Purification and characterization of ß‐xylosidase activities from the yeast Arxula adeninivorans

Abstract β‐Xylosidase activity has been detected in cell‐free extracts, in culture fluids and as cell wall‐bound enzyme of Arxula adeninivorans. With chromatographic procedures used to purify the activity two different forms of β‐xylosidase (denoted βX‐1 and βX‐2) from the cell‐free extract and from the culture medium could be separated, but only one form (βX‐2) was present in the cell wall. Both forms are glycoproteins and were deglycosylated by endoglycosidase H treatment. The molecular masses of the enzymes under native and denaturing conditions suggest that βX‐2 is the dimer of βX‐1. M r of the native βX‐1 was 60 kDa and after deglycosylation 39 kDa determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Some enzymatic properties of βX‐1 and βX‐2 and of their deglycosylated products were studied and showed, with one exception, wide similarities. The maximum activity was reached at pH 5.0 and 60 °C. The enzymes hydrolyze only β‐glycosidic bound β‐xylopyranosides and the K m values for p ‐nitrophenyl‐β‐xylopyranoside were determined to be 0.23–0.33 mM. The β‐xylosidase acitivity was inhibited competitively by xylose. The deglycosylated enzymes were, however, stronger inhibited ( K i = 2.1 mM) as their glycosylated ones ( K i = 5.8 mM).