Research Article | Volume: 10, Issue: 1, January, 2022

Purification and biochemical characterization of protease from the seeds of Cyamopsis tetragonoloba

Rajesh Kumar Rawaliya Preeti Patidar Sadhana Sharma Krishnan Hajela   

Open Access   

Published:  Jan 07, 2022

DOI: 10.7324/JABB.2021.100121
Abstract

The present study was carried out to isolate, purify, and characterize protease from the seeds of Cyamopsis tetragonoloba. The protease was precipitated by a 60% ammonium sulfate cut and further purified by elution from ion-exchange chromatography at 0.3 M NaCl. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis result showed that protease was monomeric having 69.9 kDa molecular weight. Gelatin zymography was carried out to confirm the proteolytic activity of the protease. The protease has a wide range of substrate specificity and could cleave natural substrates like casein, gelatin, bovine serum albumin (BSA), hemoglobin (Hb), and synthetic substrate like N-αBenzoyl-DL-arginine ?-nitroanilide (BAPNA). The Vmax value of the protease was 102.04 μM/minute with casein as the substrate and Km value was 56.56 μM/minute. The purified protease was completely inhibited by serine proteases inhibitors like Phenyl Methyl Sulfonyl Fluoride, soybean trypsin inhibitor, and aprotinin, and not inhibited by other protease inhibitors. This concluded that the purified protease was serine protease. The protease was highly stable at a wide range of temperatures from 20°C to 70°C. Gelatin showed the highest proteolytic activity when compared to the casein, Hb, and BSA. BAPNA showed 1.5101 U/mg specific activity. The sugar content of protease was estimated by the method of DuBois. The protease was highly glycosylated and contained 35 μg of sugar in 0.2 mg of protease.


Keyword:     Protease purification characterization Cyamopsis tetragonoloba thermostable


Citation:

Rawaliya RK, Patidar P, Sharma S, Hajela K. Purification and biochemical characterization of protease from the seeds of Cyamopsis tetragonoloba. J Appl Biol Biotech. 2022;10(01):172-180.

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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