Planta

, Volume 200, Issue 3, pp 289–295

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

  • Richard T. Mayer
  • T. G. McCollum
  • R. P. Niedz
  • C. J. Hearn
  • Roy E. McDonald
  • E. Berdis
  • H. Doostdar
Article

Abstract

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000–28000 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.

Mention of a trademark, warranty, propriety, 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.

Key words

Citrus Chitinases Chitosanase Lysozymes Pathogenesis-related proteins Plant defense 

Abbreviations

Ab

antibody

BCLVC

basic chitinase/lysozyme cv. Valencia callus

BCVC

basic chitinase cv. Valencia callus

CE

capillary electrophoresis

CM-chitin-RBV

carboxymethyl-chitin-remazol brilliant violet

GlcNAc

N-acetyl-d-glucosamine

HEWL

hen egg-white lysozyme

Mr

relativemolecular mass

pI

isoelectric point

PLC

potato leaf chitinase

PR

pathogenesis-related

SEC

size exclusion chromatography

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • Richard T. Mayer
    • 1
  • T. G. McCollum
    • 1
  • R. P. Niedz
    • 1
  • C. J. Hearn
    • 1
  • Roy E. McDonald
    • 1
  • E. Berdis
    • 1
  • H. Doostdar
    • 1
  1. 1.U.S. Department of AgricultureAgricultural Research Service, U.S. Horticultural Research LaboratoryOrlandoUSA

Personalised recommendations