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KMID : 0525619970020030007
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1997 Volume.2 No. 3 p.7 ~ p.26
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Enzyme Properties and Gene Structure of Rice Chitinases
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박희영/Park HY
김수일/Kim SI
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Abstract
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Five electrophoretic bands of crude enzyme extracted from rice leaves were found to possess both chitinase and β -1,3-glucanase activities. These chitinase/ β -1,3-glucanase were resolved into acidic and basic fractions by DEAE-cellulose and chitin affinity column chromatography. An acidic protein, RCG-2, containing chitinase and β -1,3-glucanase activity concurrently was purified from acidic fraction by chromatofocusing and gel slicing. The purified enzyme gave a single band on polyacrylamide gel electrophoresis and its molecular weight was appeared to be 29.7 kd by SDS-PAGE. This enzyme also had lysozyme activity. The optimal temperature for both enzyme activities was 40℃, optimal pH were 4.0 for chitinase activity and 7.0 for β -1,3-glucanase activity. Chitinase was induced in rice cell suspension culture with treatment of acetylchitooligosaccharides mixture. Among eleven isozymes found in 10% Native-PAGE, four isozyme were identified as induced enzymes. Acidic chitinase fraction separated with DEAE-cellulose column chromatography, includes three induced chitinase, while basic fraction contains only one induced isozyme. The basic inducible protein, ICG, containing chitinase and β -1,3-glucanase activity concomitantly was purified. The isolated ICG enzyme gave a single band on native and SDS polyacrylamide gel electrophoresis and its molecular weight was estimated to be 52.53 kd. The optimal temperature and optimal pH of both enzyme activities in ICG were 60℃, pH 6.0 for chitinase activity and 37℃ ,pH 4.0 for β -1,3-glucanase activity. TLC analysis of the chitooligosaccharide hydrolysates with RCG-2 or ICG enzyme indicated that both chitinases act as endowise. The nucleotide sequences of full-length cDNA clones for chitinase, CH6 and CH 16, whose size were about 1.1kb with signal peptides were determined and compared with those of other plant chitinase. Northern blot analysis with CH16 as a probe showed that the chitinase transcript reached maximum at 8 hour after elicitor-treatment and their size were about 1.1 kb, demonstrating that these clones were induced by elicitor.
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