Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein.

Abstract

The tissue of insects, pests, and fungi has a chitin layer followed by protein in the cell membrane. The complete biodegradation of chitin and protein-present in the waste requires the action of two enzymes, namely chitinase, and protease. Combining chitinase and protease in a single protein/enzyme will serve as a bifunctional enzyme that can efficiently degrade the chitin and protein-rich biomass. The present study was aimed to fuse these two enzymes to produce a single protein and study the kinetics of the recombinant fusion protein. A chitinase and alkaline protease genes were isolated, cloned, and expressed successfully as a fusion product in heterologous host Escherichia coli. The two native genes were successfully fused in E.coli by using flexible glycine-serine (G4S)2 linker (GGGGS, GS linker). The recombinant fusion protein in E.coli showed hydrolyzed chitin and protein on chitin and bovine serum albumin agar plates confirming the successful cloning and expression of chitinase and protease enzymes in a single fusion protein. The common pUC18-T7 mini vector with the ompA signal sequence helps the extracellular expression of fusion protein efficiently. The native gel electrophoresis revealed a molecular mass of purified protein as 92.0 kDa. The fusion protein's maximal chitinase and protease activity occurred at pH 5.0 and 8.0 and 30 0C, respectively resembling the individual enzymes'. In the kinetic studies of the fusion protein, it was observed that the presence of metal ions such as Cu2+, Na2+, and Ca2+; significantly enhanced the enzyme activities while organic solvents oxidants and chemicals have drastically affected the activities of both the enzymes in the fusion protein. No such fusion protein has been produced in a heterologous host yet. The reports on fusion protein with biomass-degrading capacity are also scarce. This is probably the first report of a bifunctional chitinase/protease expressed in E. coli.