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

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Title Purification and characterization of Limonoate-D-ring lactone hydrolase from kinnow fruits grown in Punjab, India
Purification and characterization of LDLH
 
Creator Kumar S
Kumar R
Sharma PC
Pal A
 
Subject Inhibition studies
Kinnow seeds
Limonoate-D-ring lactone hydrolase
Reverse phase HPLC
Seralose CL-6B
 
Description Not Available
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.
The authors are thankful to Indian Council of Agricultural Research for funding this research as institute project. The authors are also thankful to Dr. Dharam Singh Chopra, Retd. Professor and Ex-Head, Deptt. of Biochemistry, CCSHAU, Hisar, for language editing of the current manuscript.
 
Date 2020-07-31T10:46:17Z
2020-07-31T10:46:17Z
2018-03
 
Type Research Paper
 
Identifier Not Available
0254-8704 (Print); 2394-0379 (Online)
http://krishi.icar.gov.in/jspui/handle/123456789/38687
 
Language English
 
Relation Not Available;
 
Publisher Triveni Enterprises Lucknow, India