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Improvement of the Efficiency of Transglycosylation Catalyzed by α-Galactosidase from Thermotoga maritima by Protein Engineering

K. S. Bobrov1#, A. S. Borisova1#, E. V. Eneyskaya1, D. R. Ivanen1, K. A. Shabalin1, A. A. Kulminskaya1, and G. N. Rychkov1,2*

1Petersburg Nuclear Physics Institute, Orlova Roscha, 188300 Gatchina, Leningrad Region, Russia; fax: (81371) 32014; E-mail: georgy-rychkov@yandex.ru; rychkov@omrb.pnpi.spb.ru

2St. Petersburg State Polytechnical University, 29 Politekhnicheskaya st., 195251 St. Petersburg, Russia

# These authors contributed equally to this work.

* To whom correspondence should be addressed.

Received March 26, 2013; Revision received June 24, 2013
At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.
KEY WORDS: α-D-galactosidase, transglycosylation, enzymatic synthesis of pNP-digalactosides

DOI: 10.1134/S0006297913100052