Biochemical Characterization of Cystine Lyase from Broccoli (Brassica oleracea Var. italica)

• Published in: Journal of agricultural and food chemistry Vol. 47; no. 6; pp. 2218 - 2225
• Main Authors: Ramírez, Edna C, Whitaker, John R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.06.1999
• Subjects: amino acid composition; amino acids; Amino Acids - analysis; Biological and medical sciences; Biotechnology; Brassica - enzymology; Brassica oleracea var. italica; broccoli; chemical composition; coenzymes; cofactors; Cystathionine gamma-Lyase - chemistry; Cystathionine gamma-Lyase - isolation & purification; Cystathionine gamma-Lyase - metabolism; enzyme activity; Enzyme engineering; enzyme inhibitors; Enzyme Inhibitors - pharmacology; Enzyme Stability; Food industries; Fruit and vegetable industries; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; Kinetics; lyases; Methods. Procedures. Technologies; molecular weight; purification; Substrate Specificity; temperature; Thermodynamics; Brassica oleracea var. italica; Biochemical analysis; Purification; Enzyme; Isozyme; Active site; Lyases; Experimental study; Molecular weight; Broccoli; Enzymatic activity; Vegetable crop; Cruciferae; Dicotyledones; Substrate specificity; Angiospermae; Carbon-sulfur lyases; Optimality condition; Isolation; Spermatophyta; Kinetics; Cystathionine β-lyase; Mechanism of action; Vegetable
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf981153x

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. l-Cystine, l-cysteine-S-sulfate, l-djenkolic acid, and some S-alkyl-l-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with l-cystathionine. A K m value of 81.2 μM was found for l-cystine. Inhibition and K i determinations indicated that l-cysteine is a linear noncompetitive inhibitor with a K i of 5 mM and dl-homocysteine is a competitive inhibitor with a K i of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition. Keywords: Purification; cystine lyase; β-cystathionase; broccoli; Brassica oleracea var. italica