CN104630164A - Method for producing low-temperature lipoxygenase by fermenting marine microorganisms - Google Patents

Method for producing low-temperature lipoxygenase by fermenting marine microorganisms Download PDF

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CN104630164A
CN104630164A CN201510084631.0A CN201510084631A CN104630164A CN 104630164 A CN104630164 A CN 104630164A CN 201510084631 A CN201510084631 A CN 201510084631A CN 104630164 A CN104630164 A CN 104630164A
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lipoxygenase
low temperature
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窦少华
李晓艳
王晓辉
于爽
张庆芳
迟乃玉
张旭姣
王强
金连豆
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Dalian University
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    • 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.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11012—Linoleate 13S-lipoxygenase (1.13.11.12)

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Abstract

The invention relates to a method for producing low-temperature lipoxygenase by fermenting marine microorganisms. The method comprises the following specific steps: at first, activating a lipoxygenase generation strain, carrying out step-by-step low temperature acclimation to ensure good growth of the strain in a low temperature environment, carrying out step-by-step enlarging cultivation on the low temperature acclimated lipoxygenase generation strain at 10-18 DEG C, inoculating the lipoxygenase generation strain in a liquid fermentation culture medium according to inoculation quantity of 3-9% by volume of fermentation broth, culturing at 10-18 DEG C for 72-144 hours to obtain low-temperature lipoxygenase, and further concentrating, separating and purifying the collected crude enzyme according to different demands and application objects to prepare enzymic preparations with different activities, purities and dosage forms. The low-temperature lipoxygenase produced by the method provided by the invention has high enzyme activity and high catalytic efficiency at low temperature, thereby simplifying the processing technology, shortening the processing time, reducing the production cost, improving the production efficiency and perfecting and improving the product quality; the enzymic preparation is simple, convenient and quick to apply and operate, thus having broad development potential and application prospects.

Description

The method of marine microorganism fermentative production low temperature lipoxygenase
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 lipoxygenase marine microorganism fermentation method for producing.
Background technology
Lipoxygenase (Lipoxygenase, EC 1.13.11.12, be called for short LOX) be the dioxygenase that a class contains nonheme iron, the unsaturated fatty acids hydroperoxide that its catalysis generates, the raw materials for production of bleaching agent for flour and sweetener, antitumor drug precursor, coating and washing composition etc., be important pharmaceutical synthesis and chemosynthesis intermediate, there is the huge market requirement.Industrial application LOX is the LOX of plant origin mostly, and the overwhelming majority derives from soybean.Due to the restriction of process costs, the LOX that soybean is extracted often contains multiple isozyme, to its catalysate characteristic of control very unfavorable (Casey R, et al.Food Biotechnol, 2005).Compared with traditional extraction method, marine microorganism ferments the LOX stable performance obtained, and its good homogeneity receives increasing concern.Current Domestic is outer mainly concentrates on middle temperature LOX to the research of microbe-derived LOX, research contents comprises strain improvement, fermentation optimization (Lu Xinyao, food science and technology, 2013), aspect (the Casey R such as cloning and expression and zymoprotein molecular modification of zymologic property, structure and catalysis characteristics, immobilization, gene, et al.Trends Food Sci Tech, 1999; Koeduka T, et al.Curr Microbi-ol, 2007; Zhang Chong etc., biotechnology journal, 2012; Zhang Dongliang etc., nuclear agricultural science report, 2012; Hansen J, et al.Appl Microbiol Biot, 2013; Wang Guangsheng etc., food science and technology, 2014 etc.), cold-adapted enzyme has high enzymatic activity and high catalytic efficiency at low temperatures, can reduce complete processing, shortens the time of the course of processing and saves expensive heating or refrigeration costs, in energy-conservation, have sizable advantage; The vigor of cold-adapted enzyme can be made to lose through gentle thermal treatment, and low temperature or thermophilic process can not affect the quality (Chen Xiulan etc., Chinese agriculture science and technology Leader, 2007) of product.Therefore, the above-mentioned characteristic that low temperature LOX possesses equally will contribute to its promotion and application, have more wide commercial exploitation value and market application advantage.At present, yet there are no the relevant report about marine microorganism fermentative production low temperature LOX, low temperature LOX industrialization, large-scale production.
Summary of the invention
The object of this invention is to provide a kind of method of marine microorganism fermentative production low temperature lipoxygenase, the method mainly microorganism after domestication by low temperature, the method of fermentative production low temperature lipoxygenase in 10 ~ 18 DEG C of liquid fermentation mediums, the low temperature lipoxygenase crude enzyme liquid enzyme work that this production method obtains can reach 170U/ml, as again through abstraction and purification, the zymin of different concns and purity can be obtained.
Technical scheme of the present invention is as follows: the method that fermentable produces low temperature lipoxygenase specifically comprises the following steps:
(1) actication of culture of lipoxygenase can be produced, then through domestication by low temperature step by step, acclimation temperature from high to low, 30 DEG C → 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, makes its well-grown in low temperature environment; The domestication by low temperature substratum of this bacterial classification is: peptone 5.0 ~ 15.0g, linolic acid 2.0 ~ 10.0g, NaCl 5.0 ~ 10.0g, L-Aspartic acid 0.5 ~ 2.0g, Pidolidone 0.5 ~ 2.0g, agar 20.0g, tap water 1.0L, pH value is 6.0 ~ 8.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
Described can produce the microbe-derived in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of lipoxygenase, bacterium numbering: 1.228 or 1.239 or 1.2620;
(2) according to a conventional method by the lipoxygenase producing strains after domestication by low temperature in step (1) in liquid seed culture medium in 10 ~ 18 DEG C of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Described liquid seed culture medium: yeast extract paste 10.0 ~ 20.0g, peptone 5.0 ~ 10.0g, NaCl5.0 ~ 10.0g, L-Aspartic acid 0.5 ~ 2.0g, Pidolidone 0.5 ~ 2.0g, tap water 1.0L, pH value is 6.0 ~ 8.0, in 103kpa, 121 DEG C of high pressure steam sterilization 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 ~ 18 DEG C, namely marine microorganism fermentative production low temperature lipoxygenase terminates; Described liquid culture medium: extractum carnis 3.0 ~ 10.0g, peptone 5.0 ~ 10.0g, NaCl5.0 ~ 10.0g, L-Aspartic acid 0.5 ~ 2.0g, Pidolidone 0.5 ~ 2.0g, Tween800.1 ~ 0.4g, (NH 4) 2sO 41.0 ~ 1.5g, KH 2pO 41.0 ~ 2.0g, MgSO 40.1 ~ 0.5g, CaCl 20.1 ~ 0.3g, ZnSO 47H 2o 2.4 ~ 3.0mg, tap water 1.0L, pH value is 6.0 ~ 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
(4) by the fermented liquid of step (3) 4,000 ~ 10,000rpm collected by centrifugation thalline, ultrasonication, 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 zymin of different activities, purity and formulation.
Further, the strain activation and culture base of step (1) generation lipoxygenase is: yeast extract paste 5.0g, peptone 10.0g, NaCl 10.0g, agar 20.0g, tap water 1.0L, pH value is 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min.
The explanation that in the present invention, lipoxygenase producing strains initial activation and growth conditions provide by China Committee for Culture Collection of Microorganisms's common micro-organisms center is carried out.Bacterial strain first activates, produce low temperature lipoxygenase 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 lipoxygenase, 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, 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 main application operating of lipoxygenase of the inventive method production is simple, convenient, fast, cost is low, be applicable to medicine, food-processing, papermaking and weaving, print and dye and wash and other lipoxygenase application industry, there is wide potentiality to be exploited and application prospect.
Embodiment
Embodiment 1
(1) medium preparing
1. strain activation and culture base: yeast extract paste 5.0g, peptone 10.0g, NaCl10.0g, agar 20.0g, tap water 1.0L, pH value is 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
2. bacterial classification domestication by low temperature substratum: peptone 5.0g, linolic acid 2.0g, NaCl 5.0g, L-Aspartic acid 0.5g, Pidolidone 0.5g, agar 20.0g, tap water 1.0L, pH value is 8.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
3. liquid seed culture medium: yeast extract paste 10.0g, peptone 5.0g, NaCl 5.0g, L-Aspartic acid 0.5g, Pidolidone 0.5g, tap water 1.0L, pH value is 8.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
4. liquid culture medium: extractum carnis 3.0g, peptone 5.0g, NaCl 5.0g, L-Aspartic acid 0.5g, Pidolidone 0.5g, Tween800.1g, (NH 4) 2sO 41.0g, KH 2pO 41.0g, MgSO 40.1g, CaCl 20.1g, ZnSO 47H 2o 2.4mg, tap water 1.0L, pH value is 7.0, in 103kpa, 21 DEG C of high pressure steam sterilization 30min;
(2) can produce the bacterial classification of lipoxygenase, 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, 30 DEG C → 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 in low temperature environment;
(4), after according to a conventional method the lipoxygenase 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 5L of fermentating liquid volume 3 ~ 5%, when cultivating 120 ~ 144h for 10 ~ 12 DEG C, namely marine microorganism fermentative production low temperature lipoxygenase terminates;
(6) fermented liquid of (5) is collected thalline, ultrasonication at 4,000 ~ 10,000rpm centrifugal 20min, the supernatant liquor collected is crude enzyme liquid, and the work of crude enzyme liquid enzyme can reach 166.9U/ml;
(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 lipoxidase zymin of different activities, purity and formulation.
Embodiment 2
(1) medium preparing
1. strain activation and culture base: yeast extract paste 5.0g, peptone 10.0g, NaCl10.0g, agar 20.0g, tap water 1.0L, pH value is 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
2. bacterial classification domestication by low temperature substratum: peptone 10.0g, linolic acid 5.0g, NaCl 6.0g, L-Aspartic acid 0.8g, Pidolidone 1.0g, agar 15.0g, tap water 1.0L, pH value is 6.5, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
3. liquid seed culture medium: yeast extract paste 12.0g, peptone 8.0g, NaCl 6.0g, L-Aspartic acid 1.0g, Pidolidone 1.0g, tap water 1.0L, pH value is 6.5, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
4. liquid culture medium: extractum carnis 5.0g, peptone 6.0g, NaCl 8.0g, L-Aspartic acid 1.0g, Pidolidone 1.0g, Tween800.2g, (NH 4) 2sO 41.2g, KH 2pO 41.5g, MgSO 40.3g, CaCl 20.2g, ZnSO 47H 2o2.6mg, tap water 1.0L, pH value is 6.5, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
(2) will produce the bacterial classification of lipoxygenase, 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, 30 DEG C → 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 in low temperature environment;
(4), after according to a conventional method the lipoxygenase producing strains after domestication by low temperature being cultivated 48 ~ 72h in 12 ~ 14 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 50L of fermentating liquid volume 5 ~ 7%, when cultivating 96 ~ 120h for 12 ~ 14 DEG C, namely marine microorganism fermentative production low temperature lipoxygenase terminates;
(6) by the fermented liquid of (5) at 4,000 ~ 10,000rpm collected by centrifugation liquid, the liquid collected is crude enzyme liquid, and the work of crude enzyme liquid enzyme can reach 170.0U/ml;
(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 lipoxidase zymin of different activities, purity and formulation.
Embodiment 3
(1) medium preparing
1. strain activation and culture base: yeast extract paste 5.0g, peptone 10.0g, NaCl10.0g, agar 20.0g, tap water 1.0L, pH value is 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
2. bacterial classification domestication by low temperature substratum: peptone 15.0g, linolic acid 10.0g, NaCl10.0g, L-Aspartic acid 2.0g, Pidolidone 2.0g, agar 20.0g, tap water 1.0L, pH value is 6.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
3. liquid seed culture medium: yeast extract paste 20.0g, peptone 10.0g, NaCl10.0g, L-Aspartic acid 2.0g, Pidolidone 2.0g, tap water 1.0L, pH value is 6.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
4. liquid culture medium: extractum carnis 10.0g, peptone 10.0g, NaCl10.0g, L-Aspartic acid 2.0g, Pidolidone 2.0g, Tween800.4g, (NH 4) 2sO 41.5g, KH 2pO 42.0g, MgSO 40.5g, CaCl 20.3g, ZnSO 47H 2o 3.0mg, tap water 1.0L, pH value is 6.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
(2) will produce the bacterial classification of lipoxygenase, 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, 30 DEG C → 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 in low temperature environment;
(4), after according to a conventional method the lipoxygenase producing strains after domestication by low temperature being cultivated 48 ~ 72h in 14 ~ 16 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 500L of fermentating liquid volume 7 ~ 9%, when cultivating 72 ~ 96h for 14 ~ 16 DEG C, namely marine microorganism fermentative production low temperature lipoxygenase terminates;
(6) by the fermented liquid of (5) at 4,000 ~ 10,000rpm collected by centrifugation liquid, the liquid collected is crude enzyme liquid, and the work of crude enzyme liquid enzyme can reach 169.1U/ml;
(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 lipoxidase zymin of different activities, purity and formulation.

Claims (2)

1. a method for marine microorganism fermentative production low temperature lipoxygenase, is characterized in that, comprise the following steps:
(1) actication of culture of lipoxygenase can be produced, then through domestication by low temperature step by step, acclimation temperature from high to low, 30 DEG C → 25 DEG C → 22 DEG C → 20 DEG C → 18 DEG C → 16 DEG C → 14 DEG C → 12 DEG C → 10 DEG C, makes its well-grown in low temperature environment; The domestication by low temperature substratum of this bacterial classification is: peptone 5.0 ~ 15.0g, linolic acid 2.0 ~ 10.0g, NaCl 5.0 ~ 10.0g, L-Aspartic acid 0.5 ~ 2.0g, Pidolidone 0.5 ~ 2.0g, agar 20.0g, tap water 1.0L, pH value is 6.0 ~ 8.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
Described can produce the microbe-derived in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of lipoxygenase, bacterium numbering: 1.228 or 1.239 or 1.2620;
(2) according to a conventional method by the lipoxygenase producing strains after domestication by low temperature in step (1) in liquid seed culture medium in 10 ~ 18 DEG C of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Described liquid seed culture medium is: yeast extract paste 10.0 ~ 20.0g, peptone 5.0 ~ 10.0g, NaCl5.0 ~ 10.0g, L-Aspartic acid 0.5 ~ 2.0g, Pidolidone 0.5 ~ 2.0g, tap water 1.0L, pH value is 6.0 ~ 8.0, in 103kpa, 121 DEG C of high pressure steam sterilization 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 ~ 18 DEG C, namely marine microorganism fermentative production low temperature lipoxygenase terminates; Described liquid culture medium is: extractum carnis 3.0 ~ 10.0g, peptone 5.0 ~ 10.0g, NaCl5.0 ~ 10.0g, L-Aspartic acid 0.5 ~ 2.0g, Pidolidone 0.5 ~ 2.0g, Tween800.1 ~ 0.4g, (NH 4) 2sO 41.0 ~ 1.5g, KH 2pO 41.0 ~ 2.0g, MgSO 40.1 ~ 0.5g, CaCl 20.1 ~ 0.3g, ZnSO 47H 2o 2.4 ~ 3.0mg, tap water 1.0L, pH value is 6.0 ~ 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min;
(4) by the fermented liquid of step (3) 4,000 ~ 10,000rpm collected by centrifugation thalline, ultrasonication, 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 zymin of different activities, purity and formulation.
2. the method for marine microorganism fermentative production low temperature lipoxygenase according to claim 1, it is characterized in that, the strain activation and culture base that step (1) produces lipoxygenase is: yeast extract paste 5.0g, peptone 10.0g, NaCl 10.0g, agar 20.0g, tap water 1.0L, pH value is 7.0, in 103kpa, 121 DEG C of high pressure steam sterilization 30min.
CN201510084631.0A 2015-02-16 2015-02-16 Method for producing low-temperature lipoxygenase by fermenting marine microorganisms Pending CN104630164A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102093988A (en) * 2009-12-11 2011-06-15 大连大学 Method for producing low-temperature lipase by microbial fermentation
CN102277317A (en) * 2011-07-28 2011-12-14 江南大学 Lipoxygenase-producing pseudomonas aeruginosa and screening method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102093988A (en) * 2009-12-11 2011-06-15 大连大学 Method for producing low-temperature lipase by microbial fermentation
CN102277317A (en) * 2011-07-28 2011-12-14 江南大学 Lipoxygenase-producing pseudomonas aeruginosa and screening method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. VIDAL-MAS等: "Cloning and expression of a lipoxygenase from Pseudomonas aeruginosa 42A2", 《ANTONIE VAN LEEUWENHOEK》 *
R. E. VANCE等: "The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES》 *

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Application publication date: 20150520