Modifying the chain-length selectivity of the lipase from Burkholderia cepacia KWI-56 through in vitro combinatorial mutagenesis in the substrate-binding site.
The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator. To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis. In this paper, Phe119 and Leu167, which are located in the acyl portion of the substrate-binding pocket of the lipase of B.cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis. The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation. The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate, p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates. Two mutant lipases, L167V and F119A/L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro. It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester.
Yang
Junhao
J
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Koga
Yuichi
Y
Nakano
Hideo
H
Yamane
Tsuneo
T
D001665:Binding Sites; D016956:Burkholderia cepacia / Q000201:enzymology*; D020148:Butyric Acid / Q000737:chemistry / Q000378:metabolism*; D002847:Chromatography, Agarose / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
D017931:DNA Primers; D004952:Esters; D011994:Recombinant Proteins; D020148:Butyric Acid; D010649:Phenylalanine; D008049:Lipase