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Journal of Environmental Biology

pISSN: 0254-8704 ; eISSN: 2394-0379 ; CODEN: JEBIDP

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        Abstract - Issue Mar 2018, 39 (2)                                                                                                             Back

nstantaneous and historical temperature effects on a-pinene

Purification and characterization of Limonoate-D-ring lactone hydrolase from kinnow fruits grown in Punjab, India


S. Kumar1*, R. Kumar1, P.C. Sharma2 and A. Pal3

1Division of Horticultural Crop Processing, ICAR-Central Institute of Post Harvest Engineering & Technology, Abohar - 152 116, India

2Department of Food, Science and Technology, YS Parmar University of Horticulture and Forestry, Solan-173 230, India

3Department of Chemistry and Biochemistry, CCS Haryana Agricultural University, Hisar-125 004, India

*Corresponding Author E-mail:




Key words

Inhibition studies

Kinnow seeds

Limonoate-D-ring lactone hydrolase

Reverse phase HPLC

Seralose CL-6B




Publication Data

Paper received : 10.09.2016

Revised received : 11.04.2017

Re-revised received : 16.06.2017

Accepted : 08.08.2017          



Aim: The enzyme limonoate-D-ring lactone hydrolase (LDLH) is known to cause delayed bitterness in kinnow fruit juice. This enzyme catalyzes the conversion of non-bitter precursor, limonoate-A-ring lactone/LARL to bitter limonin in acidic medium of juice.


Methodology: Limonoate-D-ring lactone hydrolase was extracted from kinnow seeds and purified using ammonium sulphate fractionation (25-85%) followed by molecular exclusion chromatography (seralose-CL-6B). Enzyme activity was inhibited with food grade inhibitors and enzyme assay was carried out with high pressure liquid chromatography


Results: This procedure resulted in 74.57 fold purification and recovery of 5.02%. Molecular mass of the purified enzyme was found to be 224 kDa, while subunit molecular mass of 45 kDa was adjudged using gel electrophoresis suggesting its pentameric nature. Bromelain treatment inactivated the enzyme by 11.5%; sodium hexmetaphosphate, L-glutamate and 1,2-cyclohexylenedinitrilotetraacetic acid could inhibit enzyme activity by ~40%; while ethylenediamine tetraacetic acid caused 58.1% inhibition. Papain, pepsin, histidine and DL-malic acid did not have any discernable effect on its activity.    


Interpretation: Sodium hexmetaphosphate, L-glutamate, 1,2-cyclohexylene dinitrilotetraacetic acid and EDTA can be used for inhibiting LDLH activity in kinnow juice to curtail limonin generation, thereby reducing delayed bitterness.



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