An in vitro evaluation of a thermostable pectate lyase by using error-prone PCR

Thermostable pectate lyase 47 (PL 47) prepared from Bacillus sp. TS 47 was mutated by a random mutation with error-prone PCR. The mutated PL 47 gene was expressed in Escherichia coli and the variants were screened over the PL activity using a plate assay method. Three less-thermostable mutants were isolated and their enzymes (LT-PLs) were isolated and characterized. The LT-PLs were inferior to the PL 47 in their thermostability and thermoactivity. Analyses of the amino acids substituted in the LT-PLs suggested that Ala325 in the PL 47 contributes to its thermostability.