title: Rice (Oryza sativa) lipase: molecular cloning, functional expression and substrate specificity.
abstract: Lipases are important biocatalysts showing many interesting properties with industrial applications. Previously, different isoforms of lipases, Lipase-I and Lipase-II from rice (Oryza sativa) have been purified and characterized. Lipase-II identified as the major lipase in rice bran is designated as rice bran lipase (RBL). In this study, we report the cloning and expression of the RBL in Escherichia coli and Pichia pastoris. An exploration of expression in four different E. coli expression systems analyzed: BL21(DE3)pLysS, RIL(DE3)pLysS, Rosetta(DE3)pLysS and Origami(DE3)pLysS indicated that E. coli was not a suitable host. Expression with supplement of rare codons in Rosetta (DE3)pLysS and RIL(DE3)pLysS resulted in highest expression as insoluble inclusion bodies. The hurdles of expression in E. coli were overcome by expression as a secretory protein in P. pastoris X-33. The expression of lipase in shake flasks was optimized to achieve the maximum recombinant lipase activity of 152.6 U/mL. The purified recombinant lipase had a specific activity of 998 U/mg toward triacetin. The pH and temperature optimum of native and recombinant enzymes were pH 7.4 and 25 ± 2 °C, respectively. Both the lipases showed higher activity toward short chain triacylglycerol and unsaturated fatty acid enriched oils. Computational modeling and molecular docking studies reveal that the catalytic efficiency of the lipase correlates with the distance of the nucleophilic Ser(175)-OH and the scissile ester bond. The shorter the distance, the greater is the turnover of the substrate.
authors.affiliation: Department of Protein Chemistry and Technology, CSIR, Central Food Technological Research Institute, Mysore 570 020, India. kakade.viju@gmail.com
authors.forename: K R
authors.initials: KR
authors.lastname: Vijayakumar
authors.forename: Lalitha R
authors.initials: LR
authors.lastname: Gowda
mesh_terms: D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012275:Oryza / Q000201:enzymology*; D010843:Pichia / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003062:Codon; D008049:Lipase