Isolation of a thermostable legume chitinase and study on the antifungal activity.
Chitinases are listed as one class of pathogenesis-related proteins, and they have become a popular research topic because of their resistance to plant-pathogenic diseases. A chitinase with antifungal activity was isolated from the Canadian cranberry beans (Phaseolus vulgaris). The procedure included extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, CM-Sephadex C-50, and Sephadex G-75. There was an almost 108-fold increase in specific activity of the purified chitinase compared with that of the crude extract. The enzyme exhibited a molecular mass of 30.6 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and non-reducing conditions, indicating that it was a monomeric protein. The pI was determined to be 7.6 by isoelectric-focusing electrophoresis. The optimum pH and the optimum temperature for activity towards N-acetyl-glucosamine was 5.4 and 40-55 degrees C, respectively. It exerted a potent inhibitory action toward fungal species including Botrytis cinerea, Physalospora piricola, Fusarium oxysporum, and Pythium aphanidermatum. The chitinase was thermostable up to 58 degrees C in both enzymatic reaction and antifungal activity. The present findings demonstrated a thermostable chitinase from cranberry beans with potentially exploitable significance.
Wang
Shaoyun
S
College of Bioscience & Biotechnology, Fuzhou University, Fuzhou 350002, People's Republic of China. shywang@fzu.edu.cn
Shao
Biao
B
Fu
Hong
H
Rao
Pingfan
P
D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology; D020171:Botrytis / Q000187:drug effects; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D005658:Fungi / Q000187:drug effects; D005670:Fusarium / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D027805:Phaseolus / Q000201:enzymology*; D011775:Pythium / Q000187:drug effects; D013816:Thermodynamics
D000935:Antifungal Agents; D008670:Metals; D002686:Chitin; D004492:Edetic Acid; D002688:Chitinases