title: Cloning, secretory expression and characterization of recombinant β-mannanase from Bacillus circulans NT 6.7.
abstract: The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). β-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and 515 U per ml, respectively, with most of the activity in the cell extract attributed to the periplasmic fraction. In contrast, expression of mannanase was much lower when using the B. circulans NT 6.7 mannanase gene without its signal sequence. The optimum temperature of recombinant β-mannanase activity was 50°C and the optimum pH was 6.0. The enzyme was very specific for β-mannan substrates with a preference for galactomannan. Hydrolysis products of locust bean gum were various mannooligosaccharides including mannohexaose, mannopentaose, mannotetraose, mannotriose and mannobiose, while mannose could not be detected. In conclusion, this expression system is efficient for the secretory production of recombinant β-mannanase from B. circulans NT 6.7, which shows good characteristics for various applications.
authors.affiliation: Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkok, 10900 Thailand ; Center for Advanced Studies in Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, 10900 Thailand.
authors.forename: Yotthachai
authors.initials: Y
authors.lastname: Piwpankaew
authors.affiliation: Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, 10900 Thailand.
authors.forename: Supa
authors.initials: S
authors.lastname: Sakulsirirat
authors.affiliation: Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkok, 10900 Thailand ; Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, 10900 Thailand ; Center for Advanced Studies in Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, 10900 Thailand.
authors.forename: Sunee
authors.initials: S
authors.lastname: Nitisinprasert
authors.affiliation: Department of Food Sciences and Technology, Food Biotechnology Laboratory, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
authors.affiliation: Department of Food Sciences and Technology, Food Biotechnology Laboratory, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkok, 10900 Thailand ; Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, 10900 Thailand ; Center for Advanced Studies in Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, 10900 Thailand.
authors.forename: Suttipun
authors.initials: S
authors.lastname: Keawsompong
keywords: Bacillus circulans NT 6.7; Escherichia coli; Expression; Mannooligosacharides; β-mannanase