Strain Screening, Fermentation Of Thermostable α-amylase And Molecular Cloning Of The Enzyme

Thermostableα-amylse,catalyzing hydrolysis of starch at high temperatures,is one of the most important enzyme.It is widely applied in desinzing,sugar and fermentation industry.Bacillus subtilis is one of the industrial strains,which secretes thermostable a-amylase at high level.In order to obtain the thermostable desizing enzyme,which can be used in textile industry,a strain was isolated from soil,and the characteristics of its crude enzyme were studied.Based on the physiological and biochemical characteristics,and the phylogenetic analysis of 16S rDNA sequence,the strain C1 is identified preliminarily as Bacillus subtilis,temporarily named Bacillus subtilis C1.In the basal medium,the Strain C1 reaches its stationary phase within 24 hours at pH 7 and 45℃.And,at 68 h,it reaches the highest enzyme activity of 185.6u/mL.The study of the characters of the high-temperature alpha-amylase shows that the optimum pH is 6 and the optimum temperature is 70℃.This enzyme is stable to high temperature,and 81%of enzyme activity is retained when disposed at 70℃for 1h.It is significant to study further on the strain for the industrial application.In the study,the optimum medium for Bacillus subtilis C1 was systematically studied withminitab software.On the basis of single factor experiments,the prime factors affecting enzyme activity were selected by means of Plackett-Burman design.And then,the factors were optimized by response surface analysis.The result shows that the optimal medium composition was as follows:wheat bran 2.16 g,cotton seed meal 1.98 g, yeast extract 0.40 g,NaCl 1.00 g,CaCl₂ 0.40 g,starch 0.10 g.Enzyme activity under the optimal culture conditions is 2329 u·mL^-1,which is 12.6 fold of that of the original medium,this study has a significant advancement.The experimental data under various conditions have validated the theoretical values.About 1.5 kb specific fragment was obtained from genomic DNA of Bacillus subtilis C1 by PCR amplification with primers designed according to the known sequences of theα-amylase gene in the Gene Bank.And then the fragment was ligated with pPICZa A Vector, transformated into Top 10 F' competent cell successfully.Cloning of theα-amylase gene of Bacillus subtilis C1 fotmded the base for construction of the engineering strain with high-yieldα-amylase.