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Optimization of Fermentation Conditions and Partical Characteirzation of Thermostable Enzymes Activities of Crude Extracts from Geobacillus sp. CHB1

Author: WuWenZuo
Tutor: HuangZhiPeng; WangShiHua; LinXinJian
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Geobacillus sp. thermostable protease thermostable α-amylase clone
CLC: TQ925
Type: Master's thesis
Year: 2012
Downloads: 89
Quote: 0
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Geobacillus was named as a new genus in2001, and it is a kind of thermophilicaerobic. The Bacillus strain CHB1, identified belong to Geobacillus sp. by ChineseAcademy of Sciences, was isolated from the soil beside hen house by Soil andFertilizer Institute, Fujian Academy of Agricultural Sciences. Taking CHB1as theobject of this study, we optimized the conditions for the production of protease andα-amylase under high temperature, investigated the characterization of these twoenzymes and also cloned the gene encoded α-amylase.Thermostable protease produced from CHB1at60℃could be observed on theselective medium of protease. The conditions for the production of protease wereoptimized. The optimal medium combination comprised2.0%of beef extract,0.01%of NaCl and0.001%of CuSO4, and the optimal cluture conditions comprised40mLfor bottling volume, pH at7.0,5.0%for inoculum percent and60℃for culturetemperature were obtained. The maximum enzyme activity reached up to70U. Theresults of the characterization of protease showed that the maximal activity ofprotease occured at60℃and maintain70%of the maximal activity after incubation at100℃for3h, the enzyme remained stable in a wide pH range including acidic,neutral and alkaline conditions, and the Cu2+could improve the activity of protease. Itcan be deduced that there are acid protease, neutral protease, alkali protease and somemetal protease in the protease produced by CHB1. So CHB1has the potential to beused in industry for the abundance resource of protease.Thermostable α-amylase produced from CHB1at60℃could be observed on the selective medium of amylase. The conditions for the production of α-amylase wereoptimized. The optimal medium combination comprised0.3%of beef extract,0.5%ofsoy peptone,1.0%of cyclodextrin,0.2%of CaCl2,0.05%of KH2PO3and0.05%ofFeSO4, and the optimal cluture conditions comprised30mL for bottling volume, pHat7.0,5.0%for inoculum percent,60℃for culture temperaturewere obtained. Themaximum enzyme activity reached up to80U. The results of the characterization ofα-amylase showed that the maximal activity of α-amylase occured at60℃andmaintain80%of the maximal activity after incubation at100℃for1h, the enzymeremained high activity in acid and alkali condition with more stable for the former,and the low concentration of Al3+(0.01mol/L), some denaturing agent and organicsolvent could improve the activity of α-amylase. SDS-PAGE showed that themolecular weight of α-amylase extracted by ammonium sulfate fractionation wasabout67.5kDa.With genomic DNA as template and degenerate primers designed by CODEHOPmethod, the partial sequence of the gene encoded α-amylase was cloned andsequenced. The completed sequence of the gene encoded α-amylase was cloned byrepeated genomic walking PCR protocols using a DNA Walking SpeedUp premixkit. Unfortunately the clone was failed at last.

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