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.

Evaluation of Plant Growth-Promoting and Salinity Ameliorating Potential of Halophilic Bacteria Isolated From Saline Soil

“…The genomic DNA of each isolate was extracted by the LPS buffer method (Nilpa et al, 2022) and the quality of isolated DNA was checked on 1% agarose gel prepared in 1X TAE buffer. The universal PCR primers 27F (52 AGA GTT TGA TCC TGG CTC AG 3G) and 1492R (52 TAC CTT GTT ACG ACTT 3AC) were used to amplify the highly conserved ∼1,400 bp region of the 16S ribosomal RNA gene (rRNA gene).…”

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

“…The PLOS ONE Editors retract this article [ 1 ] because it was identified as one of a series of submissions for which we have concerns about authorship, competing interests, and peer review. We regret that the issues were not addressed prior to the article’s publication.…”