Purification and properties of a pectin-methyl trans-eliminase, a phospholipase and two aminopeptidases produced in culture by Phoma medicaginis var. pinodella

Abstract

A pectin-methyl trans-eliminase produced in cultured by Phoma medicaginis var. pinodella was purified using ammonium sulphate fractionation, DEAE-cellulose ion-exchange chromatography, Sephadex G-100 gel-filtration and isoelectric focusing. A purification of 32·1-fold was achieved after gel-filtration and it was shown by gel electrophoresis that the enzyme was free from protein contaminants. An examination of the enzyme's properties revealed that it existed in two forms: as a monomer of mol. wt 29 500 and as a tetramer of mol. wt 118 000. Both forms possessed a pH optimum of 7·5, were activated by Ca2+ and combined during electrofocusing to show a pI value of 7·9.Using the same purification process, a phospholipase was obtained from a culture of the fungus and purified 6·4-fold after gel-filtration. Gel electrophoresis showed that it was free from protein contaminants after isoelectric focusing. The enzyme had a molecular weight of around 140 000 and a pI value of 5·5. Gas-liquid chromatography showed palmitic acid was produced by the degradation of dipalmitate derivatives of lecithin and lysolecithin suggesting that the phospholipase of P. medicaginis var. pinodella possessed B-type activity.The fungus also produced two aminopeptidases. One was purified 493·8-fold after gel-filtration and had a mol. wt of approximately 125 000. This enzyme exhibited a pH optimum of 7·0 and a temperature optimum of 35 °C; it was activated by Mn2+, threonine, glycine and sodium thioglycolate. The other enzyme was purified 287·5-fold after gel-filtration and exhibited a mol. wt of approximately 112 000. This enzyme possessed a pH optimum of 7·0 and a temperature optimum of 30 °C; it was activated by Co2+ and MoO42−. Both enzymes showed a pI value of 5·0.