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Journal of Food Science and Technology

, Volume 54, Issue 6, pp 1577–1584 | Cite as

Two isoforms of lipoxygenase from mature grains of pearl millet [Pennisetum glaucum (L.) R. Br.]: purification and physico-chemico-kinetic characterization

  • Bunty SharmaEmail author
  • Laxman Kumar Chugh
Original Article

Abstract

This study describes the partial purification and characterization of lipoxygenase (LOX) from pearl millet mature grains of inbred HBL 0843-2. Two isoforms of LOX, i.e., LOX 1 and LOX 2, were purified using ammonium sulphate fractionation, gel filtration chromatography and ion exchange chromatography near homogeneity to 56 and 40 folds with yield of 28 and 24%, respectively. LOX 1 and LOX 2 having molecular masses of approximately 85 and 79 kDa, respectively were purified. LOX 1 and LOX 2 exhibited maximum activity at pH 4.5 and 4.8, respectively at 25 °C temperature. Both the isoforms, which showed thermostability up to 35 °C when incubated for 30 min, were stable at a pH range of 7–7.8. LOX 1 and LOX 2 had apparent K m value of 0.86 and 0.57 µM, respectively. Ascorbic acid and vitamin E inhibited 66–78 and 61–69% activity of LOX 1 and LOX 2, respectively but Na+, Zn2+ and K+ strongly inhibited the activity of these isozymes. The present information about lipoxygenase enzyme might be valuable in drafting the strategies for its inactivation, which in turn can obstruct the LOX damaging effects on food products during processing and storage.

Keywords

Lipoxygenase Purification Pearl millet Isoforms Characterization 

Notes

Acknowledgment

Bunty Sharma thanks Chaudhary Charan Singh Haryana Agricultural University, Hisar (India), for providing financial assistance in the form of merit scholarship.

References

  1. Abdullah QL, AL-Jibori MH, AL-Arrji SB (2014) Extraction, purification and characterization of lipoxygenase from Pleurotus ostreatus. Iraqi J Sci 55:61–69Google Scholar
  2. Altunkaya A, Gokemen V (2011) Purification and characterization of polyphenol oxidase, peroxidase and lipoxygenase from freshly cut lettuce (L. sativa). Food Technol Biotechnol 49:249–256Google Scholar
  3. Anonymous (2015) ICAR Trials AHT-M, AHT-L, PT-A, AHPT-E, RHVT- Kharif 2012- Identification of promising pearl millet genotypes. In Annual Abridged Report (2012-13), pp. 33-34, Bajra Section, Department of Genetics and Plant Breeding, CCS Haryana Agricultural University, HisarGoogle Scholar
  4. Babitha MP, Prakash HS, Shetty HS (2004) Purification and properties of lipoxygenase Induced in downy mildew resistant pearl millet seedlings due to infection with Sclerospora graminicola. J Plant Sci 166:31–39CrossRefGoogle Scholar
  5. Barone R, Briante R, D`Auria S, Febbraio F, Vaccaro C, Guidice LD, Borrelli GM, Fonzo ND, Nucci R (1999) Purification and characterization of a lipoxygenase enzyme from durum wheat semolina. J Agric Food Chem 47:1924–1931CrossRefGoogle Scholar
  6. Baysal T, Demirodoven A (2007) Lipoxygenase in fruits and vegetables. Enzyme Microb Technol 40:491–495CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 72:248–254CrossRefGoogle Scholar
  8. Catherine CNS, Perez GM, Vander Hijde HT (1998) Purification, product, characterization and kinetic properties of soluble tomato lipoxygenase. Plant Physiol Biochem 36:657CrossRefGoogle Scholar
  9. Chen Z, Chen D, Chu W, Zhu D, Yan H, Xiang Y (2016) Retention and molecular evolution of lipoxygenase genes in modern rosid plants. Front Genet 7:176. doi: 10.3389/fgene.2016.00176 CrossRefGoogle Scholar
  10. Daglia M, Aceti C, Giorgetti S, Papetti A, Gazzani G (2005) Purification and characterization of soluble Cichorium intybus var. silvestre Lipoxygenase. J Agric Food Chem 53:6448–6454CrossRefGoogle Scholar
  11. Dahuja A, Madaan TR (2004) Estimation of parameters responsible for the generation of off-flavour in some Indian varieties of soybeans. Plant Foods Hum Nutr 58:1–8CrossRefGoogle Scholar
  12. Galliard T, Phillips DP (1971) Lipoxygenase from potato tubers: partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid. J Biochem 124:431–438CrossRefGoogle Scholar
  13. Gardner HW (1996) Lipoxygenase as a versatile biocatalyst. J Am Oil Chem Soc 73:1347–1357CrossRefGoogle Scholar
  14. Hsieh CC, McDonald CE (1984) Isolation of lipoxygenase isoenzymes from flour of durum wheat endosperm. Cereal Chem 6:392–398Google Scholar
  15. Jacobo-Velazquez DA, Hernández- Brenes C, Cisneros-Zevallos L, Benavides J (2010) Partial purification and enzymatic characterization of avocado (Persea americana Mill, cv. Hass) lipoxygenase. Food Res Int 43:1079–1085CrossRefGoogle Scholar
  16. Khairwal IS, Yadav OP (2005) Pearl millet (Pennisetum glaucum) improvement in India and retrospect and prospects. Ind J Agri Sci 75:183–191Google Scholar
  17. Koch RB (1986) Calcium ion activation of lipoxygenase. Arch Biochem Biophys 125:303–307CrossRefGoogle Scholar
  18. Kuo JM, Hwang A, Yeh DB, Pan MH, Tsai ML, Pan BS (2006) Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position. J Agric Food Chem 54:3151–3156CrossRefGoogle Scholar
  19. Laemmili UK (1970) Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 277:680–685CrossRefGoogle Scholar
  20. Li J, Zhang Y, Yu Z, Wang Y, Yang Y, Liu Z, Jiang J, Song M, Wu Y (2007) Superior storage stability in low lipoxygenase maize varieties. J Stored Prod Res 43:530–534CrossRefGoogle Scholar
  21. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666CrossRefGoogle Scholar
  22. Lorenzi V, Maury J, Casanova J, Berti L (2006) Purification, product characterization and kinetic properties of lipoxygenase from olive fruit (Oleae uropaea L.). Plant Physiol Biochem 4:450–454CrossRefGoogle Scholar
  23. Mandal S, Dahuja A, Kar A, Santha IM (2014) In vitro kinetics of soybean lipoxygenase with combinatorial fatty substrates and its functional significance in off flavour development. Food Chem 46:394–403CrossRefGoogle Scholar
  24. NRAA (2012) Products, diversification, marketing and price discovery of Pearl Millet in India. Policy Paper No. 2. National Rainfed Area Authority, NASC Complex, DPS Marg, New Delhi, 110012, India, p 48Google Scholar
  25. Peter G, Schwarz BP, Vick AB (1991) Purification and characterization of lipoxygenase isoenzymes in germinating barley. Cereal Chem 70:589–595Google Scholar
  26. Rai KN, Velu G, Bhattacharjee R, Kulkarni VN, Muralidharan V, Longvah T, Raveendran TS (2013) Gene action for grain zinc content in pearl millet. Crop IM 35:186–191Google Scholar
  27. Ratachatachaiyos K, Theerakulkait C (2009) Isolation and some characteristics of lipoxygenase from aromatic brown rice (Oryza sativa L.) cv. Khao Dawk Mali 105. J Immunol Immunochem 30:106–117CrossRefGoogle Scholar
  28. Shiiba K, Negishi Y, Okada K, Nagao S (1991) Purification and characterization of lipoxygenase isozymes from wheat germ. Cereal Chem 68:115–122Google Scholar
  29. Siedow JN (1991) Plant lipoxygenase: Structure and function. Annu Rev Plant Physiol Mol Biol 4:145–188CrossRefGoogle Scholar
  30. Singh SD, Sihag S, Sihag ZS, Chug LK (2014) Effect of replacing maize with pearl millet on egg production and quality in layers. Ind J Anim Nutr 31:92–96Google Scholar
  31. Szymanowska U, Jakubczyka A, Baraniak B, Kur A (2009) Characterization of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.). Food Chem 116:906–910CrossRefGoogle Scholar
  32. Theerakulkait C, Barrett MD (1995) Lipoxygenase in sweet corm germ: isolation and physiochemical properties. J Food Sci 60:1029–1033 and 1040Google Scholar
  33. Wang R, Chen Y, Ren J, Guo S (2014) Aroma stability of millet powder during storage and effects of cooking methods and antioxidant treatment. Cereal Chem 91:262–269CrossRefGoogle Scholar
  34. Wennman A, Oliw EH, Karkehabadi S, Chen Y (2016) Crystal structure of manganese lipoxygenase of the rice blast fungus Magnaporthe oryzae. J Biol Chem. doi: 10.1074/jbc.M115.707380 Google Scholar
  35. Yang GS, Schwarz PB, Vick BA (1993) Purification and characterization of lipoxygenase isoenzymes in germinating barley. Cereal Chem 70:589–595Google Scholar
  36. Zhou B, Miao A, Yang Q, Liu ZL (2005) Antioxidative effects of flavonols and their glycosides against the free radical-induced peroxidation of linoleic acid in solution and in micelles. Chemistry 11:680–691CrossRefGoogle Scholar
  37. Zilic S, Dodig D, Sukalovic VHT, Maksimovic M, Saratlic G, Skrbic B (2010) Bread and durum wheat compared for antioxidant contents andlipoxygenase and peroxidase activities. Int J Food Sci Technol 45:1360–1367CrossRefGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2017

Authors and Affiliations

  1. 1.Department of Chemistry and Biochemistry, College of Basic Sciences and HumanitiesChaudhary Charan Singh Haryana Agricultural UniversityHisarIndia

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