title: Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu.
abstract: To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 110-743, Republic of Korea.
authors.forename: Phuong Lan
authors.initials: PL
authors.lastname: Tran
authors.affiliation: Division of Industrial Promotion Research, Korea Food Research Institute, 62 Anyangpangyo-ro 1201 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do 463-746, Republic of Korea.
authors.forename: Hyun-Ju
authors.initials: HJ
authors.lastname: Cha
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 110-743, Republic of Korea.
authors.forename: Jin-Sil
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Scienece, 7-45 Songdo-dong, Yeonsu-ku, Incheon 406-840, Republic of Korea.
authors.forename: Sung-Hoon
authors.initials: SH
authors.lastname: Park
authors.affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 110-743, Republic of Korea; Department of Food Science and Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: parkkwanhwa@gmail.com.
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D020240:Apraxia, Ideomotor; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D018698:Glutamic Acid / Q000378:metabolism; D006031:Glycosylation; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D018698:Glutamic Acid; D006639:Histidine; C101183:maltooctaose; D000516:alpha-Amylases; C501919:alpha-amylase, Bacillus licheniformis
keywords: Bacillus licheniformis thermostable α-amylase; Binding-subsite mapping; Site-directed mutagenesis; Substrate transglycosylation; Transfer product