Crystallization, structural characterization and kinetic analysis of a GH26 ß-mannanase from Klebsiella oxytoca KUB-CW2-3.

ß-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannan-based polysaccharides at ß-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 ß-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49-72% amino-acid sequence similarity with ß-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Šrevealed an open cleft-shaped active site. The enzyme structure is based on a (ß/α)8-barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to ß-strand 4 and at the end of ß-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency (kcat/Km) towards DP6 (2571.26 min-1 mM-1). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.

Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters.

Gene encoding for ß-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 ß-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.