Rice (Oryza sativa) lipase: Molecular cloning, functional expression and substrate specificity.

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
Ser175-OH and the scissile ester bond. The shorter the distance, the greater is the turnover of the
substrate.