Finding Novel Lipase Resources From Genome Databases Of Burkholderia Cepacia

Whole cell biocatalyst displayed many advantages, including convenient recovery from the reaction media, good operation stability and simple preparation, etc. It becomes a routine method to mine novel lipolytic enzymes from the known genomic DNA sequences in recent years.2 cell-bound lipolytic gene sequences were predicted by analysis of the genomic DNA sequences of Burkholderia cepacia J2315. The two kinds of cell-bound lipolytic enzyme, named lipC24 and lipC21, were cloned from Burkholderia sp. ZYB002 and then expressed in E. coli, respectively. The results of this research are as follows:1. Nucleotide sequencing revealed that the lipC24 gene had an open reading frame of 1317 bp, and the deduced amino acid sequence of LipC24 corresponded to 438 amino acid residues, including a conserved -G-X1-S-X2-G-motif. The expression of chaperon LipB is required for the functional expression of LipC24.The relative molecular weight of the purified LipC24 was about 45 kDa. The purified LipC24 displayed hydrolysis activity to various 4-nitrophenyl esters and substrate preference for the medium chain length 4-nitrophenyl-esters. The optimal temperature was 40℃ and the optimal pH was 7.5. The half-life was 15.75 min at 40℃. It had good stability between pH 7.0 and 8.0. Tertiary structure simulation shows that the Ser179, His367 and Asp336 amino acid residues formed the active center of catalytic triplets. Gly180 and Ala82 stabilized oxygen anion hole.2. The lipC21 gene had an open reading frame of 1287 bp, and the deduced amino acid sequence of LipC21 corresponded to 427 amino acid residues, including a conserved-G-Xi-S-X2-G-motif. The chaperon GroES/EL is required for the soluble expression of LipC21. The relative molecular weight of the purified LipC21 was about 45 kDa. However, the soluble LipC21 displayed no hydrolysis activity to various 4-nitrophenyl esters.