Purification and Characterization of α-Glucan Phosphorylase Isoform Pho 2 from Spinach Leaves
Journal Title: Biotechnology Journal International - Year 2015, Vol 5, Issue 4
Abstract
α-Glucan phosphorylase is an important enzyme of carbohydrate metabolism. In spinach leaves, it has been reported in two multiple forms viz. Pho 2 (cytosolic) and Pho 1 (plastidial). Here, we extracted and purified Pho 2 form of α-glucan phosphorylase from spinach using salting out with ammonium sulfate, desalting using Sephadex G-25 chromatography, anion exchange chromatography using DEAE-Sepharose and gel filtration chromatography using Sepharose-4-B. The purified enzyme had a specific activity, 150 units/mg protein. There was 38% recovery and 652 fold purification after final Sepharose-4B chromatography. The purified enzyme showed a single protein band on SDS sodium dodecyl sulfate polyacrylamide gel electrophoresis having molecular weight 94,000±2000 daltons. The native molecular weight is found to be 188,000±3000 daltons as determined using gel filtration chromatography over Sephadex G-200. The Pho2 exhibited optimum pH at pH 6.0 with two half pH optima at pH 5.2 and pH 7.0. The optimum temperature of Pho2 is found to be 37ºC with two half temperature optima at 30ºC and 40ºC. The Km value of the enzyme for starch and glucose-1-phosphate is found to be 116 µg/mL and 0.55 mM, respectively.
Authors and Affiliations
Anil Kumar, Ritu Jain
Keywords
Related Articles
- EP ID EP237092
- DOI 10.9734/BBJ/2015/15106
- Views 139
- Downloads 0
Establishment of Efficient System for Isolation and Manipulation of Single Protoplasts Containing Aposporous Embryo Sac Initial Cell in Guinea Grass (Panicum maximum)
Aims: In order to do the molecular analysis of mechanism of aposporous embryo sac initial cell (AIC) appearance, as the first step, we attempted to establish the system of isolating and manipulating single cells containi...
The Effect of Locally Fabricated Pelletizing Machine on the Chemical and Microbial Composition of Organic Fertilizer
In order to make organic fertilizer more quantifiable and attractive to farmers, a pelletizing machine was fabricated locally to produce pellets from powdery fertilizer. However, concerns have been raised on the potentia...
Biovalorization of Olive Mill Waste Water for the Production of Gellan Gum from Sphingomonas paucimobilis
Aims: The aim of this work was to investigate and optimize the potential of olive mill waste water (OMWW) to be utilized as a substrate for the production of gellan gum by Sphingomonas paucimobilis. Study Design: The st...
Bacterial Synthesis of Silver Nanoparticles by Culture Free Supernatant of Lactic Acid Bacteria Isolated from Fermented Food Samples
Aim: Biosynthesis of silver nanoparticles (SNPs) using culture free supernatant (CFS) of Lactic acid bacteria (LAB). Study Design: To biosynthesize, characterize and to determine the antibacterial potential of silver na...
Cell Proliferation Using Broccoli Leaf Cutting in vitro Culture: Its Biochemical and Antioxidant Properties
Significance of the study: Tissue or cell culture keeps a significant role in micro-propagation in the plant production industry. Nowadays it has been successfully performed from many species in plant. Millions of explan...