Synthesis of benzyl β-D-galactopyranoside by transgalactosylation using a p-galactosidase produced by the over expression of the Kluyveromyces lactis LAC4 gene in Arxula adeninivorans

摘要

The LAC4 gene of Kluyveromyces lactis encoding for p-galactosidase was overexpressed in the yeast Arxula adeninivorans to produce the enzyme, which can be used for the synthesis of p-D-galactosides. These compounds play a major role as precursors for the Synthesis of glycolipids and glycoproteins in medicine or for the production of tensides. The Xplor~R2 transformation/expression platform was used because it enabled stable integration of the gene in the Arxula genome and the production of high levels of the enzyme. The recombinant p-galactosidase, fused with C-terminal His-tag region (Lac4-6hp), was purified by precipitation with ammonium sulphate and FPLC using hydroxylapatite. The enzyme exhibited optimal activity at 37 to 40 °C, pH 6.5 in 50 mM sodium acetate buffer. Activity was measured by the formation of p-nitrophenol at 405 nm from the hydrolyzed chromogenic substrate, p-nitrophenyl-β-D-gal. Biochemical characterization included the calculation of K_M and apparent k_(cat) values of the enzyme. The formation of benzyl P-D-gal by 0.1 U enzyme from A. adeninivorans with transgalactosylation was six times higher than that for the prokaryotic enzyme from ￡ coll. Moreover, the partially purified enzyme was used for the selective hydrolysis of allyl β-D-gal in a mixture of allyl β-and allyl α-D-gal, with 4gl~(-1) being hydrolysed within one day by 1 U ml~(-1). Thus, the recombinant p-galactosidase produced in A adeninivorans is of potential interest for the enzymatic synthesis of benzyl β-D-gal and other galactosides as well as the selective hydrolysis of anomeric mixtures and could be used to replace difficult chemical procedures.