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Screening of a Red Mutant of Cholesterol Oxidase Producer from Brevibacterium sp. and the Study of Related Enzyme Stability

Author: SangJi
Tutor: WangWu
School: Jiangnan University
Course: Microbiology
Keywords: Cholesterol oxidase Steroidal Brevibacterium Compound Mutation Purification Stability
CLC: TQ925
Type: Master's thesis
Year: 2006
Downloads: 110
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Abstract


In this experiment, the production of cholesterol oxidase ( COD ) steroidal Brevibacterium ( Brevibacterium sp.DGCDC - 82 ) as a starting strain NTG mutagenesis by ultrasonic - assisted breeding , red mutant strain Brevibacterium sp . DGCCN - 25 , the mutant enzyme production capacity increased 140 % , the COD level reached 1.21U/ml . Red substance produced strains DGCCN - 25 high performance liquid chromatography and LC-MS chromatograms , suggesting that alcohol-soluble red pigment molecular weight of 792 , has yet to see the related research reports , tentatively scheduled to be called steroid rod orange pigment. Optimized DGCCN - 25 production COD fermentation process , the use of orthogonal experimental analysis adjusted for enzyme production medium , derived mutant strain is more appropriate conditions for enzyme production : the amount of cholesterol add from 3g / L increased to 4 g / L COD yield increased by 24.8 % , yeast extract from 8 g / L increased to 9g / L , compared with optimized medium , enzyme activity reached 1.51U/ml . The separation and purification of DGCCN - 25 production of COD . The crude extract focused on solving the problem of excessive loss of activity after the centrifugation of the fermentation broth by adding 4.4 % ( V / V ) of isopropanol in the fermentation broth after centrifugation COD yield increased from 75% to 92% . Sibiricum combination of Sephadex G-25 gel filtration chromatography and DEAE - Sephadex ion exchange chromatography combined in the enzyme solution after the crude extract was further purified . G-25 chromatography , the specific activity of 4.95U/mg purification factor of 2.58 times ; specific activity reached 13.26U/mg DEAE - Sephadex ion exchange chromatography purification factor of 2.67 times . G-25 chromatography , specific activity reached 15.31U/mg , reached a preliminary purification purposes , the total yield of 41.3% . Enzyme stability study found that the extraction of the crude enzyme solution was stored at 4 ° C conditions , the loss of activity ; purified COD in more than 40 ° C heat treatment , the stability is poor ; vacuum freezing process loss of activity . Adding a stabilizing agent sodium potassium tartrate - mannitol helps to improve the stability of the COD : 4 ° C storage 350 Days enzyme activity retention rate was 70 % , while the control group was only 20 % ; 3h 40 ° C heat treatment , compared with control group , the activity retention rate increased from 70% to 90% ; vacuum freeze- drying treatment , the loss of activity was 10 %, less than 30% of the control .

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CLC: > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )
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