CHARACTERIZATION OF AN 'ENDOPOLYGALACTURONASE' OF THE YEAST KLUYVEROMYCES MARXIANUS

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Abstract
Analytic isoelectric focusing showed that the ‘endopolygalacturonase’ from Kluyveromyces marxianus consists of 21 multiple enzyme components. Neither changes in cultivation conditions, enzyme substrate or reaction conditions resulted in any quantitative or qualitative differences in the enzyme patterns. Two of these multiple enzyme forms, the main band, (IP = pH 5.8) which accounted for approximately 95% of the total activity, and an ‘acid-band’ (IP = pH 2.7), were purified by means of preparative isoelectric focusing and their molecular weights and amino acid compositions were determined. The molecular weight of the main band was established as 76,000 daltons (2 subunits of 47,900 and 28,100), The molecular weight of the ‘acid-band’ was 32,200. Both amino acid analyses and molecular weight determinations suggested that the proteins are chemically and physically different. The pH optimum was 4 for the pure enzyme on different pectin substrates, e.g., high molecular weight pectic acid, low molecular weight pectic acid and highly methylated pectins B and C. The temperature optimum obtained for pure enzyme with high or low molecular-weight pectic acid as substrate was 40° C. Vmax and Km-value determination at different pH values with low molecular weight pectic acid as a substrate was used to identify the catalytically active groups at the active site. They were tentatively identified as an unprotonated α-carboxyl group and a protonated carboxyl group of aspartic acid.

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