Purification, characterization and gene structure of (1→3)‐β‐glucanase isoenzyme GIII from barley ( Hordeum vulgare )

A new member of the barley (1→3)‐β‐glucan glucanohydrolase family of enzymes has been purified from extracts of germinated grain and young seedlings by fractional precipitation with ammonium sulphate, ion‐exchange chromatography, chromatofocussing and gel‐filtration chromatography. The enzyme, which has been designated (1→3)‐β‐glucanase isoenzyme GIII, is a basic protein with an apparent molecular mass of 32 000 Da. Oligosaccharide products released by the enzyme during hydrolysis of the (1→3)‐β‐glucan, laminarin, indicate that the enzyme is an endohydrolase. A 2349‐bp fragment of barley genomic DNA has been isolated and identified as the gene encoding the (1→3)‐β‐glucanase isoenzyme GIII. The open reading frame encoding the isoenzyme is interrupted by a single intron of 180 bp that splits a codon in the putative signalpeptide region. Northern‐blot analyses with gene‐specific probes indicate that the (1→3)‐β‐glucanase isoenzyme GIII mRNA accumulates in developing leaves; no mRNA transcripts were detected in the aleurone or scutellum of germinated grain, or in mature vegetative tissues. Although plant (1→3)‐β‐glucanases are generally classified as ‘pathogenesis‐related’ proteins, the physiological function of the barley (1→3)‐β‐glucanase isoenzyme GIII is unclear.