%0 Journal Article
%T Cloning and Expression of Randomly Mutated Bacillus subtilis -Amylase Genes in HB101
%A Mohammed Rabbani
%A Hamid MirMohammad Sadeghi
%A Fatemeh Moazen
%A Mehran Rahimi
%A Golnaz Salehi
%J Biotechnology Research International
%D 2011
%I Hindawi Publishing Corporation
%R 10.4061/2011/305956
%X The aim of this study was to isolate and express the randomly mutated ¦Á-amylase gene from B. subtilis strain 168. BS168F: 5¡ä-gtgtcaagaatgtttgc-3¡ä and BS168R: 3¡ä-gttttgttaaaagatga-5¡ä primers were used to amplify the amylase gene using the following cycle in error-prone PCR method: 94¡ãC for 30£¿s, 40¡ãC for 2 min, and 72¡ãC for 2 min in 30 cycles that were followed with 72¡ãC for 2 min as a post cycle. E. coli XL1 blue was used as host for plasmid construction. Amylase enzyme activity assay was performed using continuous spectrophotometric procedures. Results of sequencing showed that sequence was cloned from the first ATG and with the correct open reading frame. Having confirmed the integrity of the insert, the gene was ligated into expression vector pET-15b and then further confirmed using digestion analysis. Amylase activity showed 3 clones with higher enzymatic activity compared with the wild type. Error-prone PCR method produced a mutated gene that provides amylase activity much higher than that of wild type. Sequencing the mutated genes should shed light on the important region of the genes that could be manipulated in future studies. 1. Introduction -Amylases are extracellular enzymes which randomly cleave the -1,4 linkages between adjacent glucose units in the linear amylase chain, generating glucose, maltose, and maltotriose units [1]. According to the type of bonds they hydrolyze and their products, they are classified as ¦Á, ¦Â, and amylases [2]. They are among the most important commercial enzymes, with wide applications in starch processing, brewing, alcohol production, textile, paper, detergent, clinical, medicinal, and analytical chemistry [1]. Amylase enzymes make up approximately 25% of the enzyme market. Although -amylases can be derived from plants and animals, they are the enzymes from microbial sources (typically Bacillus spp.) that are generally used to meet the expanding industrial demands [3]. Mesophilic and thermophilic fungi are also good source of amylolytic enzymes due to their working conditions and reaction specificity properties [4]. The Gram-positive bacterium B. subtilis naturally thrives in the soil and the plant rhizosphere. Due to its life in unfavorable conditions, Bacillus produces and secretes a wide variety of enzymes with industrial applications [5]. B. subtilis is preferred organism, as it transports proteins across the cytoplasmic membrane into the growth medium [6]. Being an excellent model for investigating the mechanisms of gene regulation, differentiation, and metabolism, B. subtilis has been extensively studied in
%U http://www.hindawi.com/journals/btri/2011/305956/