Functional Expression and Characterization of a Lipase from Oryza sativa.

Lipases (acylglycerol acylhydrolases EC 3.1.1.3) principally catalyze the
hydrolysis of ester bonds in mono-, di- and triacylglycerols. In addition they also
catalyze novel reactions such as inter-esterification and esterification with
enantioselectivity and regioselectivity. Two lipases, Lipase-I and Lipase-II in rice
(Oryza sativa) are reported. Lipase-II identified as the major lipase in rice bran is
designated as rice bran lipase (RBL). In this study, the purification, cloning and
expression of RBL in Escherichia coli and Pichia pastoris are reported. RBL was
purified to apparent homogeneity using conventional protein purification techniques.
The exact molecular mass of purified RBL was 35,293 Da. A BLAST search of the
RBL NH2-terminal sequence indicated that it was identical to a putative
triacylglycerol lipase of Oryza sativa (GenBank Acc. No. BAC83592.1). A 987 bp
open reading frame of RBL gene was amplified and cloned. The deduced amino acid
sequence of 328 amino acids showed 100 % sequence identity to the putative
triacylglycerol lipase. RBL gene was cloned into pRSET A vector to generate a
recombinant vector pRSET ALip. Expression in four different E. coli expression
systems: BL21(DE3)pLysS, Origami(DE3)pLysS, Rosetta(DE3)pLysS and
RIL(DE3)pLysS were analysed. Insignificant expression in BL21(DE3)pLysS and
Origami(DE3)pLysS indicated that they were not suitable hosts. Expression with
supplementation of rare codons in Rosetta(DE3)pLysS and RIL(DE3)pLysS resulted
in higher levels of expression with low activity. Despite the compensation in reduced
temperature and cultivation time, rRBL was obtained as insoluble inclusion bodies.
Refolding in solution did not increase the lipase activity. The hurdles of expression in
E. coli were overcome by expressing RBL as a secretory protein in P. pastoris X-33.
Expressed rRBL from shake flasks showed an activity of 152.6 ± 5.8 U/mL. Purified
rRBL had a specific activity of 998 ± 17.6 U/mg and a molecular mass of 36,928 Da.
A comparative study of chain length and fatty acid specificity of native and
recombinant RBL showed higher preference for triglycerides containing short chain
and unsaturated fatty acids. Among the natural oils, maximum activity was observed
with rice bran oil, which is of physiological advantage as this facilitates release of
energy during germination. The determined kinetic properties demonstrate that both
native and recombinant lipase followed Michaelis-Menten kinetics and display similar
catalytic behaviour. The difference in hydrolytic rates for various triglycerides was
explained by molecular docking studies. In the absence of three-dimensional structure
of RBL, a molecular model based on the atomic coordinates of Rhizomucor miehei
lipase (RML) was generated. The 3D structures of RBL and RML superimposed with
an overall RMSD of 0.1 Å. Molecular docking studies revealed that the catalytic
efficiency of the lipase correlates with the distance between the nucleophilic
Ser175:OH and the scissile ester bond. The shorter the distance, greater is the turnover
of the substrate.
Lipases play a prominent role in mobilization of stored lipids during plant
growth and development. Functional analysis of lipase and protein expression in
combination with quantitative real-time PCR was used to study the role of two lipases
during germination, growth and development of rice caryopsis. The results reveal that
the two lipases: Lipase-I and RBL appear to be differentially expressed. Both the
lipases are expressed during seed germination and embryogenesis. Changes in
specific activity revealed that the lipase activity increased significantly until the end
of germination. During rice caryopsis development, lipase expression increased
gradually and reached maximum as the grain matured. The expression and enzyme
activity profiles lead to the conclusion that RBL (Lipase-II) is responsible for lipid
mobilization during reproductive growth and Lipase-I mobilizes lipid essential for
growth maintenance, which is not restricted to germination only.