestication by low temperature step by step, acclimation temperature from high to low, 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures; The domestication by low temperature substratum of this bacterial classification is: glucose 20.0 ~ 50.0g, peptone 1.0 ~ 5.0g, (NH
4)
2sO
42.0 ~ 3.0g, CaCO
31.0 ~ 5.0g, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min;
The described bacterial classification that can produce glucose oxidase derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), bacterium numbering: 1.10601 or 1.6446 or 1.10904;
(2) according to a conventional method by the glucose oxidase producing strains after domestication by low temperature in step (1) in liquid seed culture medium in 10 ~ 16 DEG C of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Described liquid seed culture medium is: peptone 7.0 ~ 12.0g, extractum carnis 2.0 ~ 7.0g, NaCl 8.0 ~ 11.0g, and pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min.
(3) pressed by liquid two stage seed in the inoculum size access liquid culture medium of fermentating liquid volume 3 ~ 9%, when cultivating 72 ~ 144h for 10 ~ 16 DEG C, namely marine microorganism fermentative production low temperature glucose oxidase terminates; Described liquid culture medium is: glucose 75.0 ~ 85.0g, peptone 1.0 ~ 5.0g, KH
2pO
41.4 ~ 2.0g, MgSO
47H
2o 0.7 ~ 0.8g, KCl 0.2 ~ 0.6g, pH value is 5.6, in 103kpa, 121 DEG C of autoclaving 30min.
(4) by the fermented liquid of (3) at 4,000 ~ 8,000rpm collected by centrifugation liquid, the supernatant liquor collected is crude enzyme liquid.
(5) different with use object according to difference needs, the crude enzyme liquid that (4) can also be obtained is concentrated, separation and purification further, is prepared into the low temperature glucose oxidase preparation of different activities, purity and formulation.
2. the method for marine microorganism fermentative production low temperature glucose oxidase according to claim 1, it is characterized in that, the strain activation and culture base producing glucose oxidase in step (1) is: peptone 10.0g, yeast extract paste 5.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value is nature, in 103kpa, 121 DEG C of autoclaving 30min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795892A (en) * | 2018-06-19 | 2018-11-13 | 张宝华 | A method of preparing, detach and purify glucose oxidase |
| CN109652390A (en) * | 2019-02-25 | 2019-04-19 | 大连大学 | A kind of marine low temperature glucose oxidase and its application |
| CN109749967A (en) * | 2019-02-28 | 2019-05-14 | 大连大学 | The marine bacteria of one plant of malaga carbohydrate oxidase and its application |
| CN109864188A (en) * | 2019-02-28 | 2019-06-11 | 大连大学 | A kind of feed addictive of the glucose oxidase containing low temperature and application thereof |
| CN109880809A (en) * | 2019-02-28 | 2019-06-14 | 大连大学 | A kind of genetic engineering bacterium and preparation method thereof producing low temperature glucose oxidase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093990A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low temperature amylases through microbial fermentation |
-
2015
- 2015-02-16 CN CN201510084670.0A patent/CN104630167A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093990A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low temperature amylases through microbial fermentation |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795892A (en) * | 2018-06-19 | 2018-11-13 | 张宝华 | A method of preparing, detach and purify glucose oxidase |
| CN108795892B (en) * | 2018-06-19 | 2021-01-12 | 北京天一辉远生物科技有限公司 | Method for preparing, separating and purifying glucose oxidase |
| CN109652390A (en) * | 2019-02-25 | 2019-04-19 | 大连大学 | A kind of marine low temperature glucose oxidase and its application |
| CN109749967A (en) * | 2019-02-28 | 2019-05-14 | 大连大学 | The marine bacteria of one plant of malaga carbohydrate oxidase and its application |
| CN109864188A (en) * | 2019-02-28 | 2019-06-11 | 大连大学 | A kind of feed addictive of the glucose oxidase containing low temperature and application thereof |
| CN109880809A (en) * | 2019-02-28 | 2019-06-14 | 大连大学 | A kind of genetic engineering bacterium and preparation method thereof producing low temperature glucose oxidase |
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