Broccoli Processing Wastes as a Source of Peroxidase

• Published in: Journal of agricultural and food chemistry Vol. 55; no. 25; pp. 10396 - 10404
• Main Authors: Duarte-Vázquez, Miguel A, García-Padilla, Sandra, García-Almendárez, Blanca E, Whitaker, John R, Regalado, Carlos
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12.12.2007
• Subjects: Biological and medical sciences; Brassica - enzymology; broccoli; broccoli processing wastes; enzyme activity; Enzyme Stability; extraction; Food Chem/Biochem; Food Handling; Food industries; food processing wastes; Fruit and vegetable industries; Fundamental and applied biological sciences. Psychology; heat treatment; Industrial Waste; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Peroxidase; Peroxidase - chemistry; Peroxidase - isolation & purification; Peroxidase - metabolism; Plant Stems - enzymology; protein secondary structure; Protein Structure, Secondary; purification; reverse micellar extraction; reverse micelles; stems; Substrate Specificity; value-added products; waste utilization; Peroxidase; reverse micelles; purification; broccoli processing wastes; Broccoli; Vegetables; Purification; Peroxidases; Enzyme; Waste treatment; Oxidoreductases; Reverse micelle
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf072486+

A peroxidase isozyme (BP) was purified to homogeneity from broccoli stems (Brassica oleraceae var. maraton) discarded from industrial processing wastes. BP specific activity was 1216 ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] units/mg, representing 466-fold that of crude extract. BP is a monomeric glycoprotein containing 16% carbohydrates, with a molecular mass of 49 kDa and an isoelectric point close to 4.2. From kinetic data it showed a two-substrate ping-pong mechanism, and the catalytic efficiency measured as the rate-limiting step of free BP regeneration was 3.4 × 106 M−1 s−1. The ABTS K m value was 0.2 mM, which was about 20 times lower than that reported for acidic commercial horseradish peroxidase (HRP). Assessment of BP secondary structure showed 30% helical character, similar to HRP and cytochrome c peroxidase. BP lost only 25% activity after 10 min of heating at 55 °C and pH 6; it was stable in the pH range from 4 to 9 and showed an optimum pH of 4.6 using ABTS as substrate. BP was active on substrates normally involved in lignin biosynthesis, such as caffeic and ferulic acids, and also displayed good catechol oxidation activity in the presence of hydrogen peroxide. Reverse micellar extraction was successfully used as potential large-scale prepurification of broccoli peroxidase, achieving a purification factor of 7, with 60% activity yield. Stems from the broccoli processing industry have a high potential as an alternative for peroxidase purification.