New Assay Method for Allene Oxide Cyclase, an Important Enzyme in Jasmonic Acid Biosynthesis
Journal Title: Biotechnology Journal International - Year 2015, Vol 9, Issue 2
Abstract
Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method. Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC. Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan. Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system. Results: We found in the presence of 50 nmol of purified AOS together with 20 M 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system. Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.
Authors and Affiliations
Keimei Oh, Yoichiro Shimura
Keywords
Related Articles
- EP ID EP237316
- DOI 10.9734/BBJ/2015/20089
- Views 88
- Downloads 0
Bio-bleaching of Anthocephalus cadamba Kraft Pulp through Direct Fungal Treatment by FEQP Sequence
Aims: The study aims at mitigating pulp kappa number before bleaching to minimize pollution load. Study Design: An experimental study. Methodology: The various parameters of direct fungal (Coprinellus disseminatus MLK01)...
Genetic and Phenotypic Identification of Vancomycin-Resistant Staphylococcus aureus Isolates from Retail Poultry Carcasses in Omu-Aran, North-Central Nigeria
Staphylococcus aureus is a well-known agent of zoonotic infections. Livestock-associated methicillin-resistant S. aureus (LA-MRSA) had been receiving public health attention for over a decade. Recently, the genomes of so...
Biovalorization of Olive Mill Waste Water for the Production of Gellan Gum from Sphingomonas paucimobilis
Aims: The aim of this work was to investigate and optimize the potential of olive mill waste water (OMWW) to be utilized as a substrate for the production of gellan gum by Sphingomonas paucimobilis. Study Design: The st...
Characterization of Trypsin-Like Serine Protease from Lethocerus indicus Salivary Venom and its Cytotoxic Effect against Human Epidermoid Carcinoma Cell, A431
Objective: Lethocerus indicus salivary venom characterization and evaluation of extracellular degradation activity and cytotoxic effect against native human collagen type 1 and epidermoid carcinoma cell, A431. Method: Sa...
Effect of Arsenic (As) on the Spermatogenesis of Black Bengal Goat (Capra hircus) Reared at the Arsenic Prone Area of Mymensingh District in Bangladesh
Aim: Arsenic (As) alters the spermatogenic process as well as testicular histology has been reported in different species of domestic, wild, aquatic life, and laboratory animals. The present study was conducted to invest...