Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila

• Published in: Biochemistry (Moscow) 2012-11, Vol.77 (11), p.1303-1311
• Main Authors: Dotsenko, G. S., Semenova, M. V., Sinitsyna, O. A., Hinz, S. W. A., Wery, J., Zorov, I. N., Kondratieva, E. G., Sinitsyn, A. P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Cloning; Cloning, Molecular; Enzymes; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene expression; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Hydrolases; Kinetics; Life Sciences; Microbiology; Molecular Sequence Data; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sordariales - enzymology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Temperature
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297912110090

Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K m and k cat values are 0.4 mM and 15 sec −1 for p -nitrophenyl-β- D -mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K m and k cat values are 1.3 mg/ml and 67 sec −1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p -nitrophenyl-α- D -galactopyranoside (PNPG) as well as GM of various structures. The K m and k cat values are 0.08 mM and 35 sec −1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.