CN104694504A - Method for producing heat-resistant superoxide dismutase (SOD) by using moorella thermoautotrophica - Google Patents

Method for producing heat-resistant superoxide dismutase (SOD) by using moorella thermoautotrophica Download PDF

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CN104694504A
CN104694504A CN201510070705.5A CN201510070705A CN104694504A CN 104694504 A CN104694504 A CN 104694504A CN 201510070705 A CN201510070705 A CN 201510070705A CN 104694504 A CN104694504 A CN 104694504A
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sod
moorella thermoautotrophica
superoxide dismutase
fermentation
thermoautotrophica
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丁彦蕊
王雪芹
饶榕
牟兆琳
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Jiangnan University
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Jiangnan University
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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.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

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  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
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Abstract

The invention relates to a method for producing superoxide dismutase (SOD) by using moorella thermoautotrophica and belongs to the technical field of bioengineering. According to the method, SOD is produced from moorella thermoautotrophica under the condition of liquid-state fermentation and is an ectoenzyme. The yield of heat-resistant SOD can reach 2000U/ml to the maximum, so that the method has a good industrial application prospect.

Description

Moorella thermoautotrophica produces the method for heat-resistant superoxide dismutase (SOD)
Technical field
Utilize Moorella thermoautotrophica to produce superoxide-dismutase (SOD), belong to technical field of bioengineering.The present invention relates to and utilize bacterial strain Moorella thermoautotrophica preserving number to be ATCC33924, obtain high temperature resistant SOD by liquid state fermentation.
Background technology
Superoxide-dismutase (superoxide dismutase, be called for short SOD) has the effect of good defence oxygen toxicity, can the ability of enhancing body Antiradiation injury.In recent years, it has become the novel anti-inflammatory drug for the treatment of inflammation, tumour and autoimmune disease; For sun-proof, anti-skin aging and the additive of anti-inflammatory and the biochemical reagents for some disease probe in makeup and food service industry.SOD is extensively present in animal, plant, all aerobic microbiologicals and minority anaerobion body.Current, SOD mainly extracts from animal blood (as pig blood or ox blood) (Yuan Ye. the preparation method [P] of superoxide-dismutase. application number, 02147888.0,2002-12-19; Xu Wenzhong, Yan Jiaqi. large-scale production process of superoxide dismutase [P]. application number, 01144922.5,2001-12-24), output is very limited, and is easily subject to the impact of the factors such as raw material sources, security and quality instability.Due to animal, plant particularly animal blood source difficulty, and microorganism can artificial culture, is conducive to realizing suitability for industrialized production.According to current report, can pass through Production by Microorganism Fermentation SOD, but be mainly intracellular enzyme (Diao Zhimin, Yao Yinxia, Zhang Wenjing. the research [J] of yeast SOD fermentation condition. Qinghai Normal University's journal (natural science edition), 2002,4:48-51; Yang Mingyan, Guo Ailian, Shen Jian, etc. the saccharomycetic mutagenic and breeding of high yield SOD and Fermentation Conditions [J]. Food science, 2005,26 (10): 147-150), extraction process is comparatively loaded down with trivial details.
Natural SOD thermostability is poor, easily by heat inactivation in processing and use procedure.How effectively to solve the stable problem of SOD enzyme activity under high temperature process conditions, how to make the product containing SOD need not scruple the key that high temperature is efficiency utilization SOD in the technological process of production.Utilize biotechnology to produce high stability of new generation, the SOD of high heat resistance is that current SOD develops most important approach.
The present invention adopts Moorella thermoautotrophica to produce SOD, Moorella thermoautotrophica is a kind of thermoduric bacteria, resistant to elevated temperatures SOD can be produced, this bacterium fermentative production SOD is adopted to have fermentation period short, in fermented liquid, SOD vigor is higher, and can be high temperature resistant, DEVELOPMENT PROSPECT is wide.
Summary of the invention
The Strain comparison that can produce SOD is at present few, is mainly some yeast, and is intracellular enzyme, and needing after broken wall could extraction and isolation, and running cost is high, is unfavorable for prepared by scale operation.The object of the invention is for current problem, utilize bacterial strain Moorella thermoautotrophica preserving number to be ATCC 33924, obtain the outer heat-resisting SOD of born of the same parents by liquid state fermentation.
Technical scheme of the present invention is as follows: liquid state fermentation substratum composition is carbon source 0.5-100g/l, nitrogenous source 0.5-100g/l, K 2hPO 41-4g/l, MgSO 47H 2o 0.1-0.5g/l, NaCl 1-2g/l.The substratum of fermentation volume 60-70% (v/v) is loaded in the fermentor tank of certain volume, access Moorella thermoautotrophica after sterilizing cooling, inoculum size is 1-10%, initial pH and fermenting process pH is nature, leavening temperature is 20-60 DEG C, and fermentation period is 2-6 days.In final fermented liquid, SOD vigor reaches as high as 2000U/ml.
The present invention's all seed culture based formulas is identical with fermention medium.After slant strains is inoculated into the triangular flask that seed culture medium is housed, the incubation time of seed is 48 hours, and the culture temperature of seed is 45 DEG C.
Fungi preservation slant culture based formulas is: glucose 20g/l, MgSO 47H 2o 0.2g/l, yeast extract paste g/l, agar 20g/l, pH nature.
The measuring method that SOD lives suppresses nitroblue tetrazolium(NBT) (NBT) reductive action under light based on SOD.SOD vigour-testing method and the definition of SOD vigor refer to: Chin-Wen Lin, Jeng-Huh Yang and Lieh-Chi Su, The extraction and properties of superoxide dismutase from porcine blood Chin-Wen Lin, Jeng-Huh Yang and Lieh-Chi Su, Meat Science, 1997,46 (3): 303-312.
The present invention has following feature:
(1) Moorella thermoautotrophica is used for the carbon source of SOD fermentation can be glucose, fructose, maltose, wood sugar, sucrose, lactose, semi-lactosi.Nitrogenous source can be (NH 4) 2sO 4, NH 4cl, NH 4nO 3, NaNO 3, peptone, yeast extract paste, extractum carnis, urea, corn steep liquor, soybean cake powder.
(2) fermenter volume that Moorella thermoautotrophica fermentation SOD adopts is 5-50L.Mixing speed is 70-150r/min, ventilation 1:0.1-1:0.5v/v/m.Basic general knowledge in conjunction with this area can adjust fermentor tank size according to the condition of production.
Beneficial effect of the present invention: adopt Moorella thermoautotrophica to carry out the fermentation of SOD first, not only enzyme is lived high, and the SOD produced has good heat-resisting effect, the method is applicable to the heat-resisting SOD of industrialization scale operation, not only solve the heat labile shortcoming of current SOD, and provide a large amount of SOD sources.
Embodiment
Embodiment 1
Slant strains be inoculated into and be equipped with in the 250ml triangular flask of 50ml substratum, substratum consists of: glucose 20g/l, extractum carnis 10g/l, peptone 10g/l, NH 4nO 310g/l, corn starch 12g/l, pH nature.45 DEG C, ferment 72 hours.After fermentation ends, by centrifugal for 50ml fermented liquid 10000 turns/min, remove thalline.Supernatant crude enzyme liquid is for measuring pH and the temperature parameter of enzyme.Get supernatant crude enzyme liquid 0.5ml, be placed in pH 4 respectively, 5,6,7, in the citrate phosphate buffer 3.5ml of 8, at 55 DEG C, measure SOD vigor be respectively 1600,1700,2000,1700,1600U/ml.Therefore the optimal reaction pH of this SOD is 6.Get supernatant crude enzyme liquid 0.5ml respectively, mix with the citrate-phosphate salt buffer of 3.5ml pH 6,25, recording enzyme work at 35,45,55,65 DEG C is respectively 1600,1700,1800,2000,1800U/ml, and therefore the optimum temperuture of this enzyme is 55 DEG C.Get supernatant crude enzyme liquid 0.5ml respectively, mix with the citrate-phosphate salt buffer of 3.5ml pH 6,25,35,45,55,65, be incubated 48 hours at 75 DEG C, then mensuration enzyme is lived and is respectively 2000,2000,2000,2000,2000,1900U/ml, result shows that this enzyme has good thermotolerance.The compound method of the citrate-phosphate salt buffer of different pH is see Zhu Gejian, industrial microorganism experimental technique handbook, China Light Industry Press, 1994.
Embodiment 2
Slant strains be inoculated in the 250ml triangular flask of the seed culture medium that 50ml is housed, seed culture medium consists of: glucose 20g/l, extractum carnis 10g/l, peptone 10g/l, NH 4nO 310g/l, corn starch 12g/l, pH nature, 121 sterilizing 20min, shaking speed 150 revs/min, cultivates 48 hours at 45 DEG C.This process has 10 identical triangular flasks, obtains 500ml seed liquor altogether.
Inoculate 500ml seed liquor into 50L fermentor tank, fermention medium consists of: glucose 20g/l, extractum carnis 10g/l, peptone 3g/l, NH 4nO 310g/l, corn starch 6g/l, pH nature, leavening temperature is 45 DEG C, liquid amount 70%, and ventilation is 1:1.5v/v/m.Fermentation time is 3 days.Under this condition, in fermented liquid, final SOD vigor is 1800U/ml.
Embodiment 3
Cultured 1L seed liquor is inoculated in 10L fermentor tank.Seed and fermention medium composition are: fructose 30g/l, extractum carnis 10g/l, corn starch 20g/l, K 2hPO 44g/l, MgSO 47H 20.5g/l, pH nature.Leavening temperature is 20 DEG C, liquid amount 70%, and ventilation is 1:0.3v/v/m, and fermentation time is 5 days, and under this condition, in fermented liquid, final SOD vigor is 1400U/ml.
Embodiment 4
Cultured 2L seed liquor is inoculated in 20L fermentor tank.Seed and fermention medium consist of: lactose 20g/l, NH 4nO 310g/l, peptone 10g/l, pH nature.Inoculum size is 10%, and culture temperature is 60 DEG C, liquid amount 60%, and ventilation is 1:0.5v/v/m, and mixing speed is 150r/min, and fermentation time is 6 days, and under this condition, in fermented liquid, final SOD vigor is 1300U/ml.
Embodiment 5
Cultured 300ml seed liquor is inoculated in 5L fermentor tank.Seed and fermention medium composition are: sucrose 20g/l, NH 4nO 310g/l, peptone 10g/l, pH nature.Leavening temperature is 40 DEG C, liquid amount 65%, and ventilation is 1:0.1v/v/m, and mixing speed is 70r/min, and fermentation period is 3 days.Under this condition, in fermented liquid, final SOD vigor is 1800U/ml.
Embodiment 6
Cultured 3.5L seed liquor is inoculated in 50L fermentor tank.Seed and fermention medium composition are: maltose 15g/l, yeast extract paste 5g/l, peptone 10g/l, soybean cake powder 5g/l, pH nature, leavening temperature is 28 DEG C, liquid amount 70%, ventilation is 1:0.4v/v/m, and mixing speed is 150r/min, and fermentation period is 5 days.Under this condition, in fermented liquid, final SOD vigor is 1950U/ml.
Embodiment 7
Cultured 600ml seed liquor is inoculated in 10L fermentor tank.Seed and fermention medium composition are: semi-lactosi 15g/l, yeast extract paste 5g/l, peptone 5g/l, urea 5g/l, pH nature.Leavening temperature is 30 DEG C, liquid amount 60%, and ventilation is 1:0.3v/v/m, and mixing speed is 120r/min, and fermentation time is 4 days.Under this condition, in fermented liquid, final SOD vigor is 1650U/ml.
Embodiment 8
Cultured 250ml seed liquor is inoculated in 5L fermentor tank.Seed and fermention medium composition are: wood sugar 10g/l, yeast extract paste 5g/l, NaCl 1g/l, K 2hPO 41g/l, MgSO 47H 2o 0.1g/l, pH nature.Leavening temperature is 45 DEG C, liquid amount 50%, and ventilation is 1:1.2v/v/m, and fermentation time is 2 days.Under this condition, in fermented liquid, final SOD vigor is 800U/ml.

Claims (3)

1. the present invention utilizes Moorella thermoautotrophica to produce heat-resisting superoxide-dismutase (SOD) by liquid state fermentation.
2. Moorella thermoautotrophica according to claim 1, is characterized in that the carbon source of available product SOD is glucose, fructose, maltose, wood sugar, sucrose, lactose, semi-lactosi, whey.
3. Moorella thermoautotrophica according to claim 1, is characterized in that the nitrogenous source of available product SOD is inorganic ammonium salt, inorganic nitrate, peptone, yeast extract paste, urea, corn steep liquor, soybean cake powder.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1814753A (en) * 2005-02-01 2006-08-09 中国科学院微生物研究所 Heat-resistant superoxide dismutase and its conding gene and use
CN101275144A (en) * 2008-03-13 2008-10-01 浙江清华长三角研究院 High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase
CN101368163A (en) * 2008-09-25 2009-02-18 山东农业大学 Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod
CN102770445A (en) * 2009-11-11 2012-11-07 赢创罗姆有限公司 Use of a protein homologous to a Meab protein for increasing the enzymatic activity of a 3-hydroxycarboxylic acid-Coa mutase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1814753A (en) * 2005-02-01 2006-08-09 中国科学院微生物研究所 Heat-resistant superoxide dismutase and its conding gene and use
CN101275144A (en) * 2008-03-13 2008-10-01 浙江清华长三角研究院 High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase
CN101368163A (en) * 2008-09-25 2009-02-18 山东农业大学 Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod
CN102770445A (en) * 2009-11-11 2012-11-07 赢创罗姆有限公司 Use of a protein homologous to a Meab protein for increasing the enzymatic activity of a 3-hydroxycarboxylic acid-Coa mutase

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MELIKE BALK ET AL.: "(Per)chlorate Reduction by the Thermophilic Bacterium Moorella perchloratireducens sp. nov., Isolated from Underground Gas Storage", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 *
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