Modulation of hydrolysis and transglycosylation activity of Thermus maltogenic amylase by combinatorial saturation mutagenesis

• Published in: Journal of microbiology and biotechnology Vol. 18; no. 8; pp. 1401 - 1407
• Main Authors: Oh, S.W. (Chungbuk National University, Cheongju, Republic of Korea), Jang, M.U. (Chungbuk National University, Cheongju, Republic of Korea), Jeong, C.K. (Chungbuk National University, Cheongju, Republic of Korea), Kang, H.J. (Chungbuk National University, Cheongju, Republic of Korea), Park, J.M. (Chungbuk National University, Cheongju, Republic of Korea), Kim, T.J. (Chungbuk National University, Cheongju, Republic of Korea), E-mail: tjkim@cbnu.ac.kr
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.08.2008; 한국미생물·생명공학회
• Subjects: Acarbose - metabolism; acarbose hydrolysis; Amino Acid Motifs; Amino Acid Sequence; Base Sequence; Biological and medical sciences; Biotechnology; Carbohydrate Sequence; combinatorial saturation mutagenesis (CSM); DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Glycosylation; Hydrolysis; Molecular Sequence Data; MUTACION; Mutagenesis; MUTATION; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Thermus - enzymology; Thermus - genetics; Thermus - metabolism; Thermus maltogenic amylase; transglycosylation activity; Trisaccharides - metabolism; 생물학; Acarbose; Thermus; Amylase; Enzyme; transglycosylation activity; Hydrolysis; Transglycosylation; Mutagenesis; acarbose hydrolysis; Modulation; Substrate specificity; Bacteria; combinatorial saturation mutagenesis (CSM); Thermus maltogenic amylase
• ISSN: 1017-7825

The roles of conserved amino acid residues (Val329-Ala330-Asn331-Glu332), constituting an extra sugar-binding space (ESBS) of Thermus maltogenic amylase (ThMA), were investigated by combinatorial saturation mutagenesis. Various ThMA mutants were firstly screened on the basis of starch hydrolyzing activity and their enzymatic properties were characterized in detail. Most of the ThMA variants showed remarkable decreases in their hydrolyzing activity, but their specificity against various substrates could be altered by mutagenesis. Unexpectedly, mutant H-16 (Gly-Leu-Val-Tyr) showed almost identical hydrolyzing and transglycosylation activities to wild type, whereas K-33 (Ser-Gly-Asp-Glu) showed an extremely low transglycosylation activity. Interestingly, K-33 produced glucose, maltose, and acarviosine from acarbose, whereas ThMA hydrolyzed acarbose to only glucose and acarviosine-glucose. These results propose that the substrate specificity, hydrolysis pattern, and transglycosylation activity of ThMA can be modulated by combinatorial mutations near the ESBS.