Journal of Environmental Biology

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: sunilsaini2007@gmail.com

 

 

 

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          

 

Abstract

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.

 

 

Copyright © 2018 Triveni Enterprises. All rights reserved. No part of the Journal can be reproduced in any form without prior permission. Responsibility regarding the authenticity of the data, and the acceptability of the conclusions enforced or derived, rest completely with the author(s).