Directed Evolution Of Lipase Of Bacillus Pumilus YZ02

Directed evolution, belonging to"irrational"design of protein, is a new strategy for protein engineering. By mimicking natural evolution mechanism, an enzyme is reconstructed in vitro under particular evolutionary conditions created artificially without any knowledge about spatial structure and catalytic mechanism of the enzyme in advance. The mutant enzymes with desired properties are screened. The generally used evolution strategies are error-prone PCR, staggered extending process and DNA shuffling, etc.In this study, the Bacillus pumilus YZ02 lipase gene previously cloned by our group was directedly evolved in vitro by error-prone PCR. Two rounds of error-prone PCR were conducted, and in every round approximately 6,000 colonies were screened. 4 mutants with lipase activity improved more than 2 folds were obtained, among which one mutant BpL2-1369 was enhanced 6 folds, and was sequenced. The results showed that four nucleotide substitutions, T61C, C147T, A334G and T371A occurred, and three of them caused amino acid changes, i.e. position 21 Ser→Pro, position 112 Arg→Gly, position 124 Leu→His. Based on the two rounds of error-prone PCR, a staggered extending process was performed, but no recombinant with lipase activity futher inproved was screened.The BpL and BpL2-1369 gene were ligated into pET28-a vector, and transferred into E.coli BL21. The recombinants were induced by IPTG.. Then, the lipase was purified and characterized. The results showed that the specific activity of the evolved lipase was 1.31-fold than that of the wild lipase, and the Km decreased from 8.24mmol/L to 7.17mmol/L. The pH stability of the evolved lipase was better than wild lipase when pH>8.0, but a little inferior while pH<8.0.Based on the above work, the structure of Bacillus pumilus lipase was mimicked. With this simulant structure, the mutant lipase was analyzed and compared with the wild lipase, we found that the three mutated amino acids: position 21 Ser→Pro, position 112 Arg→Gly and position 124 Leu→His, were located at the third amino acid of the firstα-helix, at the turn of the fourth and fifthβfold, and at the first amino acid of the fifthβfold, respectively.