Functional Expression and Characterization of a Lipase from Oryza sativa.

Vijayakumar, K. R. (2013) Functional Expression and Characterization of a Lipase from Oryza sativa. PhD thesis, University of Mysore.

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Abstract

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.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Lipases, Oryza sativa, rice
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry 600 Technology > 08 Food technology > 21 Cereals > 01 Rice
Divisions:	Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	04 Mar 2014 06:25
Last Modified:	04 Mar 2014 06:25
URI:	http://ir.cftri.res.in/id/eprint/11364

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