Purification and characterization of lipoxygenase from mung bean (Vigna radiata L.) germinating seedlings

3 Biotech (2016) 6:113 DOI 10.1007/s13205-016-0427-5 ORIGINAL ARTICLE Purification and characterization of lipoxygenase from mung bean (Vigna radiata L.) germinating seedlings Raveendra Aanangi1 • Kasi Viswanath Kotapati2 • Bhagath Kumar Palaka2 • Thyagaraju Kedam1 • Nirmala Devi Kanika1 • Dinakara Rao Ampasala2 Received: 7 November 2015 / Accepted: 26 April 2016 / Published online: 17 May 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Abstract This study reports purification and characterization of lipoxygenase protein from mung bean germinating seedlings. Lipoxygenases (LOXs) are key enzymes in seed germination. The purified mung bean LOX has resolved into two peaks by chromatofocusing, one has highest LOX activity with an isoelectric point of 5.84 and the other has lowest LOX activity with an isoelectric point of 5.52. The purified LOX has molecular mass of approximately 97 kDa and showed high activity with linoleic acid than linolenic acid and arachidonic acid. The optimal activity of LOX was observed at pH 6.5 and temperature 35 °C. Far-UV circular dichroism (CD) studies revealed that the purified mung bean LOX possess secondary structural elements with significant a-helix and b-strands. Further, the secondary structure of mung bean LOX was stable up to 60 °C at pH 6.5. Biophysical and chemical properties of the mung bean LOX are similar to the other legume LOXs and may be considered as type-1 LOX. Keywords Chromatofocusing  Circular dichroism  Mung bean  Plant lipoxygenases  Protein purification Abbreviations LOX Lipoxygenase PUFAs Polyunsaturated fatty acids ETYA Eicosatetraenoic acid DE-52 Diethyl amino ethyl cellulose & Dinakara Rao Ampasala 1 Department of Biochemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India 2 Centre for Bioinformatics, School of Life sciences, Pondicherry University, Puducherry 605014, India NDGA CD TBA TCA MDA X-Gal PMSF Nordihydroguaiaretic acid Circular dichroism Thiobarbituric acid Trichloro acetic acid Malondialdehyde 5-Bromo-4-chloro-3-indolyl b-Dgalactopyranoside Phenylmethylsulfonyl fluoride Introduction Lipoxygenases (LOXs, linoleate: oxygen oxidoreductases, EC: 1.13.12.11) represent a large gene family of non-heme, non-sulfur iron or manganese containing dioxygenases which are ubiquitously distributed throughout nature indicating the biological and evolutionary importance of these enzymes (Joo and Oh 2012). LOXs catalyze the regio- and stereo-specific dioxygenation of polyunsaturated fatty acids (PUFAs) containing (1Z, 4Z)-pentadiene system (Kuhn and Thiele 1999). Evidences suggested that the primary products generated by these enzymes are called as hydroperoxides, which act as substrates for the synthesis of short chain carbonyl compounds, involved in plant growth, host– pathogen interactions, defense, development and senescence (Brash 1999; Gfeller et al. 2010; Siedow 1991). LOXs are also known to play a major role in production of volatile substances, which influence the flavor and aroma of food from the plants and have wide applications in food industry as they play important role in maintaining food quality and the aroma of food grains (Suzuki et al. 2010). Germination is the most critical step in the life cycle of spermatophytes and represents the entry of plants into the 123 113 Page 2 of 8 ecosystem, during which a seed from a dormant embryonic state enters into a highly dynamic phase leading to seedling establishment (Weitbrecht et al. 2011). Germination begins with imbibition and terminates with seed coat rupture and radicle protrusion through the endosperm (Kong et al. 2015). During germination, lipid bodies are degraded in seeds by a new set of proteins, among which, LOXs are playing an important role (Feussner et al. 2001). During the past few decades, many isoforms of LOX were identified from different plants and their physical and enzymatic properties are characterized. Although occurrence of LOXs in vegetables and fruits is known, it has been reported that legumes are rich source for LOXs, among them soybean seed LOXs were well studied at molecular level (Feussner and Wasternack 2002; Kolomiets et al. 2001). Three isoforms of LOX were identified and characterized from soybean seeds based on their pH optimum, substrate specificity, product formation, kinetic parameters and enzyme stability (Axelrod et al. 1981; Clemente et al. 2000; Kolomiets et al. 2001). Apart from soybean, other legumes have also been reported as good source of LOX proteins. Legumes are inexpensive source of LOX proteins and particularly high level of LOX activities were identified from various legume seeds (Rao et al. 1998). Mung bean was identified as a novel source of LOX in the natural production of green-note aroma compound, hexanal, it is also considered as a cheap and readily available staple food in Asia (Chow et al. 2007). Further, mung bean and its seedlings have been used as a source of hydroperoxide lyase enzyme (Rehbock et al. 1998). Previously, high levels of LOX activity have been reported in germinating seedlings of mung bean (Devi et al. 2005; Rao et al. 1998). Earlier we reported a full-length LOX from mung bean germinating seedlings, during developmental stage more level of LOX expression was observed (Kotapati et al. 2015). However, no reports were available to date on purification and characterization of LOX from mung bean. In the present study, considering the importance of LOX proteins in food industry, a report on the isolation and biochemical properties of purified LOX from mung bean seedlings is presented. Materials and methods 3 Biotech (2016) 6:113 Acrylamide, Bis-acrylamide, Coomassie brilliant blue, Lauryl sulphate (SDS) and protein size markers were procured from Sigma Chemicals Co (St. Louis, MO, USA). NDGA was a generous gift from department of chemistry, S.V. University. All other chemicals were reagent grade procured from Merck, Mumbai, India. LOX protein extraction and purification Extraction and purification methods for mung bean LOX was adopted from Clemente et al. (2000) and Roopashree et al. (2006) with minor modifications. In brief, finely ground powder of mung bean seedlings was extracted with ice cold hexane to make it defatted. About 30 g of defatted fine powder was extracted with 300 ml of 50 mM sodium phosphate buffer, pH 6.8, at 4 °C for 3–4 h and centrifuged at 10,0009g for 30 min. The fine supernatant was dialyzed against 25 mM sodium phosphate buffer (pH 6.8) for 24 h with three buffer changes and centrifuged at 25,0009g for 20 min. The supernatant was dialyzed against 40 % poly ethylene glycol 20,000 for 16 h and then centrifuged at 25,0009g for 20 min. The dialyzed sample was applied to Sephadex G-150, gel filtration column (100 9 2.5 cm) and fractions were collected with a fraction size of 2.5 ml per tube at a flow rate of 20 ml/h. The active fractions were pooled and further purified by ion exchange (...truncated)