title: Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters.
abstract: Gene encoding for β-mannanase (E.C 3.2.1.78) from Klebsiella oxytoca KUB-CW2-3 was cloned and expressed by an E. coli system resulting in 400 times higher mannanase activities than the wild type. A 3314bp DNA fragment obtained revealed an open reading frame of 1164bp, namely kman-2, which encoded for 387 amino acids with an estimated molecular weight of 43.2kDa. It belonged to the glycosyl hydrolase family 26 (GH26) exhibited low similarity of 50-71% to β-mannanase produced by other microbial sources. Interestingly, the enzyme had a broad range of substrate specificity of homopolymer of ivory nut mannan (6%), carboxymethyl cellulose (30.6%) and avicel (5%), and heteropolymer of konjac glucomannan (100%), locust bean gum (92.6%) and copra meal (non-defatted 5.3% and defatted 7%) which would be necessary for in vivo feed digestion. The optimum temperature and pH were 30-50°C and 4-6, respectively. The enzyme was still highly active over a low temperature range of 10-40°C and over a wide pH range of 4-10. The hydrolysates of konjac glucomannan (H-KGM), locust bean gum (H-LBG) and defatted copra meal (H-DCM) composed of compounds which were different in their molecular weight range from mannobiose to mannohexaose and unknown oligosaccharides indicating the endo action of mannanase. Both H-DCM and H-LBG enhanced the growth of lactic acid bacteria and some pathogens except Escherichia coli E010 with a specific growth rate of 0.36-0.83h(-1). H-LBG was more specific to 3 species of Weissella confusa JCM 1093, Lactobacillus reuteri KUB-AC5, Lb salivarius KL-D4 and E. coli E010 while both H-KGM and H-DCM were to Lb. reuteri KUB-AC5 and Lb. johnsonii KUNN19-2. Based on the nucleotide sequence of kman-2 containing two open reading frames of 1 and 2at 5' end of the +1 and +43, respectively, removal of the first open reading frame provided the recombinant clone E. coli KMAN-3 resulting in the mature protein of mannanase composing of 345 amino acid residues confirmed by 3D structure analysis and amino acid sequence at N-terminal namely KMAN (GenBank accession number KM100456). It exhibited 10 times higher extracellular and periplasmic total activities of 17,600 and 14,800 units than E. coli KMAN-2. With its low similarity to mannanases previously proposed, wide range of homo- and hetero-polysaccharide specificity, negative effect to E. coli and most importance of high production, it would be proposed as a novel mannanase source for application in the future.
authors.affiliation: Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand; Center for Advanced Studies for Agriculture and Food, KU Institute for Advanced Studies, Kasetsart University (CASAF, NRU-KU), Bangkok 10900, Thailand; Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERD O-CHE), Bangkok 10900, Thailand.
authors.forename: Nawapan
authors.initials: N
authors.lastname: Pongsapipatana
authors.affiliation: Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand.
authors.forename: Piyanat
authors.initials: P
authors.lastname: Damrongteerapap
authors.affiliation: Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Thailand.
authors.forename: Sudathip
authors.initials: S
authors.lastname: Chantorn
authors.affiliation: Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand.
authors.forename: Wilawan
authors.initials: W
authors.lastname: Sintuprapa
authors.affiliation: Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand; Center for Advanced Studies for Agriculture and Food, KU Institute for Advanced Studies, Kasetsart University (CASAF, NRU-KU), Bangkok 10900, Thailand; Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERD O-CHE), Bangkok 10900, Thailand.
authors.forename: Suttipun
authors.initials: S
authors.lastname: Keawsompong
authors.affiliation: Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand; Center for Advanced Studies for Agriculture and Food, KU Institute for Advanced Studies, Kasetsart University (CASAF, NRU-KU), Bangkok 10900, Thailand; Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERD O-CHE), Bangkok 10900, Thailand. Electronic address: fagisnn@ku.ac.th.
authors.forename: Sunee
authors.initials: S
authors.lastname: Nitisinprasert
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D041121:Klebsiella oxytoca / Q000201:enzymology* / Q000235:genetics*; D008351:Mannans / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008351:Mannans; D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: Characters; E. coli; Molecular cloning; β-mannanase