Properties of Chlorophyllase from Capsicum annuum L. Fruits

Abstract

Chlorophyll, Chlorophyllase, Capsicum annuumThe in vitro properties of semi-purified chlorophyllase (chlorophyll-chlorophyllido hy drolase, EC 3.1.1.14) from Capsicum annuum fruits have been studied. The enzym e showed an optimum of activity at pH 8.5 and 50 °C. Substrate specificity was studied for chlorophyll (Chi) a, Chi b, pheophytin (Phe) a and Phe b, with K m values of 10.70, 4.04, 2.67 and 6.37 ^im respectively. Substrate inhibition was found for Phe b at concentrations higher than 5 ^m. Chlorophyllase action on Chi a' and Chi b' was also studied but no hydrolysis was observed, suggesting that the mechanism of action depends on the configuration at C-132 in the chloro phyll molecule, with the enzyme acting only on compounds with R132 stereochemistry. The effect of various metals (Mg2+, Hg2+, Cu2+, Zn2+, Co , Fe2+ and Fe3+) was also investigated, and a general inhibitory effect was found, this being more marked for Hg2+ and Fe2+. Func tional groups such as -SH and -S-S-seem ed to participate in the formation o f the enzymesubstrate complex. Chelating ion and the carbonyl group at C3 appeared to be important in substrate recognition by the enzyme. The method for measuring Chlase activity, including HPLC separation of substrate and product, has been optimized.

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

“…In this study, the inhibition of Chlase activity was affected by DEPC, a chemical compound responsible for histidine modification, which suggests the importance of this amino acid in maintaining the catalytic activity of studied enzymes. Similarly, Hornero-Méndez and Mínguez-Mosquera (2001) demonstrated the activatory effect of 2-ME and DTT towards Chlase from C. annuum. Addition of these substances to the reaction medium might contribute to reducing of disulphide bonds into thiol groups and increasing of substrate binding to the active site of the enzyme.…”

“…Chlorophyllase (Chlase, chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) is a chloroplast membrane protein that has been considered to be involved in catalyzing dephytilation reaction of chlorophyll molecules into specific chlorophyllide (Chlide) forms (Arkus et al, 2005;Okazawa et al, 2006;Harpaz-Saad et al, 2007;Yi et al, 2007). This enzyme was isolated and partially purified from tissues of a broad range of higher plants and algae, and its catalytic activity under in vitro conditions was intensively studied by many researchers (Hornero-Méndez & Mínguez-Mosquera, 2001;Todorov et al, 2003;Tsuchiya et al, 2003;Arkus et al, 2005;Okazawa et al;2006;Lee et al, 2010;Gupta et al, 2011;Gupta et al, 2012). It has been elucidated that young leaves of tea and tobacco possess higher levels of Chlase activity when compared to their respective mature organs (Kuroki et al, 1981;Todorov et al, 2003b).…”

The effects of ozonated water on the microbial counts and the shelf life attributes of fresh-cut spinach

“…The effectiveness of ozonated water to control yellowing of fresh‐cut spinach may be direct, due to its strong oxidation potential (Pryor &and Rice, ) or indirect by reducing the pH from 8.4 to 4.2 for the ozonated water in our experiment. The optimum pH for chlorophyllase, the first enzyme involved in chlorophyll degradation pathway (Thomas, Ougham, & Hortensteiner, ) is 8.5 (Hornero‐Mendez & Minguez‐Mosquera, ). Moreover, the optimum pH for the cofactor Mg‐dechelating substance and Mg‐dechelatase, which are involved in the second step of the path of chlorophyll degradation (Thomas et al, ) is 7.2 (Buchert, Civello, & Martinez, ).…”

“…The optimum pH for chlorophyllase, the first enzyme involved in chlorophyll degradation pathway (Thomas, Ougham, & Hortensteiner, 2001) is 8.5 (Hornero-Mendez & Minguez-Mosquera, 2001). Moreover, the optimum pH for the cofactor Mg-dechelating substance and Mgdechelatase, which are involved in the second step of the path of chlorophyll degradation (Thomas et al, 2001) is 7.2 (Buchert, Civello, & Martinez, 2011).…”

Chlorophyll disappearance and chlorophyllase activity during ripening of Capsicum annuum L fruits

“…This fact could be explained by a highly efficient action of chlorophyllase and other chlorophylldegradative enzymes for chlorophyll a, as already described in many higher plant species and algae. 5,[20][21][22] Profiles for chlorophyllase activity were clearly different for red and chlorophyll-retaining cultivars (Fig 5). Initially, when the fruit was not yet developed (NDG), an increase in chlorophyllase activity was noticed, coinciding with a period of net chlorophyll synthesis (Figs 3 and 4), which is in accordance with previous observations in Olea europaea fruits.…”