ASPERGILLUS NIGER ENDOPOLYGALACTURONASE.

Abstract

A purified endopolygalacuronase (endo PG) from Aspergillus niger has been characterized. The optimum temperature was 45°C and the optimum pH was 4.1–4.2 with a highest stability at pH 6. The purified enzyme was a typical endo PG: the enzyme hydrolyzes about 56.7% and 7% of the glycosidic linkages of polygalacturonic acid and pectin (DE = 72.8%), respectively, and a loss of 50% in viscosity of polygalacturnoic acid was reached when only 2.3% of the glycosidic linkages of the substrate were split. The products at the maximum hydrolysis were mono-, di- and trigalacturonic acids. Endo PG has a V_max of 239 μ mole reducing groups per min per mg of protein and a K_m of 0.44 mg per ml of polygalacutronic acid (DP_n= 44). Sodium, potassium and ammonium ions stimulated endo PG activity, while divalent ions, or ethylene — diamine – tetracetic acid had no effect. Pectinolytic enzymes (saponifying enzymes and depolymerases) are widespread in nature; they are produced by bacteria, fungi and higher plants (Rombouts and Pilnik 1972; MacMillan and Sheiman 1974). They are involved in natural processes, as fruit physiological changes (Pilnik and Voragen 1970) and they are responsible for the phytopathogenicity of many microorganisms (Rexova-Benkova and Markovic 1976). These enzymes are very useful for their applications in food processing. Commercial preparations of fungal pectinolytic enzymes are employed in the production of fruit and vegetable juices (Rombouts and Pilnik 1978). These preparations contain mainly pectinesterases (pectin-pectyl hydrolase, E.C. 3. 1. 1. 11 and endopolygalacturonase (poly (1, 4 α D galacturonide) glycanohydrolase, E.C. 3. 2. 1. 15); for this reason, special attention was paid to endopolygalacturonase (endo PG (ENDO, 1964a, 1964b, 1964c; Yamasaki et al. 1966). Purified endo PG was also used as a tool for specific degradations of pectic substances (Talmadge et al. 1973) or material containing galacturonic acids (Kikuchi and Yokotsuka, 1973; Kikuchi and Sugimoto, 1976). In a previous paper (Thibault and Mercier 1978) we have described a rapid method for the purification of endo PG from a commercial preparation of pectolytic enzymes. This method is based on a remark of English et al. (1972) who observed that endo PG was delayed during chromatography on agarose gel (Bio-Gel A 0.15 m). We demonstrated that dialyzed endo PG from a crude preparation can be optimally bound by ion-exchange (Thibault 1978) to the agarose gel (Sepharose 6B) when the column is eluted at pH 4.3–4.4 (20 mM acetate buffer). The endo PG released by a 0–0.15 M NaCl gradient is therefore purified 62 times if the activity is determined by a viscosimetric method and 36 times if the activity is expressed in n. katals (cf. enzyme assay methods for the definitions). The present paper reports characterization and some physico-chemical properties of the purified enzyme.