A Simple Method for On-Gel Detection of Myrosinase Activity

Molecules

Zoltán Cziáky¹  Zsolt Szűcs²  Sándor Gonda²  Tamás Plaszkó²  Gábor Vasas²  Márta M-Hamvas²  Attila Kiss-Szikszai³ 

[1] Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, Sóstói str. 31/b, H-4400 Nyíregyháza, Hungary;Department of Botany, Division of Pharmacognosy, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary;Department of Organic Chemistry, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary;

关键词: myrosinase;    thioglucosidase;    sulfatase;    on-gel detection;    desulfo-sinigrin;    LC-ESI-MS;   

DOI  :  10.3390/molecules23092204

来源: DOAJ

【 摘 要 】

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4− is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R2 = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.

【 授权许可】

Unknown