Comprehensive in silico structural-functional analysis of Enterobacter GH19 class I chitinase (chiRAM) gene: cloning and heterologous expression

Abstract I. Background. Present study aims to clone and express the gene-encoding chitinase / GH19 family from Enterobacter sp. in E.coli with in silico sequence analyses.. II. Methods and results. The putative open reading frame of GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM-T and pET-28a + vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, Genbank accession no.: MK533791.2) was translated to chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). chiRAM in silico protein sequence analysis revealed GH19 class I chitinase: N-terminus signal peptide (Met1-Ala23), catalytic domain (Val83-Glu347 & catalytic triad Glu149, Glu171, Ser218), proline-rich hinge (Pro414 -Pro450), (polycystic kidney disease protein motif (Gly 465-Ser 533), C-terminus chitin-binding domain (Ala553- Glu593), and class I conserved motifs (NYNY and AQETGG). Three dimensional model was constructed by LOMETS MODELLER, PDB template: 2dkvA (Oryza sativa L. japonica class I chitinase). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72kDa; SDS-PAGE) in 1.0 mM IPTG induced E.coli BL21 (DE3) Rosetta at room temperature, 18 hrs post induction. Optimized expression yielded active chiRAM with 1.974 U/mL ± 0.0002, on shrimp colloidal chitin (SCC), in induced E.coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified the 72 kDa band in the soluble fraction with 52.3% coverage sequence exclusive to Enterobacter cloacae chitinase/GH19 (WP_063869339.1). III. Conclusions. Despite the successful cloning and expression of chiRAM of Enterobacter sp. in E.coli with an appreciable chitinase activity, prospective studies would focus on minimizing IBs to facilitate chiRAM purification and characterization.