Inducible thermoalkalophilic polygalacturonate lyase from Thermomonospora fusca.
A thermostable polygalacturonate lyase (PL; EC 4.2.2.2) was secreted by Thermomonospora fusca during stationary phase in pectin-mineral salts medium at 52 degrees C. Biosynthesis was induced by addition of pectic substances to cultures growing on glucose or cellulose but not cellobiose; the disaccharide repressed enzyme synthesis and triggered inactivation of enzyme previously secreted. The PL, purified to electrophoretic and serologic homogeneity, had a molecular size of 56 kilodaltons and an isoelectric point at pH 4.16. The amino acid composition closely resembled that of the major extracellular endoglucanases of the actinomycete. The enzyme had six cystine residues but no detectable sulfhydryl groups. It was inactivated by mild reducing agents and activated by oxygenation, indicating the necessity for disulfide bond maintenance. Temperature and pH optima for the PL reaction were 60 degrees C and 10.45, respectively. Calcium was essential for activity but not stability; calcium dependence curves were altered by low concentrations of toxic metals. The Km for pectin increased 30,000-fold as the percent esterification (methoxylation) of that substrate was increased from 0 to 60%. The size of the minimal susceptible site for PL attack on the pectin molecule was calculated as being equivalent to 10 unesterified residues, based on the correlation of Km values at various degrees of esterification with the percentage of cleavable bonds predicted by a random-number-generating computer program.
D000192:Actinomycetales / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D004790:Enzyme Induction; D004794:Enzyme Repression; D007700:Kinetics; D008970:Molecular Weight; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D002118:Calcium