Purification, Characterization And Gene Clone Of Chitinase From Aeromonas Caviae CB101 And The Study On Chitinase

Aeromonas caviae CB101 can secrete multiple chitinases into the culture supernatant as revealed by chitinase activity staining.One gene chi1 has been cloned and sequenced. It encodes protein of 865 amino acid resides. Structural analysis of the gene revealed that it contains an N-terminal signal peptide, a polycystic-kidney-disease(PKD)-like domain, a triosephosphate-isomerase(TIM) catalytic region,a receptor-for egg jelly(REJ)-like domain and two chitin-binding domains(chBDs) located from the N-terminal to C-terminal. It almost contains all kinds of domains which are already reported in chitinases.So,it is a good material for us to study the function of different domains of chitinase.To study these chitinases,we constructed the CB101's genomic library,we obtained nine positive cloones when the genomic library was screened on medicum containing 1% colloidal chitin and antibiotic.All these positive clones were confirmed for the presence of chi1 gene,by using the three pairs of primers which were specially designed for chi1 gene.We deleted catalytic domain of chi1 gene to study the role of this domain in catalytic activity. We use Southern blotting to confirm the single copy of chi1 gene in the genome of CB101.Sequence analysis of the all positive clones revealed that they all contain chi1 gene.Chitinase enzyme from the culture supernatant was purified by Ammonium sulfate precipitation method followed by the DEAE-Sepharose and Mono Q anion exchange chromatography column .The result of it's N-terminal sequencing approves that this chitinase is the truncation off the signal peptide and some C-terminal domains of the cloned chitinase Chi1. these results suggest that the Aeromonas caviae CB101 can only secrete one kind of chitinases ,which are Chi1 and its truncates.