Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.).
Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23,000-28,000 and isoelectric points 10.3-10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. 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 inhibited by histamine, imidazole, histidine and the N-acetyl-D-glucosamine oligosaccharide (GlcNAc)3. The basic chitinase from cv. Valencia callus, 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 hydrolyzing 13.8-100% acetylated chitosans and (GlcNAc)4-6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)2-6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (AbPLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.
Mayer
R T
RT
U.S. Department of Agriculture, Agricultural Research Service, U.S. Horticultural Research Laboratory, Orlando, FL 32803-1419, USA. rmayer@ix.netcom.com
McCollum
T G
TG
Niedz
R P
RP
Hearn
C J
CJ
McDonald
R E
RE
Berdis
E
E
Doostdar
H
H
D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002957:Citrus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014462:Ultrafiltration
D006026:Glycoside Hydrolases; C019868:chitosanase; D002688:Chitinases; D009113:Muramidase