Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of a method for treating hyperproliferative diseases and in specific, relates to a method that optimizes L-Glutaminase from Streptomyces Luteogriseus isolated from marine sediment.
Background of the invention:
[0002] Cancer is a major public health concern and one of the leading causes of death worldwide. Cancer is not a single disease, it is a class of diseases each of which presents a metabolism that shifts to support the hyper proliferation that is characteristic of the cancer group. Although different cancers may appear in disparate tissues and cancer cells may migrate from one tissue to another, at their root each cancer cell cohort involves a shift in normal metabolism so that rather than supporting duties to maintain survival of the host organism the cell had differentiated to perform, the cancer cell's metabolism alters pathways, down-regulating several, up-regulating others, to improve the cancer cell's hyper proliferative activities. Cancer cells are characterized by uncontrolled proliferation and rapid growth.

[0003] Till date, cancer treatment has been unsatisfactory. Despite, the large number of anticancer therapies are investigated in clinical trials and the advances made in chemotherapeutic regimens, cancer treatment today is inadequate: it is not very effective and has major side effects. There is a significant unmet medical need for clinically effective, nontoxic treatments of cancer that overcome the drawbacks of conventional therapies. We need therapies with increased efficacy and reduced toxicity that can completely eliminate malignancies, ensure survival and provide patients with a better quality of life during and after treatment.

[0004] Later, Antiangiogenic therapies prevent the formation of new blood vessels in and around tumors. They normalize the tumor vasculature and increase its efficiency, thus increasing the tumor uptake of drugs and oxygen, and distributing them to a larger fraction of the tumor cells. Increased penetration of drugs throughout the tumor enhances the outcome of therapy, and increased levels of oxygen enhance the efficacy of radiation and chemotherapeutic agents.

[0005] In existing technology, L-Glutaminase synthesis by marine halomonas meridian is known. L-Glutaminase is an important L-glutaminase is an important anticancer agent that is used extensively worldwide by depriving cancer cells of L-glutamine. The marine bacterium, Halomonas meridian was isolated from the Red Sea and selected as the more active L-glutaminase-producing bacteria. L-glutaminase fermentation was optimized at 36 h, pH 8.0, 37 °C, and 3.0% NaCl, using glucose at 1.5% and soybean meal at 2%. The purified enzyme showed a specific activity of 36.08 U/mg, and the molecular weight was found to be 57 kDa by the SDS-PAGE analysis. The enzyme was highly active at pH 8.0 and 37° C. However, L-Glutaminase does not contain anti-oxidative properties and anti-retroviral properties.

[0006] Therefore, there is a need for L-Glutaminase that contain anti-oxidative properties and anti-retroviral properties. There is also a need for L-Glutaminase that has great quality for the proliferation of immature lymphocytes and other macromolecules. There is also a need for L-Glutaminase that uses ATP as an energy source to change L-glutamate into L-glutamine in normal healthy cells.
Objectives of the invention:
[0007] The primary objective of the invention is to provide a method for isolating L-Glutaminase from streptomyces Luteogriseus in marine sediment.

[0008] Another objective of the invention is to provide a method for isolating L-Glutaminase that is used for cancer therapy, as malignant and carcinoma cells require external sources of amino acids to proliferate and multiply, unlike healthy tissues.

[0009] The other objective of the invention is to provide a method for isolating L-Glutaminase from strain streptomyces Luteogriseus to exhibit high L-Glutaminase production.

[0010] Yet another objective of the invention is to provide a method of cost-effective L-Glutaminase that selectively target cancer cells.

[0011] Further objective of the invention is to provide a method of L-glutamine to treat cancer cells, which is fifteen folds greater than that of normal cells.
Summary of the invention:
[0012] The present disclosure proposes a method for optimizing L-Glutaminase from Streptomyces Luteogriseus isolated from marine sediment. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0013] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method that optimizes L-Glutaminase from Streptomyces Luteogriseus isolated from marine sediment.

[0014] According to an aspect, the invention provides a method for isolating L-Glutaminase from Streptomyces Luteogriseus. At one step, around 0.1 mL of each dilution is plated on starch casein agar plates for preparing a medium. In specific, the each dilution is prepared by collecting and blending plurality of sediment sample for obtaining a soil sample that is around 1 gram. Next, the soil sample is dissolved in 9 mL of 0.9% of NaCl solution for preparing a sediment suspension. Later, the sediment suspension is filtered through a sterile mesh for removing gravel and plant debris. Finally, serial dilutions are performed from the sediment suspension that includes 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6.

[0015] At one step, about 5 μg/mL of rifampicin and 75 μg/mL of cycloheximide are added to the medium for inhibiting bacterial and fungal contamination. At one step, pH value of about 7.9 is adjusted by using 0.1 M NaOH and allowed to sterilize in an autoclave at a temperature of about 121°C for a time period of about 15 minutes. At one step, about 1 mL of the each dilution is injected on the starch casein agar plates using a spread plate method. At one step, the starch casein agar plates are allowed to incubate for about 14 days at about 28°C.

[0016] At step, morphological characteristics of colonies with different colony characteristics are selected for displaying abilities to produce L-Glutaminase that assists to inhibit cancer cell proliferation. In specific, the morphological characteristics of colonies are identified easily due to their behavior such as filamentous, compact, chalky, firm, dry surface, leathery and conical appearance. The morphological characteristics of colonies are selected and isolated on the starch casein agar plates by using recurrent streak plate technique. The morphological characteristics of colonies growth is recorded on each plate of the starch casein agar plates after incubation.

[0017] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0018] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0019] FIG.1 illustrates a flowchart of an exemplary method for isolating L-Glutaminase from Streptomyces Luteogriseus, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0020] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0021] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method that optimizes L-Glutaminase from Streptomyces Luteogriseus isolated from marine sediment.

[0022] According to an exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of an exemplary method of optimization of optimization of L-Glutaminase from Streptomyces Luteogriseus isolated from marine sediment. At step 102, around 0.1 mL of each dilution is plated on starch casein agar plates for preparing a medium. In specific, the each dilution is prepared by collecting and blending plurality of sediment sample for obtaining a soil sample that is around 1g.

[0023] Next, the soil sample is dissolved in 9 mL of 0.9% of NaCl solution for preparing a sediment suspension. Later, the sediment suspension is filtered through a sterile mesh for removing gravel and plant debris. Finally, serial dilutions are performed from the sediment suspension that includes 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6.

[0024] At step 104, about 5 μg/mL of rifampicin and 75 μg/mL of cycloheximide are added to the medium for inhibiting bacterial and fungal contamination. At step 106, pH value of about 7.9 is adjusted by using 0.1 M NaOH and allowed to sterilize in an autoclave at 121°C for about 15 minutes. At step 108, about 1mL of the each dilution is injected on the starch casein agar plates using a spread plate method. At step 110, the starch casein agar plates are allowed to incubate for about 14 days at about 28°C.

[0025] At step 112, morphological characteristics of colonies with different colony characteristics are selected for displaying abilities to produce L-Glutaminase that assists to inhibit cancer cell proliferation. In specific, the morphological characteristics of colonies are identified easily due to their behavior such as filamentous, compact, chalky, firm, dry surface, leathery and conical appearance. The morphological characteristics of colonies are selected and isolated on the starch casein agar plates by using recurrent streak plate technique. The morphological characteristics of colonies growth is recorded on each plate of the starch casein agar plates after incubation.

[0026] In one embodiment herein, the L-Glutaminase enzyme is evaluated by performing multiple experiments, which includes UV-absorption spectrum, effect of pH, temperature, metal ions, additives and substrate specificity. In one example embodiment herein, to perform absorption studies 2 mg of lyophilized L-glutaminase enzyme is dissolved in 0.1 M Tris-HCl buffer at pH 8.0. Later, the scanning is performed by a 0.2 cm path length cuvette at a scan speed of 20 nm/min with a bandwidth of 1 nm, and the wavelength is set in the range of 200 to 800 nm. The final absorption spectrum is collected by averaging three independent scans on a spectrophotometer at 10°C.

[0027] The effect of pH on L-glutaminase activity is determined by measuring the enzyme activity at various pH levels varying from 2.0 to 13.0. The effect of temperature on the activity of L-glutaminase is determined by measuring the enzyme activity at various temperatures ranging from 10 to 80°C. The effect of metal ions on the activity of L-glutaminase is determined by measuring the enzyme activity at four different concentrations of metal ions, such as 5 mM, 10 mM, 15 mM and 20 mM.

[0028] The effect of additives on the activity of L-glutaminase is determined by measuring the enzyme activity in the presence of various additives, such as SDS, L- cysteine, L-histidine, 2-mercaptoethanol and EDTA. The effect of substrate specificity on L-glutaminase activity is determined by measuring the enzyme activity in the presence of various substrates that include L- Glutamine, D-glutamine and L-asparagine.

[0029] In one example embodiment herein, the protein concentrations in the crude enzyme extract from isolated L- Glutamine vary significantly across the four ammonium sulphate saturations, such as 20, 40, 60 and 80%. The quantities of total protein is observed as 756.32±12, 536.42±9, 246.75±7 and 72.26±4 μg/mg with respect to the 20, 40, 60 and 80% ammonium sulphate fractions of crude enzyme extracts. Determination of protein concentration is performed by using a standard calibration curve. These determination revealed that various ammonium sulphate saturations that varies protein perceptibility percentages.

[0030] The overall evaluation of different saturations revealed that 20% of ammonium sulphate saturation provides the maximum protein quantity, whereas 80% of saturation provides the lowest protein quantity. Based on these results, it was observed that the ammonium sulphate gradually increases and saturation decreases the protein yield from the crude extract. Therefore, the protein quantification for different ammonium sulphate result fractions of crude enzyme extract.

[0031] The activity of L-glutaminase in the crude enzyme ammonium sulphate fractions is determined by estimating the amount of NH4+ released from the substrate glutamine by using Nessler’s method. The concentration of liberated NH4+ in the samples are directly proportional to the L-glutaminase activity. A substantial difference is found in the activities of L-glutaminase among the four ammonium sulphate fractions (20, 40, 60 and 80%) of bacterial isolate KLP-08 crude enzyme extracts.

[0032] From these results, 60 and 80% fractions of crude enzyme had the significant L-glutaminase activity whereas, 20 and 40% crude enzyme fractions does not exhibit enzyme activity. The L-glutaminase activities in the samples containing crude enzyme extracts of 20, 40, 60 and 80% ammonium sulphate fractions are found to be 0.00, 0.00, 20212±15 and 21012±19 U/mg respectively.

[0033] As well as the specific activity of L-glutaminase for the crude enzyme extracts of 20, 40, 60 and 80% ammonium sulphate fractions are calculated as 0.00, 0.00, 1.638 and 5.776 U/mg respectively. Based these results, the greatest L-glutaminase activity is found with 80% crude enzyme fraction. Due to the maximum enzyme concentration, a very high quantity of NH4+ is determined in the test samples containing 80% ammonium sulphate crude enzyme fraction.

[0034] While the amidohydrolase activity of L- glutaminase is not found in the test samples that contains 20 and 40% ammonium sulphate crude enzyme fractions. Due to the absence of amidohydrolase activities of 20 and 40% fractions, gave negative result in presence of NH4+. As a result, 60 and 80% ammonium sulphate fraction of crude enzyme is extracted and further analyzed for purification of potential L-glutaminase enzyme.

[0035] In one example embodiment herein, the Enzymes are derived from bacterial sources that metabolize amino acids have a broad range of applications in amino acid depletion therapy due to their accessibility, faster production rate and relatively easy enzyme modification. In specific, L-Glutaminase, an enzyme that depletes glutamine, is a crucial factor in inhibiting cancer cell proliferation. In addition to its anti-cancer properties, L-Glutaminase possesses anti-oxidative and anti-retroviral properties. Amino acid deprivation-based anticancer therapy induces the famine of amino acids, leading to the conversion of tumor cells into auxotrophic forms to specific amino acids, thereby inhibiting the proliferation of tumor cells.

[0036] Despite the greater need for L-Glutaminase in cell proliferation, both healthy and cancerous cells are unable to synthesize it, and thus considered as a non-essential amino acid. However, L-Glutamine is required in greater quantities, especially for the proliferation of immature lymphocytes and for the synthesis of nitrogen-containing molecules such as hexosamines, proteins and other macromolecules, and can also act as a substrate for respiration.

[0037] L-Glutamine synthetises use ATP as an energy source to change L-Glutamate into L-Glutamine in normal healthy cells. Malignant cells are needed for a significant amount of L-Glutamine, but the lack of the L-Glutamine synthetises enzyme, they are unable to produce sufficient endogenous L-glutamine and must rely on serum glutamine content for survival and growth. The amount of L-glutamine needed by cancer cells is fifteen folds greater than that of normal cells. Injection of the L-Glutaminase enzyme triggers apoptosis since malignant cells no longer need an external source of glutamine. However, normal healthy cells have the advantage of producing L-glutamine via a de-novo pathway that makes use of the L-glutamine synthetises enzyme.

[0038] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a method for optimizing isolated from marine sediment is disclosed. The proposed method for isolating L-Glutaminase from streptomyces Luteogriseus in marine sediment. The proposed method for isolating L-Glutaminase that is used for cancer therapy, as malignant and carcinoma cells require external sources of amino acids to proliferate and multiply, unlike healthy tissues.

[0039] The proposed method for isolating L-Glutaminase from strain streptomyces Luteogriseus to exhibit high L-Glutaminase production. The proposed method of cost-effective L-Glutaminase selectively target cancer cells. The proposed method of L-glutamine treat cancer cells, which is fifteen folds greater than that of normal cells.

[0040] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I / We Claim:
1. A method for isolating L-Glutaminase from Streptomyces Luteogriseus, comprising:
plating around 0.1 mL of a diluted solution on starch casein agar plates for preparing a medium;
adding 5 μg/mL of rifampicin and 75 μg/mL of cycloheximide to the medium for inhibiting bacterial and fungal contamination;
adjusting a pH value of the medium of 7.9 using 0.1 M sodium hydroxide (NaOH) and allowing to sterilize in an autoclave at a temperature of 121°C for a time period of 15 minutes;
injecting 1 mL of the diluted solution on the starch casein agar plates using a spread plate method;
allowing the starch casein agar plates to incubate for a time period of at least 14 days at a temperature of 28°C; and
selecting the morphological characteristics of colonies with different colony characteristics for displaying abilities to produce L-Glutaminase.
2. The method as claimed in claim 1, wherein the diluted solution is prepared by:
collecting and blending plurality of sediment samples for obtaining a soil sample;
dissolving the soil sample in 9 mL of 0.9% of Sodium chloride (NaCl) solution for preparing a sediment suspension;
filtering the sediment suspension through a sterile mesh for removing gravel and plant debris; and
performing serial dilutions from the sediment suspension to obtain the diluted solution.
3. The method as claimed in claim 2, wherein the soil sample weight is 1 gm.
4. The method as claimed in claim 2, wherein the sediment suspension includes 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6.
5. The method as claimed in claim 1, wherein the morphological characteristics of colonies are identified easily due to their behavior such as filamentous, compact, chalky, firm, dry surface, leathery and conical appearance.
6. The method as claimed in claim 1, wherein the morphological characteristics of colonies are selected and isolated on the starch casein agar plates by using recurrent streak plate technique.
7. The method as claimed in claim 1, wherein the morphological characteristics of colonies growth is recorded on each plate of the starch casein agar plates after incubation.
8. The method as claimed in claim 1, wherein the produced L-Glutaminase assists to inhibit cancer cell proliferation.