Cloning, expression and characterization of a cDNA encoding a lipase from Rhizopus delemar

Area of Science:

• Molecular Biology
• Biochemistry
• Enzymology

Background:

• Lipases are crucial enzymes for hydrolyzing ester bonds in triglycerides.
• Understanding lipase gene function and structure is vital for biotechnological applications.
• Rhizopus delemar lipase is a commercially significant enzyme.

Purpose of the Study:

• To clone and characterize the lipase gene (LIP cDNA) from Rhizopus delemar.
• To express the lipase in Escherichia coli and analyze its enzymatic properties.
• To determine the nucleotide sequence and deduce the amino acid sequence of the lipase.

Main Methods:

• Construction of a lambda gt11 cDNA library in E. coli using Rhizopus delemar mRNA.
• Phenotypic screening for lipase-producing clones.
• DNA sequencing of the isolated LIP cDNA.
• Analysis of lipase substrate selectivity and comparison with authentic fungal lipase.

Main Results:

• A 1287-bp LIP cDNA insert was identified, encoding a putative preprolipase.
• The expressed lipase hydrolyzed triglycerides at sn-1 and sn-3 positions, but not sn-2.
• The deduced amino acid sequence showed homology to other lipases and contained a conserved pentapeptide.
• The lipase was found to accumulate in the cytoplasm of E. coli.

Conclusions:

• The LIP cDNA represents a full-length analogue of the Rhizopus delemar lipase gene.
• The cloned lipase exhibits specific substrate selectivity characteristic of fungal lipases.
• Expression in E. coli provides a system for studying lipase structure-function relationships.