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

Summary We have purified a β-1,3-endoglucanase (EC 3.2.1.39) 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 32,000, a pl > pH 10 and is serologically similar to a potato leaf glucanase induced by Phytophthora infestans infection. The enzyme hydrolyzes laminarin (Lam- inaria digitata) optimally at pH 5 and 50 °C. The enzyme will hydrolyze pachyman and laminarin extensively and yeast glucan slightly, but does not hydrolyze lichenin, barley glucan, cellulose, or starch. Product characterization by thin-layer chromatography indicates that the enzyme is an endohydrolase. The protein is N-terminal blocked, however, partial internal amino acid sequence analysis revealed that the peptide shared homology with a number of β-1,3-endoglucanases. Antibody to the purified protein was raised in a rabbit and used to screen an amplified cDNA library prepared from Citrus sinensis (L.) Osbeck cv. Valen- cia callus. A positive clone (pBGVC-1) containing a 1,249 by insert was isolated. A full length sequence of the clone was obtained and it contained a 1,229 by open reading frame starting at nucleotide 20. Sequence analysis indicated that the clone is homologous to other 0-1,3-endoglucanase genes. The predicted amino acid sequence was homologous with other 0-1,3-glucanases, contained both N- and C-terminal signal sequences, the glycosyl hydrolase family 17 signature sequence, and the sequence identical to the peptide that was sequenced from the purified protein.