Thermostable recombinant β-(1→4)-mannanase from C. thermocellum: biochemical characterization and manno-oligosaccharides production.
Functional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length CtManf and truncated CtManT were cloned in the pET28a(+) vector and expressed in E. coli BL21(DE3) cells, exhibiting 53 kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, CtManf and CtManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca(2+) and Mg(2+) salts. Plausible mannanase activity of CtManf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of manno-oligosaccharides. Notably highest protein concentrations of CtManf and CtManT were achieved in tryptone yeast extract (TY) medium, as compared with other defined media. Both CtManf and CtManT displayed stability at 60 and 50 °C, respectively, and Ca(2+) ions imparted higher thermostability, resisting their melting up to 100 °C.
Department of Biotechnology, Indian Institute of Technology Guwahati , Guwahati-781 039, Assam, India.
Arabinda
A
Ghosh
Ana Sofia
AS
Luís
Joana L A
JL
Brás
Carlos M G A
CM
Fontes
Arun
A
Goyal
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008358:Mannose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D044902:beta-Mannosidase; D008358:Mannose