CN104694497A

CN104694497A - Method for producing heat-resistant superoxide dismutase (SOD) by using thermobifida fusca

Info

Publication number: CN104694497A
Application number: CN201510069874.7A
Authority: CN
Inventors: 丁彦蕊; 牟兆琳; 王雪芹; 饶榕
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-02-10
Filing date: 2015-02-10
Publication date: 2015-06-10

Abstract

The invention relates to a method for producing superoxide dismutase (SOD) by using thermobifida fusca and belongs to the technical field of bioengineering. According to the method, SOD is produced from thermobifida fusca under the condition of liquid-state fermentation and is an ectoenzyme. The yield of heat-resistant SOD can reach 2500U/ml to the maximum, so that the method has a good industrial application prospect.

Description

Brown is thermophilic splits the method that spore bacterium produces heat-resistant superoxide dismutase (SOD)

Technical field

Utilize brown thermophilic split spore bacterium produce superoxide-dismutase (SOD), belong to technical field of bioengineering.The present invention relates to and utilize bacterial strain Thermobifida fusca preserving number to be ATCC 66938, 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 that brown is thermophilic to be split spore bacterium and produce SOD, and brown is thermophilic, and to split spore bacterium be a kind of thermoduric bacteria, and can produce resistant to elevated temperatures SOD, adopt this bacterium fermentative production SOD to have fermentation period short, in fermented liquid, SOD vigor is higher, and can be high temperature resistant, and 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 Thermobifida fusca preserving number to be ATCC 66938, 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 ₂00.1-0.5g/l, NaCl 1-2g/l.The substratum of fermentation volume 60-70% (v/v) is loaded in the fermentor tank of certain volume, after sterilizing cooling, brown is thermophilic splits spore bacterium in access, and inoculum size is 1-10%, and initial pH and fermenting process pH is nature, leavening temperature is 20-45 DEG C, and fermentation period is 2-6 days.In final fermented liquid, SOD vigor reaches as high as 2500U/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 ₂00.2g/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) thermophilic to split spore bacterium for the carbon source that SOD ferments can be glucose, fructose, maltose, wood sugar, sucrose, lactose, semi-lactosi to brown.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) the thermophilic fermenter volume splitting spore bacterium fermentation SOD employing of brown 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 that brown is thermophilic to be split spore bacterium and carried 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 45 DEG C, measure SOD vigor be respectively 2200,2300,2500,2300,2200U/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 2100,2300,2500,2000,1900U/mL, and therefore the optimum temperuture of this enzyme is 45 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 2500,2500,2500,2500,2300,2200U/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 2450U/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 2500U/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 45 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 2050U/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 2450U/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 2500U/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 2050U/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 ₂00.1g/l, pH nature.Leavening temperature is 45 DEG C, liquid amount 50%, and ventilation is 1:1.2v/v/m, and fermentation time is 4 days.Under this condition, in fermented liquid, final SOD vigor is 1800U/ml.

Claims

1. the present invention utilizes that brown is thermophilic to be split spore bacterium (Thermobifida fusca) and produce heat-resisting superoxide-dismutase (SOD) by liquid state fermentation.

2. brown according to claim 1 is thermophilic splits spore bacterium, it is characterized in that the carbon source of available product SOD is glucose, fructose, maltose, wood sugar, sucrose, lactose, semi-lactosi, whey.

3. brown according to claim 1 is thermophilic splits spore bacterium, it 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.