Structural study of the mutants of FDTS fromT. maritima

Abstract

present in microorganisms, plants and animals. From a biotechnological point of view, Kluyveromyces lactis β-galactosidase is suitable for many applications due to its neutral optimum pH and for the fact that K. lactis is a GRAS organism (Generally Recognized As Safe). Interestingly, β-galactosidases are being used in lactose intolerance treatments and in food industry. Moreover, yeast expressing this enzyme can be used to improve the valorization of the cheese whey, a cheese industry byproduct by coupling the degradation of lactose with ethanol production, biomass production, etc. [1]. On the basis of their sequence, β-galactosidases are classified within families 1, 2, 35 and 42 of glycosyl hydrolases in the CAZy database [2]. Those from eukaryotic organisms are grouped into family 35 with the only exceptions of K. lactis and K. marxianus β-galactosidases which belong to family 2, together with the prokaryotic β-galactosidases from Escherichia coli and Arthrobacter sp. Whereas the structures of these last two prokaryotic enzymes have been determined [3], [4], none of the eukaryotic β-galactosidase structures have been reported to date. Although their sequence similarity with the prokaryotic enzymes is significant (48% vs. E. coli and 47% vs. Arthrobacter) there are many differences, particularly some long insertions and deletions, which play an important role in protein stability and in substrate recognition and specificity. Gaining insight into the structural features that determine its stability and understanding the specificity determinants and catalytic mechanism should lead to improvements of its biotechnological applications by rational protein engineering. In this study, we describe X-ray crystallographic studies and an analysis of Kluyveromyces lactis β-galactosidase structure.