Plant and Cell Physiology, 1994, Vol. 35, No. 5 811-820
© 1994
Identification and Characterization of Acidic Hydrolases with Chitinase and Chitosanase Activities from Sweet Orange Callus Tissue1
U.S. Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture 2120 Camden Road, Orlando, Florida 32803-1419, U.S.A.
3To whom inquiries should be addressed.
Acidic chitinases (EC 3.2.1.14 [EC] ) were isolated and characterized from 4-week-old nonembryogenic Citrus sinensis L. Osbeck cv ‘Valencia’ callus tissue. The enzymes were purified using size exclusion, anion exchange, and chromatofocusing HPLC techniques. Eleven isoforms were isolated with M1s between 26,000 and 37,400. Eight of the isoforms were purified to homogeneity, and all but one cross-reacted with a polyclonal antibody raised against a basic class I potato leaf chitinase. The isoelectric points (determined by chromatofocusing) were from pH 4.5 to 5.4. All hydrolases degraded chitin and four were capable of hydrolyzing solubilized shrimp shell chitosan suggesting they may be chitosanases (EC 3.2.1.99 [EC] ). Apparent chitosanase activity generally decreased with decreasing acetylation of the chitosan (i.e. from 20% to 0% acetylation). The chitinases and chitinases/chitosanases are predominantly endochitinases. Chitosanase activity was optimal at pH 5 while the pH optimum for chitinase activity ranged between pH 3.5 and 5.5. The chitinases and chitinases/chitosanases wer stable up to 60°C and showed their highest enzyme activity at that temperature. N-terminal sequences were obtained on three of the isoforms. One of the isoforms was identified as a class II chitinase and the other two as class III chitinases.
1Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
2Present address: TU-Munich, Department of Phytopathology, 85350 Freising-Weihenstephan, Germany
(Received February 17, 1994; Accepted May 7, 1994)
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