Biochemical and Molecular Characterization of a β-1,3-Endoglucanase from Valencia Orange Callus

Abstract

Inhibition of the growth of fungal pathogens has been related to levels of a β-1,3-endoglucanase (EC 3.2.1.39) (GLU) in citrus as well as other plant species. Our long-term objective is to transform Citrus spp. to express enhanced levels of GLU with the aim of increasing resistance to fungal pathogens. We have purified a β-1,3-endoglucanase from nonembryogenic Citrus sinensis (L.) Osbeck cv. Valencia callus to electrophoretic homogeneity by means of pH precipitation and ion exchange chromatography. The protein has an apparent Mr of 32.5 and a pI > 10. The enzyme hydrolyzes laminarin (Laminaria digitata) optimally at pH 5 and 50°C. The enzyme will hydrolyze any glucan polymer with a β-1,3 linkage whether soluble or insoluble and the rate of hydrolysis is proportional to the relative abundance of β-1,3 linkages. The enzyme does not hydrolyze cellulose or starch. Product characterization by thin-layer chromatography indicates that the enzyme is an endohydrolase. Initial attempts to sequence the protein indicated that it was N-terminally blocked. Therefore the protein was hydrolyzed using AspN, the fragments separated by SDS-PAGE, blotted onto nitrocellulose, and one of the fragments was sequenced. Amino acid sequence analysis indicated that the protein shared homology with a number of β-1,3-endoglucanases. Antibody to the purified protein was raised in rabbits and used to screen an amplified cDNA library prepared from Citrus sinensis (L.) Osbeck cv. Valencia callus. One of the positive clones was selected and sequence analysis indicated that the clone was homologous with other β-1,3-endoglucanases.