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

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Molecular Biology

Background:

• Lipases are crucial biocatalysts with diverse industrial applications.
• Rice bran lipase (RBL), a major lipase in rice, has shown potential.
• Previous characterization of rice Lipase-I and Lipase-II laid the groundwork.

Purpose of the Study:

• To clone and express rice bran lipase (RBL) in microbial hosts.
• To overcome expression challenges encountered in Escherichia coli.
• To optimize RBL expression and characterize the recombinant enzyme.

Main Methods:

• Cloning and expression of RBL in four different E. coli systems.
• Secretory expression of RBL in Pichia pastoris X-33.
• Optimization of shake flask conditions and enzyme purification.
• Enzyme activity assays, pH and temperature optimum determination.
• Computational modeling and molecular docking studies.

Main Results:

• E. coli was unsuitable for RBL expression, yielding insoluble inclusion bodies.
• Pichia pastoris facilitated secretory expression, achieving a maximum activity of 152.6 U/mL.
• Purified recombinant RBL exhibited a specific activity of 998 U/mg toward triacetin.
• Optimal activity at pH 7.4 and 25°C; preference for short-chain triacylglycerols and unsaturated fatty acids.
• Catalytic efficiency correlates with the distance between Ser(175)-OH and the ester bond.

Conclusions:

• Pichia pastoris is a suitable host for efficient secretory expression of rice bran lipase.
• Optimized recombinant RBL possesses industrially relevant enzymatic properties.
• Understanding structure-activity relationships aids in predicting and enhancing lipase efficiency.