The actinomycetes strain was isolated from mangrove soil of south Indian costal region of India. The isolates were identified as actinomycetes by colony morphology, biochemical test and 16s rRNA sequencing. These isolates were capable of producing high amount of L-Glutaminase enzyme E. The potential application of the L-Glutaminase in pharmaceutical, food industries, chemical industries and L-Glutaminase is also used in enzyme therapy especially for cancer and acute lymphocytic leukaemia as antileukemic.
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E-mail: moc. This article has been cited by other articles in PMC. It has The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8. It maintained its stability at wide range of pH from 5. It has high affinity and catalytic activity for L-glutamine Km 0.
The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell IC50, 6. Key words: L-glutaminase, anti-cancer, cytotoxicity, MTT assay Introduction Microbial sources like actinomycetes are well recognized to produce a variety of chemical structures, several of which are most valuable pharmaceuticals, agrochemicals and industrial products like enzymes Thadikamala and Reddy, Actinomycetes are considered to be preferred enzymes sources due to their production of extracellular enzymes.
Many enzymes produced by actinomycetes and have been used as drugs like wise L-glutamine amidohydrolase E. Since the discovery of its anti-tumor properties, L-glutaminases have been in prime focus Lazarus and Panasci, Nowadays, L-glutaminase is used as enzyme therapy for cancer especially for acute lymphocytic leukemia Robert et al.
Where, high rate of glutamine consumption is a characteristic nature of some types of cancerous cells Lazarus and Panasci, Based on this character experimental therapies have been developed to deprive L-glutamine to tumor cells Iyer and Singhal, Inhibition of the tumor cell uptake of glutamine is one of the possible ways to stop the growth and this is the best accomplished by the use of L-glutaminase. This in fact, results in a selective starvation of the tumor cells because unlike normal cells lack properly functioning glutamine biosynthetic machinery Tanaka et al.
Microbial therapeutic enzymes have a broad variety of specific uses as oncolytics, thrombolytics or anticoagulant, and largely as anticancer Sabu, ; Sabu et al. The production of enzyme was influenced by a variety of physicochemical and nutritional factors. The factors affecting the production in recent years had received attention as of its great demand in clinical application and also in food industries.
It is known that, the factors involved in the process of production, would not only enhance the quantity, but also quality of enzyme because of which it becomes more suitable for a specific application. Optimization of parameters can in turn influence enzyme synthesis and cell yield Okami, The strain of actinomycetes was used for glutamic acid production under optimum growth conditions Divya Teja et al.
From the compatibility perspective in mass production and as well as beneficial application aspect extracellular enzyme producer as choice of source is always attractive Pandey, The objective of this study was to utilize Streptomyces canarius FR with good ability to produce extracellular L-glutaminase and characterize the purification. Biochemical, kinetics and in vitro anticarcenogenic properties of L-glutaminase will be also examined. Materials and Methods Collection, isolation and identification of S.
The strain was preliminary tested for L-glutaminase production by streaking on minimal glutamine agar medium MGA plates. Formation of pink zones around the microbial growth indicated the positive reaction Balagurunathan and Subramanian, ; Balagurunathan et al. The culture was harvested and centrifuged at 10, rpm for 30 min the obtained cell free filtrate was used as crude enzyme according to Dura et al. Activity assay and Protein determination of L-glutaminase The activity of glutaminase enzyme is determined by estimating the amount of NH3 liberated from glutamine Borek et al.
Protein concentration was determined by Lowery et al. L- Glutaminase purification Two liters from the nutritionally optimized submerged 5 days culture of S. The collected precipitate was dissolved in phosphate buffer 0. After column equilibration the enzyme was eluted by gradient NaCl mM dissolved in phosphate buffer 0. The most active homogenous fractions were gathered and loaded to pre-equilibrated column of Sephadex G gel-filtration chromatography using the same buffer for elution.
For each fraction activity was assessed as above. The most active fractions were pooled and concentrated by dialysis against buffer Nagendra Prabhu, Biochemical and kinetic properties of the purified L-glutaminase The biochemical properties of purified S.
The Tm was calculated from the linear equation of different preincubation temperature at 60 min. Stability of L-glutaminase was examined after preincubation of the enzyme for 2 h at pH from 4. Acetate 0. The activity of the enzyme was determined for each pH. To assay the metal ions effect, the purified enzyme was pre-incubated with each metal ion separately for 30 min before adding glutamine 40 mM. The kinetic parameters of L-glutaminase as Vmax, Km and Kcat were estimated using different concentrations of glutamine, asparagine and aspartic acid, separately mM.
Catalytic efficiency Kcat was expressed by the specific activity per mol enzyme. Cytotoxicity of L- glutaminase Cytotoxic effect of the L-glutaminase was evaluated using 5 New Zealand rabbits as experimental group and one rabbit as control. Experimental group were injected intravenous with 1 mL of L-glutaminase After the two weeks, blood samples were collected 10; 25; 40 and 50 day of the last injection.
Hemolytic activity of the purified L-glutaminase was evaluated using a blood agar assay Tay et al. Histopathological examination Histological examination for the experimental group and control was carried out on liver and kidney, 50 days of the last injection, according to Roy and Maity D of control cells X The efficiency of L-glutaminase was determined using sigmoidal dose response curve-fitting models Graphpad Prizm Software, version 3.
The tested strain was characterized by formation of a pink zone around colonies using MGA medium due to breakdown of amide bond in L-glutamine and ammonia liberation Ranjekar and Sridhar, Dura et al. The result obtained by Krishnakumar et al. They showed that production of L-glutaminase from Streptomyces sp.
Balagurunathan et al. Divya Teja et al. On the other hands, the optimum conditions pH and Temperature for L-glutaminase family is varied from one member to another Usha-Kiranmayi et al. Moreover, Suresh Kumer et al. Purification of L-glutaminase L-glutaminase was purified from 5 days S. The enzyme overall purification profile from S. The crude extract contained At all purification steps, the specific activity increased compared to crude.
The maximum specific activity The partial purified was increased more than 17 fold compared with crude.
These results were in the agreement with that reviewed by Mohana Priya et al. Also, Kumar et al. On the other hand, Elshafei et al. So the method used in the present study proved to be a good method in producing and purification of L-glutaminase.
Table 1 Summary of purification steps of Streptomyces canarius L- glutaminase. Purification steps.
From Krebs to clinic: glutamine metabolism to cancer therapy
Search Menu ABSTRACT Glutamine is the most abundant free amino acid in the human body; it is essential for the growth of normal and neoplastic cells and for the culture of many cell types. Cancer has been described as a nitrogen trap. The presence of a tumor produces great changes in host glutamine metabolism in such a way that host nitrogen metabolism is accommodated to the tumor-enhanced requirements of glutamine. To be used, glutamine must be transported into tumor mitochondria. Thus, an overview of the role of glutamine in cancer requires not only a discussion of host and tumor glutamine metabolism, but also its circulation and transport.
GLUTAMINASE THESIS PDF
A Corrigendum to this article was published on 14 October A Corrigendum to this article was published on 11 November Key Points Cancer cells show increased consumption of and dependence on glutamine. Glutamine metabolism fuels the tricarboxylic acid TCA cycle, nucleotide and fatty acid biosynthesis, and redox balance in cancer cells. Glutamine activates mTOR signalling, suppresses endoplasmic reticulum stress and promotes protein synthesis. Activation of oncogenic pathways and loss of tumour suppressors reprogramme glutamine metabolism in a tissue-dependent manner.
Kazrakazahn The 40 kDa molecular weight L-glutaminase by P. Though cancer cells thesie more amounts of glutamine, they are incapable of producing their own glutamine de novo Wise, et al. KLM9 Nathiya, et al. It is universal from the presence point of opinion in plants, animals and microbes together in prokaryotes and eukaryotes. Subscribe to this RSS feed. There are UK writers just like me on hand, waiting to help you.
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