Abstract: The two rounds of errorprone PCR was used to directly evolve Bacillus subtilis lipaseA gene with highthroughput screening of nitrobenzene palmitate （pNPP） in 96pore plate. The results showed that the first round of errorprone PCR had no random mutation. After the second round of errorprone PCR reaction with Mn2+ concentration of 0.2 mmol·L-1， 124 mutant strains were screened. The absorbance value A405 of mutant strain 4B2 was 1.395， which was significantly different from nonmutant strain PET32alipaseA （A405=0448）. Mmutant strain 4B2 lipaseA gene was found to have five mutant nucleotide sites， three of which were synonymous mutations， two were missense mutations with 82 site of asparagine （AAU） changed to tyrosine （UAU） and 143 site of lysine （AAG） changed to threonine （ACG）. The transesterification efficiency of biodiesel of the mutant strain 4B2 was significantly improved contrast to that of nonmutant strain. The former was 79.5% and the latter was 49.72%. These results laid the foundation for the study of the key sites related to lipaseA transesterification activity of Bacillus subtilis.

Key words: Bacillus subtilis, lipase, errorprone PCR, pnitrophenylpalmitate, transesterification efficiency