title: Purification, characterization, and overexpression of an endo-1,4-β-mannanase from thermotolerant Bacillus sp. SWU60.
abstract: Endo-β-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the β-1,4-linkage in the mannan backbone and release short β-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60 °C, respectively. The enzyme was stable up to 60 °C for 1 h and at pH 5-9 at 4 °C for 16 h. Its enzyme activity was inhibited by Hg2+. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok, 10110, Thailand.
authors.forename: Weeranuch
authors.initials: W
authors.lastname: Seesom
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok, 10110, Thailand.
authors.forename: Polphet
authors.initials: P
authors.lastname: Thongket
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, 599-8531, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, 598-8531, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Takenaka
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, 599-8531, Japan. sakamoto@biochem.osakafu-u.ac.jp.
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok, 10110, Thailand. wasanas@swu.ac.th.
authors.forename: Wasana
authors.initials: W
authors.lastname: Sukhumsirichart
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives; D008351:Mannans / Q000378:metabolism; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008351:Mannans; C012990:galactomannan; D044902:beta-Mannosidase; D005690:Galactose
keywords: Bacillus sp.; Endo-β-1,4-mannanases; Galactomannan; Glucomannan; Mannan; Recombinant enzyme