Molecular cloning and characterization of ech46 endochitinase from Trichoderma harzianum

In the present study, endochitinase of T. harzianum isolate-ThHP3 induced against mycelium of F. oxysporum was cloned, sequenced and characterized. The complete nucleotide sequence contained an ORF of 1293 bp corresponding to 430 amino acids with 46 kDa molecular weight and theoretical pI 5.59. The precursor protein contained 22 amino acids long signal peptide at N terminus. The domain architecture of endochitinase showed low complexity regions, presence of 1W9P domain specific to cyclopentapeptide and lack of carbohydrate binding modules. The ligand binding site of ech46 endochitinase was constituted by 10 amino acids. The cDNA encoding ech46 endochitinase was ligated into pET28a vector and transformed to E. coli BL21. The predicted molecular weight of recombinant endochitinase without signal peptide was 49.4 kDa with a theoretical pI 6.67. SDS-PAGE analysis of purified 6xHis tagged protein showed a single band of 49 kDa. The refolded enzyme was active under acidic conditions with a temperature and pH optima of 50 °C and 4. Km and Vmax for recombinant endochitinase using 4-pNP-(GlcNAc)3 were 315.2 ± 0.36 μM and 0.140 ± 0.08 μM min−1, respectively and the calculated kcat was 6.44 min−1. The RT-qPCR revealed induction of ech46 by phytopathogenic fungi.