Optimization of inducer concentration in the medium for production of Glutaminase from Erwinia

Author Name(s): *Pushpinder Paul
Author Email: pushpinder_pushp@yahoo.co.in

Abstract

The purpose of study was to determine effect of inducer concentrations in the medium for production of Glutaminase enzyme from Erwinia. For production of glutaminase enzyme from free cells of Erwinia, various physical and chemical parameters were optimized. The microorganism Erwinia which is a source of glutaminase was procured from Institute of Microbial Technology, Chandigarh and was grown on specified medium. The media was sterilized by autoclaving for 15 minutes at 15 psi/cm2. The microorganism was maintained on agar slants and kept in refrigerator at 4.0 + 0.5 0C for further use and was sub-cultured after every 20 days. The viability check of microorganism was done by gram staining and tryphan blue. The work utilized Beef extract, Peptone, Yeast extract, Sodium chloride and Water as composition of growth medium for the production of glutaminase enzyme. The parameters such as Temperature, pH, & Time were optimized. By using these parameters Glutaminase was produced by adding different concentration of inducers such as Ammonium sulphate, Inorganic phosphate & L- Glutamine. It was concluded that as we increase the concentration of inducers, enzyme activity increases up to a certain concentration. The enzyme activity was determined by Nessler’s reagent method. So it is clear from this study that Inducers are very helpful to increase the production of glutaminase enzyme from Erwinia and after adding inducers Ammoniumsulphate 100mM, Inorganic phosphate 200ìL & L- Glutamine 12.5% the enzyme production was maximum.

KeyWords

Glutaminase Enzyme, Erwinia, Inducers

Introduction

The protein glutaminase is traditionally considered as a mitochondrial enzyme, playing a key role in the energy and nitrogen metabolism of mammalian cells. The microbial enzymes are found to play a major role in the diagnosis, cure, biochemical investigation, and in the monitoring of many dreadful diseases. Microorganisms represent an excellent source of many therapeutic enzymes owing to their broad biochemical diversity and their susceptibility to genetic manipulation. Glutamine, out of the 20 amino acids is commonly found in animal proteins which supplies energy and substrate to the host after severe stress [1] Only the l -stereoisomer is found to occur in mammalian protein. The liver shows net glutamine uptake after a protein-containing meal during uncontrolled diabetes, sepsis and short-term starvation but changes to net release during long-term starvation and metabolic acidosis [2]. Glutamine structure is identical to that of glutamic acid except that the acidic side-chain carboxyl group of glutamine has been coupled with ammonia, yielding an amide. The glutamic acid-glutamine inter conversion is of central importance to the regulation of the level of toxic ammonia in the body. Thus, it is not surprising that when the concentration of the amino acids of blood plasma is measured, glutamine is found to have the highest value. The role of glutamine in the prevention of chemotherapy and radiation-induced toxicity is evolving. The glutamine supplementation is inexpensive and is found to reduce the incidence of gastrointestinal, neurologic, and possibly cardiac complications during cancer therapy [3]. By enhancing the level of L-glutamine, the maximum glutaminase activity was obtained. Several mammalian and microbial glutaminase enzymes have been purified and characterized which appeared to be best for therapeutic use because of its low K M for glutamine (micro molar range), good stability its activity and long plasma half-life in tumor-bearing hosts. The administration of enteral or parenteral glutamine seems to be safe and also potentially efficacious in some patient groups undergoing intensive treatment for cancer [4]. Glutaminase can be isolated from different microbial, plant and animal sources. The microbial sources from which glutaminase has been extracted are Bacillus licheniformis A5, Micrococcus luteus K-3, Erwinia species. Erwinia is a genus of Enterobacteriaceae containing mostly plant pathogenic species which was named by the first phytobacteriologist, Erwin Smith. It is a gram negative bacterium related to E.coli, Shigella, Salmonella and Yersinia [5].

Conclusion

It was concluded from the above study that there was a continuous increase in enzyme production with increase of inducer concentration in the medium up to a certain level. After that there is slight decrease in enzyme production. The optimum concentration of inducers at which enzyme production was maximum was found as Glutamine 12.5%, ammonium sulphate 100mM & 200μL inorganic phosphate. As Glutaminase has many industrial and medicinal applications, this study must be helpful for further research

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