title: Molecular cloning and characterization of ech46 endochitinase from Trichoderma harzianum.
abstract: 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 1293bp corresponding to 430 amino acids with 46kDa 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 49.4kDa with a theoretical pI 6.67. SDS-PAGE analysis of purified 6xHis tagged protein showed a single band of 49kDa. The refolded enzyme was active under acidic conditions with a temperature and pH optima of 50°C and 4. Km and Vmaxfor recombinant endochitinase using 4-pNP-(GlcNAc)3were 315.2±0.36μM and 0.140±0.08μMmin-1, respectively and the calculated kcatwas 6.44min-1. The RT-qPCR revealed induction of ech46 by phytopathogenic fungi.
authors.affiliation: Department of Plant Pathology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India; Department of Microbiology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India. Electronic address: ankvivek@gmail.com.
authors.forename: Vivek
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Department of Veterinary Microbiology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: Richa
authors.initials: R
authors.lastname: Salwan
authors.affiliation: Department of Plant Pathology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: P N
authors.initials: PN
authors.lastname: Sharma
authors.affiliation: Department of Microbiology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India.
authors.forename: S S
authors.initials: SS
authors.lastname: Kanwar
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007438:Introns / Q000235:genetics; D007477:Ions; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008970:Molecular Weight; D010802:Phylogeny; D000072417:Protein Domains; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D040681:Structural Homology, Protein; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D005656:Fungal Proteins; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D002688:Chitinases
keywords: Endochitinase; Hydrolase; Lytic enzymes