Characterization of a novel cyclomaltodextrinase expressed from environmental DNA isolated from Bor Khleung hot spring in Thailand

A novel gene belonging to the alpha-amylase family was isolated directly from community DNA obtained from soil sediments collected from Bor Khleung hot spring in Thailand. Partial sequences harboring four conserved regions of the alpha-amylase family were amplified by PCR using degenerate primers. Upstream and downstream sequences of these fragments were obtained by a genome walking approach to identify a full-length gene (Env cda13A) encoding 619 amino acids. Amino acid sequence alignments of Env Cda13A with other enzymes suggested that this enzyme was a cyclomaltodextrinase. The Env cda13A gene was expressed in Pichia pastoris as a secreted functional protein of 68 kDa. The partially purified enzyme was shown to be monomeric and hydrolyzed various maltodextrins from maltotriose to maltoheptaose and cyclomaltodextrins to give maltose and glucose as the main products. The enzyme also hydrolyzed pullulan and soluble starch to yield glucose, but the rate of hydrolysis was slow. This study demonstrated the possibility of isolating potentially novel enzymes directly from natural environments and opens an unexplored biodiversity resource in Thailand for future novel gene discoveries.