Purification and properties of chitinase from cabbage stems with roots

Abstract

Chitinase has been purified from the extract of cabbage stems with roots through successive steps of ammonium sulfate fractionation, Sephadex G‐75 gel filtration, chromatofocusing and Sephacryl S‐200 HR gel filtration. By these steps, the purity of the enzyme increased by 63 fold and the recovery of the enzyme activity was 18%. The purified enzyme was homogeneous when analyzed by SDS‐PAGE#. It showed an optimal pH of 6 and optimal temperature of 60°C for hydrolysis of ethylene glycol chitin (EGC). The molecular mass of the enzyme was 41 kDa, as determined by SDS‐PAGE. Heavy metal ions (1.5 mM) Ag+, Hg2+ and Fe2+, and chemical modification agents NAI (1 mM), NBS (0.5 mM) and CHD (0.5 mM) significantly or completely inhibited the activity of the enzyme. Substrate EGC at high concentrations also inhibited the activity. BSA (0.05%), Triton X‐100 (0.5%) and glycerol (50%) provided significant protection of the enzyme from freezing inactivation.

Chitinase from Leaves of Beta vulgaris and other Higher Plants

“…Result of the screening experiments indicated that the enzymatic activity was varied not only with the species of the tested plants, but also according to the morphology of the examined parts. These results are to great extent in accordance to those found by Powning and lrzykiewicz (1965), Abeles et al (1971), Hirano et al (1990), Leah et al (1991) and Chang et al (1996). The increase in activity of chitinase during germination may be due to activation of the latent form and for increase in the rate of de novo synthesis of enzymes.…”

“…The molecular weight of the purified chitinase was determined to be 64×10 3 . Concerning the M,s of chitinase enzyme isolated from different sources, it was found to be different, as chitinase isolated from bean leaves, soybean seeds, cabbage stem with root and tomato stem tissue have M,s range from 27×10 3 to 31×10 1 Dalton (Pegg and Young, 1982;Wadsworth and Zikakis, 1984;Chang et al, 1996). It was found that the M,s of stomach of Japanese eel, Bacillus, cereus, Serratia marcescens and S. peculiarities chitinase were 59×10 3 , 68.52×10 3 , 58×10 3 and 45×10 3 Dalton respectively (Kono et al, 1990;Zhang et al, 1996: Porfir'eva et al, 1997.…”

“…of wheat grain and carrot roots chitinase (30 and 25EC) isolated by Molano et al (1979) and Zhang et al (1996) and lower than optimum temp. of cabbage stem and Bacillus cereus chitinase (60EC) isolated by Chang et al (1996) and Trachuk et al (1996). The molecular weight of the purified chitinase was determined to be 64×10 3 .…”

“…The optimum pH for chitinase activity from sugar beet was measured to be 4.5 which are in accordance to those reported by Wadsworth and Zikakis (1984), Kono et al (1990) and Zhang et al, (1996) for soybean seeds Japanese eel and carrot roots chitinase respectively. While Molano et al (1979) and Chang et al (1996) isolated chitinase from wheat grain and cabbage stem with an optimum pH 6.0. Chitinase isolated from Bacillus cereus showed broad optimum pH from 4.0 to 10.0 (Trachuk et al, 1996).…”

“…As it is an agridulture waste and a source of pollution, trials were done to use it in production of beneficial enzyme and consequently aid in consuming the pollution. Pure chitinase was isolated from sugar beet leaves with relatively high recovery of activity ( 27.4%) as compared to that of soybean seeds and cabbage stem (12%) and (18%) isolated by Wadsworth and Zikakis (1984) and Chang et al (1996) respectively, but lower than those from thorn apple (48%) (Broekaert et al, 1988). The optimum pH for chitinase activity from sugar beet was measured to be 4.5 which are in accordance to those reported by Wadsworth and Zikakis (1984), Kono et al (1990) and Zhang et al, (1996) for soybean seeds Japanese eel and carrot roots chitinase respectively.…”