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

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• 作者：Marion Rauter ; Maria Schwarz ; Karin Becker ; Keith Baronian ; R眉diger Bode ; Gotthard Kunze ; H.-Matthias Vorbrodt
• 关键词：尾-Galactosidase ; LAC4 ; Kluyveromyces lactis ; Arxula adeninivorans ; Transgalactosylation ; Benzyl 尾-d-galactopyranoside ; Selective hydrolysis
• 刊名：Journal of Molecular Catalysis B: Enzymatic
• 出版年：15 December, 2013
• 年：2013
• 卷：97
• 期：Complete
• 页码：319-327
• 全文大小：1596 K

文摘

The LAC4 gene of Kluyveromyces lactis encoding for 尾-galactosidase was overexpressed in the yeast Arxula adeninivorans to produce the enzyme, which can be used for the synthesis of 尾-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^庐2 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 尾-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 掳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-尾-d-gal. Biochemical characterization included the calculation of K_M and apparent k_cat values of the enzyme. The formation of benzyl 尾-d-gal by 0.1 U enzyme from A. adeninivorans with transgalactosylation was six times higher than that for the prokaryotic enzyme from E. coli. Moreover, the partially purified enzyme was used for the selective hydrolysis of allyl 尾-d-gal in a mixture of allyl 尾- and allyl 伪-d-gal, with 4 g l^鈭? being hydrolysed within one day by 1 U ml^鈭?. Thus, the recombinant 尾-galactosidase produced in A. adeninivorans is of potential interest for the enzymatic synthesis of benzyl 尾-d-gal and other galactosides as well as the selective hydrolysis of anomeric mixtures and could be used to replace difficult chemical procedures.