Purification and characterization of arginase from neurospora sp for the development of arginine biosensor

Abstract

L-arginine is a naturally occurring basic amino acid that participates in many important biochemical reactions associated with normal physiology of the organism. It has been considered the most potent Nutraceutical ever discovered, due to its powerful healing properties, and is referred to by scientists as the Miracle Molecule. Since this compound is essential for protein synthesis and has a variety of other important advantages apart from being necessary for a host of other biochemical reactions, it is important to detect L-arginine in physiological fluids. In foods, Arginine is used as a flavor-providing agent. Its detection in foods is imperative as a control measure for quality check in foods such as beverages, juices & wine. The present research work was carried out for the development of potentiometric and fiber-optic biosensors for detecting Arginine in clinical and food samples. The enzyme Arginase was extracted from fungus Neurospora crassa and recombinant enzyme was obtained by expression in E. coli. The recombinant enzyme production was carried out at shake-flask level followed by optimizing media for its production and later production was scaled up to fermentor scale. Following this, the enzyme was purified and later immobilized by various immobilization techniques and employed as biocomponent for development of Biosensors (Potentiometric and Fiber-optic) for monitoring Arginine. Sol-gel modified potentiometric biosensor was developed by means of semi-quantitative analysis on nylon membranes and later by coupling them to NH4 + ISE as a potentiometric transducer for quantitative analysis. Validation studies were carried out by spiking the samples and differences obtained were found to be statisticslly insignificant. Both clinical and food samples were monitored for arginine content. The developed biosensor is sensitive, rapid, easy-to-use and portable and the detection range (nano-level detection) achieved is way better than existing arginine biosensors. The storage stability of the biocomponent is superior and enhanced than conventional arginine biosensor techniques.