title: Physicochemical study of a novel chimeric chitinase with enhanced binding ability.
abstract: Chitinases are slow-reacting but important enzymes as they are anticipated to have diverse applications. The role of a chitin-binding domain (ChBD) in enhancing the quality of binding is essential information for purposeful engineering of chitinases. The idea of making hybrid chitinases by fusing a known ChBD to a chitinase, which naturally lacks ChBD is of interest especially for bio-controlling purposes. Therefore, in the present study, the ChBD of Serratia marcescens chitinase B was selected and fused to the fungal chitinase, Trichoderma atroviride Chit42. Both Chit42 and chemric Chit42 (ChC) showed similar activity towards colloidal chitin with specificity constants of 0.83 and 1.07 min(-1), respectively, same optimum temperatures (40°C), and similar optimum pH (4 and 4.5, respectively). In the presence of insoluble chitin, ChC showed higher activity (70%) and obtained a remarkably higher binding constant (700 times). Spectroscopic studies indicated that chimerization of Chit42 caused some structural changes, which resulted in a reduction of α-helix in ChC structure. Chemical and thermal stability studies suggested that ChC had a more stable structure than Chit42. Hill analysis of the binding data revealed mixed-cooperativity with positive cooperativity governing at ChC concentrations below 0.5 and above 2 µM in the presence of insoluble chitin. It is suggested that the addition of the ChBD to Chit42 affords structural changes which enhance the binding ability of ChC to insoluble chitin, improving its catalytic efficiency and increasing its thermal and chemical stability.
authors: National Institute of Genetic Engineering and Biotechnology, PO Box: 149651/161, Tehran, Iran.
authors: Soheila
authors: S
authors: Matroodi
authors: Mohammadreza
authors: M
authors: Zamani
authors: Kamahldin
authors: K
authors: Haghbeen
authors: Mostafa
authors: M
authors: Motallebi
authors: Saeed
authors: S
authors: Aminzadeh
mesh_terms: D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology; D014242:Trichoderma / Q000201:enzymology
chemical_list: D011993:Recombinant Fusion Proteins; D002686:Chitin; D002688:Chitinases
keywords: binding; chimeric chitinase; chitin-binding domain; kinetic; structure