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Purification, Characterization and Application of Lipoxygenase Isoenzymes from Lasiodiplodia theobromae

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Abstract

Lipoxygenase oxidizes linoleic acid into hydroperoxy octadecadienoic acid (HPOD), which is important in food and flavour industries for production of bread and flavouring compounds. As Lasiodiplodia theobromae is an unexplored, good source of lipoxygenase, it was purified from it by size-exclusion (Sephadex G100) and ion-exchange (DEAE–cellulose) chromatography and characterized. Upon purification, L. theobromae was found to contain two different lipoxygenases, one of 93 kDa (LOX1) and another of 45 kDa (LOX2). Both the isoenzymes were having optimum pH 6.0 and optimum temperatures 50 and 40 °C, respectively. The catalytic efficiency of LOX1 and LOX2 was found to be 1300 and 1.67 × 109, respectively. The catalytic efficiency of LOX2 is higher than the catalytic efficiency of soya bean LOX1 that is 10.9 × 106. Both the isoenzymes of LOX oxidized linoleic acid to produce 9-HPOD and 13-HPOD both; however, LOX1 produced more of 9-HPOD and LOX2 produced more of 13-HPOD. Both the LOXes were not inhibited by jasmonic acid. Addition of LOX1 and LOX2 altered the elasticity as well as viscosity of dough prepared from bleached wheat flour.

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Abbreviations

LOX:

Lipoxygenase

HPOD:

Hydroperoxy octadecadienoic acid

DEAE:

Diethyl aminoethyl

SDS:

Sodium dodecyl sulphate

PAGE:

Polyacrylamide gel electrophoresis

kDa:

Kilodalton

JA:

Jasmonic acid

PMSF:

Phenylmethane sulphonyl fluoride

BSA:

Bovine serum albumin

LA:

Linoleic acid

HPLC:

High-performance liquid chromatography

LC–MS:

Liquid chromatography–mass spectroscopy

NDGA:

Nordihydroguaiaretic acid

APS:

Ammonium persulphate

LB plot:

Lineweaver–Burk plot

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Acknowledgments

The authors are grateful to CSIR for funding and to the BRD School of Biosciences for the necessary facilities. We are also thankful to the DST-sponsored PURSE Central Facility for providing the LC–MS and rheometer facilities.

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Authors and Affiliations

  1. Lab#302B, BRD School of Biosciences, Sardar Patel University, Sardar Patel Maidan, Vadtal Road, Post Box No. 39, Vallabh Vidhyanagar, Gujarat, 388120, India

    Disha D. Patel, Ravi R. Patel & Vasudev R. Thakkar

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  1. Disha D. Patel
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  2. Ravi R. Patel
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  3. Vasudev R. Thakkar
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Correspondence to Vasudev R. Thakkar.

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Patel, D.D., Patel, R.R. & Thakkar, V.R. Purification, Characterization and Application of Lipoxygenase Isoenzymes from Lasiodiplodia theobromae . Appl Biochem Biotechnol 175, 513–525 (2015). https://doi.org/10.1007/s12010-014-1278-3

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