Cloning, Purification, and Characterization of Galactomannan-Degrading Enzymes from Myceliophthora thermophila

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G. S. Dotsenko^1,2*, M. V. Semenova², O. A. Sinitsyna^1,2, S. W. A. Hinz³, J. Wery³, I. N. Zorov^1,2, E. G. Kondratieva², and A. P. Sinitsyn^1,2

¹Chemical Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 932-8846; E-mail: info@rector.msu.ru; gsdotsenko@gmail.com

²Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33/2, 119071 Moscow, Russia; fax: (495) 954-2732; E-mail: inbi@inbi.ras.ru

³Dyadic Nederland BV, Nieuwe Kanaal 7s, 6709 PA, Wageningen, The Netherlands

^* To whom correspondence should be addressed.

Received April 25, 2012; Revision received May 18, 2012

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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 galactomannans (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.

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KEY WORDS: Myceliophthora thermophila, β-mannosidase, β-mannanase, α-galactosidase, enzymatic hydrolysis

DOI: 10.1134/S0006297912110090

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