CHARACTERIZATION OF 7 BASIC ENDOCHITINASES ISOLATED FROM CELL-CULTURES OF CITRUS-SINENSIS (L.)

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000-28 000 and isoelectric points 10.3-10.4) were purified from nonembryogeni c Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chit inase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitin ase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0. 2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhi bited by histamine, imidazole, histidine and the N-acetyl-D-glucosamin e oligosaccharide (GlcNAc)(3). The basic chitinase from cv. Valencia c allus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolysing 13. 8-100% acetylated chitosans and (GlcNAc)(4-6) oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes e xpressed varying degrees of hydrolytic capabilities, Experiments with (GlcNAc)(2-6) suggest that BCLVC hydrolysis occurs in largely tetrasac charide units whereas hydrolysis by the other chitinases occurs in dis accharide units. Cross-reactivities of the purified proteins with anti bodies for a potato leaf chitinase (Ab(plc)), BCLVC, BCVC-3, and tomat o AP24 indicate that these are separate and distinct proteins.