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

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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.

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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

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Authors and Affiliations

  1. U.S. Department of Agriculture, Agricultural Research Service, U.S. Horticultural Research Laboratory, 2120 Camden Road, FL 32803-1419, Orlando, USA

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

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  1. Richard T. Mayer
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  2. T. G. McCollum
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  3. R. P. Niedz
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  4. C. J. Hearn
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  5. Roy E. McDonald
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  6. E. Berdis
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  7. H. Doostdar
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Corresponding author

Correspondence to Richard T. Mayer.

Additional information

We thank Mr. M. Burkhart, Ms. T.-T. Ho, and Ms. M. Doherty for their valuable technical assistance. A portion of the funding for this work was made available from the Citrus Production Research Marketing Order by the Division of Marketing and Development, Florida Department of Agriculture and Consumer Services, Bob Crawford, Commissioner.

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Mayer, R.T., McCollum, T.G., Niedz, R.P. et al. Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.). Planta 200, 289–295 (1996). https://doi.org/10.1007/BF00200295

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  • DOI: https://doi.org/10.1007/BF00200295

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