Biochemical, molecular genetic, and immunological characterization of a beta-1,3-endoglucanase from 'Valencia' orange callus

We have purified a beta-1,3-endoglucanase (EC 3.2.1.39) from nonembryogenic Citrus sinensis (L.) Osbeck cv. Valencia callus to electrophoretic homogen eity by means of pH precipitation and ion exchange chromatography. The prot ein has an apparent M-r of 32,000, a pI>pH 10 and is serologically similar to a potato leaf glucanase induced by Phytophthora infestans infection. The enzyme hydrolyzes laminarin (Laminaria digitata) optimally at pH 5 and 50 degrees C. The enzyme mill hydrolyze pachyman and laminarin extensively and yeast glucan slightly, but does not hydrolyze lichenin, barley glucan, cel lulose, or starch. Product characterization by thin-layer chromatography in dicates that the enzyme is an endohydrolase. The protein is N-terminal bloc ked, however, partial internal amino acid sequence analysis revealed that t he peptide shared homology with a number of beta-1,3-endoglucanases. Antibo dy to the purified protein was raised in a rabbit: and used to screen an am plified cDNA library prepared from Citrus sinensis (L.) Osbeck cv. Valencia callus. A positive clone (pBGVC-1) containing a 1,249 bp insert was isolat ed. A full length sequence of the clone was obtained and it contained a 1,2 29 bp open reading frame starting at nucleotide 20. Sequence analysis indic ated that the clone is homologous to other beta-1,3-endoglucanase genes. Th e predicted amino acid sequence was homologous with other beta-1,3-glucanas es, contained both N- and C-terminal signal sequences, the glycosyl hydrola se family 17 signature sequence, and the sequence identical to the peptide that was sequenced from the purified protein.