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Screening of a Alkaline Protease Producing Strain and Cloning and Expression of Its Apr Gene

Author: LiLinZuo
Tutor: MaXiangDong
School: Henan Agricultural University
Course: Microbiology
Keywords: Bacillus subtilis Protease gene cloning Protease gene expression
CLC: Q78
Type: Master's thesis
Year: 2006
Downloads: 234
Quote: 7
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Abstract


In this study, the use of traditional microbiological methods , from Zhengzhou dairy soil sample screening to an alkaline protease producing strains of A - 109 , morphology , physiological and biochemical reactions , preliminary identification of the strain of Bacillus subtilis . Strain A-109 of total DNA as a template, the protease coding sequence was amplified using the method of landing PCR (Touchdown PCR, TD-PCR) of total DNA sample , to obtain the desired fragment . Sequencing of the amplified fragment , sequencing and homology analysis showed that: the fragment containing 1200 base pairs contained in the reading frame containing 1096 base pairs of the fragment for the protease DNA fragment homologous proteases , can be found in the database gene sequence. DNA fragment was cloned into the protease and alkaline protease E (GenBank No: AJ539133) the coding region of homology of up to 99%, and the coding region sequence of the neutral protease (GenBank No: NC003997) up to a 94 % similarity , at least there are 20 amino acid differences . Cloned into E. coli expression vector PET obtained recombinant plasmid for expression in E. coli BL21 . The results show that : the culture temperature at 30 ℃ after 4h by the inducer IPTG (0.3mmol / L) induced expression of proteases produced a large amount of expression of the product can not be directly secreted into the medium , the colony after cleavage by the Lysates expression product hydrolysis ring can be produced in the milk plates , but the protease activity of the expression produces far less than the protease activity of wild bacteria produce ; hydrolysis ring according to the mature protease can be formed in the milk plates , can initially determine the enzyme having a proteolytic activity , and further the enzymatic Properties detection that the protease optimum temperature of 50 ° C , the optimum PH of 8.5 .

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