DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See sections 10; rule 13)

TITLE OF THE INVENTION

EXOCHELIN SIDEROPHORE PRODUCTION METHOD

APPLICANT

Savitribai Phule Pune University
An Indian Entity, having address as:
Ganeshkhind, Pune 411007, Maharashtra, India

The following specification describes the invention and the manner in which it is to be performed.

CROSS-REFERENCE TO RELATED APPLICATIONS
The present application does not claims priority any patent application.
BACKGROUND
TECHNICAL FIELD
The embodiments herein generally relates to a method of producing Exochelin siderophore, and more particularly to a method for producing mycobacterial exochelin siderophore from Pseudomonas stutzeri SGM-1 .
DESCRIPTION OF THE RELATED ART
Mycobacterium is one of the genus of Actinobacteria phylum and it is classified into its own family Mycobacteriacae. This genus of Actinobacteria consists of over 190 different species. Serious diseases causing pathogens like Mycobacterium tuberculosis causing tuberculosis and Mycobactrium leprae causing leprosy are the members of this genus. In 2018, Multidrug-resistant tuberculosis (MDR-TB) remains a public health crisis and a health security threat. WHO estimates that there were 484 000 new cases with resistance to rifampicin, the most effective first-line drug of which 78% had MDR-TB. The MDR-TB burden largely falls on 3 countries India, China and the Russian Federation which together account for half of the global cases. About 6.2% of MDR-TB cases had extensively drug-resistant TB (XDR-TB) in 2018.
Requirement of iron is universal in all living forms but, the solubility of metal ions at biological pH is less. Salicylic acid, citric acid, mycobactin and exochelin are the four different types of iron chelating molecules involved in iron acquisition in mycobacteria. Exochelins are lipid- and water-soluble siderophores primarily produced by and restricted to pathogenic mycobacterial species like M. tuberculosis (primary agent of tuberculosis in humans); M. bovis (primary agent of tuberculosis in cattle and other domesticated animals); M. avium (one of the most prominent opportunistic pathogens in patients with AIDS) and saprophytic mycobacterial species like M. neoaurum (Exo-MN) and M. smegmatis (Exo-MS) and M. vaccae. All the above listed pathogenic bacteria requires the element iron to multiply in host and cause disease. To avail this elemental the pathogens of genus mycobacteria produce the iron chelating siderophore exochelin or pirate from other mycobacterial species producing it.
Thus, there is a need for production of high affinity, iron binding siderophore exochelin from Pseudomonas stutzeri SGM-1 to decipher its role and possibility for effective treatment of the multidrug-resistant tuberculosis (MDR-TB), treatment of cancer and other iron dependant pathological phenomena or diseases.
SUMMARY
This Summary is provided to introduce a method of producing mycobacterial siderophore exochelin by Pseudomonas stutzeri SGM-1 and a method of use thereof, the concepts are further described in the detail description. This summary is not intended to identify essential features of the claimed subject matter nor it is intended to use in determining or limiting the scope of claimed subject matter. In one implementation a method of producing mycobacterial siderophore exochelin is described in accordance to the present subject matter. The method may compromise growing Pseudomonas Stutzeri SGM-1 in a succinate medium to obtain a cell culture wherein growing of the Pseudomonas Stutzeri SGM-1 is carried out by adding 2% inoculum to the Succinate medium and keeping it in a shaking incubator at 37°C at 120 rpm for 24 hours. The method may further compromise collecting the cell culture and centrifuging the cell culture to obtain a supernatant of the cell culture wherin the the centrifugation is carried out in a centrifuge at 8000 rpm at 4°C.
. The method may further compromise checking the presence of siderophore in the supernatant of the cell culture by adding a CAS reagent. The method may further compromise checking the presence of siderophore in the supernatant of the cell culture by adding a CAS reagent and extracting. The method may further compromise purifying the siderophore to obtain purified siderophore wherein the extraction and purification of siderophore is carried out by using ethyl acetate and evaporating the collected ethyl acetate in a rotary evaporator to obtain the purified siderophore. In one embodiment the Succinate medium preparation may compromise method of preparing a solution D2 wherin the solution D2 is prepared by dissolving 1 g of yeast extract and 2 g of tryptone in 50ml of water. The method may further compromise preparing a solution D1 wherein the solution D1 is prepared by dissolving 4 g of dextrose in 20ml of water. The method may further compromise preparing a solution C wherein the solution C is prepared by dissolving 0.15 g of calcium chloride in 1 ml of water. The method may further compromise preparing a solution B wherein the solution B is prepared by dissolving 0.5 g of magnesium sulphate hepta hydrate in 10 ml of water. The method may further compromise preparing a solution A wherein the solution A is prepared by dissolving 0.20 g of dipotassium hydrogen phosphate and 0.50 g of ammonium sulphate in 17.4 ml water.
The method may further compromise autoclaving of the solutions A, B, C, D1 and D2 separately at a temperature of 121°C for 20 minutes at a pressure of 15 psi.
The method disclosed herein is the first report of its kind that islolates exochelin from the Pseudomonas Stutzeri SGM-1. The Pseudomonas Stutzeri SGM-1 belongs to Proteobacteria and not to Actinobacteria still it has the ability to produce siderophore Exochelin. The siderophore exochelin is primarily produced by and restricted to pathogenic and saprophytic mycobacterial species of actinobacteria. As the exochelin from bacteria other than mycobacterial species have not been reported & tested against MDR-TB pathogens, there’s an opportunity to discover anti-tuberculosis & anti-leprosy potential of the mycobacterium siderophore exochelin as a drug in alone or in combination of other drugs.
These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications
BRIEF DESCRIPTION OF THE DRAWINGS
The embodiments herein will be better understood from the following detailed description with reference to the drawings, in which:
FIG.1 is a flow diagram illustrating a method for producing Mycobacterial siderophore Exochelin according to an embodiment herein.
FIG.2 is a flow diagram illustraring a method for preparing Succinate medium as a growth medium for the production of Exochelin from the Pseudomonas stutzeri SGM-1 according to an embodiment.
FIG.3 is a graphical representation of high-resolution mass spectrum (HRMS) in positive electrospray ionization mode according to an embodiment herein.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
As mentioned, there is a need for production of high affinity, iron binding siderophore exochelin from Pseudomonas stutzeri SGM-1 to decipher its role and effective treatment of the multidrug-resistant tuberculosis (MDR-TB) and other iron dependant pathological phenomena or diseases. The method disclosed herein is the first report of its kind that islolates exochelin from the Pseudomonas Stutzeri SGM-1. The Pseudomonas Stutzeri SGM-1 belongs to Proteobacteria and not to the Actinobacteria still it has the ability to produce siderophore Exochelin. The siderophore exochelin is primarily produced by and restricted to pathogenic and saprophytic mycobacterial species of actinobacteria. As the exochelin from bacteria other than mycobacterial species have not been reported & tested against MDR-TB pathogens, there’s an opportunity to discover anti-tuberculosis & anti-leprosy potential of the mycobacterium siderophore exochelin produced by Pseudomonas Stutzeri SGM-1 as a drug in alone or in combination of other drugs.
Referring now to the drawings, and more particularly to FIG. 1 to 3, where similar reference characters denote corresponding features consistently throughout the figures, preferred embodiments are shown.
FIG.1 is a flow diagram illustrating a method for producing mycobacterial siderophore exochelin by Pseudomonas Stutzeri SGM-1 according to an embodiment herein. At step 101 Pseudomonas Stutzeri SGM-1 is grown in a succinate medium at 37°C at 120 rpm for 24hrs to form a cell culture. At step 102 the cell culture is collected in a sterile centrifuge tube and centrifuged at 8000 rpm at 4°C to pellet down the cells. At step 103 the presence of siderophore is checked qualitatively using CAS reagent assay in the supernatant of the cell culture. At step 104 0.5 ml of CAS reagent is added to the 1 ml supernatant of the cell culture, mixed properly and kept for 2 minutes for quantifying the siderophore. At step 105 the siderophore is extracted and purified using ethyl acetate in a separating funnel by vigorous shaking and allowed to separate overnight. The collected ethyl acetate from the separating funnel is evaporated using in a rotary evaporator.
In one embodiment absorption spectra (OD630) of the CAS treated supernantent of step 104 are obtained. In another embodiment the mico-organism when grown in iron limited medium could produce siderophore which was secreted into the extracellular surrounding and collected into the supernatant.
In yet another embodiment mycobacterial siderophore exochelin is produced by an indigenous salt tolerant isolate Pseudomonas stutzeri SGM-1. In yet another embodiment the Pseudomonas stutzeri SGM-1 belongs to Proteobacteria (gram negative) and not to the Actinobacteria (gram positive) still it has the ability to produce mycobacterial siderophore exochelin using the method thereof.
In yet another embodiment high resolution mass spectrum (HRMS) of extracted and purified sample was done by using the Thermo Scientific double quadrupole mass spectra in positive electrospray ionization mode to identify purified siderophore exochelin.
In yet another embodiment to identify the type of siderophore synthesized thin layer chromatography (TLC) is done. The supernatant spot development as wine color indicates presence of hydroxamate type siderophore.
In yet another embodiment the Mycobacterial siderophore Exochelin produced by Pseudomonas stutzeri SGM-1 can be used as a drug or in combination of drugs and drug delivery vehicle for anti-tuberculosis activity against multiple drug resistant Mycobacterium tuberculosis in-vitro.
In yet another embodiment the siderophore exochelin produced by Pseudomonas stutzeri SGM-1 can be used as treatment of cancer by preventing proliferation of and to kill cancer cells in-vitro.
FIG.2 is a flow diagram illustraring a method for preparing Succinate medium for the growth of Pseudomonas stutzeri SGM-1 for exochelin production according to an embodiment. At step 201 solution D2 is prepared by dissolving 1 g of Yeast extract and 2 g of Tryptone in 50ml of water. At Step 202 solution D1 is prepared by dissolving 4 g of dextrose was dissolved in 20mL water. At step 203 solution C is prepared by dissolving 0.15 g of calcium chloride (CaCl2.2H2O) in 1 mL of water. At step 204 solution B is prepared by dissolving 0.5 g of Magnesium sulphate hepta hydrate (MgSO4.7H2O) in 10 ml of water. At step 205 solution A is prepared by dissolving 0.20 g of dipotassium hydrogen phosphate and 0.50 g of ammonium sulphate [(NH4)2SO4] was added mixed in 17.4 mL water. At step 206 the solutions A, B, C, D1 and D2 are autoclaved separately at 121°C for 20 mins, 15 psi and mixed in D2, D1, C, B, A, order to obtain the succinate medium.
In one embodiment 2% inoculum was added to the autoclaved and cooled Succinate medium. The flask was kept in a shaking incubator at 37°C at 120 rpm for 24 h.
FIG.3 is a graphical representation of high-resolution mass spectrum (HRMS) in positive electrospray ionization mode according to an embodiment herein. Purified siderophore exochelin is analyzed on a High Resolution Mass Spectrometer (HRMS) System with electrospray ionization in positive ionization mode. The mass peaks of exochelin proton adduct [M + H] 663.4525 a.m.u. and sodium adduct [M + Na] 685.4339 a.m.u. were identified by METLIN metabolite mass spectral database. From METLIN library search it is confirmed that the isolate produced the siderophore which is the Mycobacterial limited or restricted peptide ‘Exochelin’.
The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the application.
,CLAIMS:WE CLAIM:
1) A method of producing mycobacterial siderophore exochelin, said method comprising the steps of:
a) growing Pseudomonas Stutzeri SGM-1 in a succinate medium to obtain a cell culture;
b) collecting the cell culture and centrifuging the cell culture to obtain a supernatant of the cell culture;
c) checking the presence of siderophore in the supernatant of the cell culture by adding a CAS reagent;
d) checking the presence of siderophore in the supernatant of the cell culture by adding a CAS reagent; and
e) extracting and purifying the siderophore to obtain purified siderophore.
2) The method as claimed in claim 1, wherein the growing of Pseudomonas Stutzeri SGM-1 in a succinate medium in step a) is carried out by adding 2% inoculum to the Succinate medium and keeping it in a shaking incubator at 37°C at 120 rpm for 24 hours.
3) The method as claimed in claims 1 or 2, wherein the Succinate medium preparation process comprises the steps of:
preparing a solution D2;
preparing a solution D1;
preparing a solution C;
preparing a solution B;
preparing a solution A; and
autoclaving the solutions D2, D1, C, B, A obtained in steps (i) to (v) separately; and mixing the autoclaved solutions D2, D1, C, B, A to obtain the Succinate medium.
4) The method as claimed in claim 3, wherein the solution D2 in step i) is prepared by dissolving 1 g of yeast extract and 2 g of tryptone in 50ml of water.
5) The method as claimed in claim 3, wherein the solution D1 in step ii) is prepared by dissolving 4 g of dextrose in 20ml of water.
6) The method as claimed in claim 3, wherein the solution C in step iii) is prepared by dissolving 0.15 g of calcium chloride in 1 ml of water.
7) The method as claimed in claim 3, wherein the solution B in step (iv) is prepared by dissolving 0.5 g of magnesium sulphate hepta hydrate in 10 ml of water.
8) The method as claimed in claim 3, wherein the solution A in step (v) is prepared by dissolving 0.20 g of dipotassium hydrogen phosphate and 0.50 g of ammonium sulphate in 17.4 ml water.
9) The method as claimed in claim 3, wherein the autoclaving of the solutions A, B, C, D1 and D2 in step vi) are carried out separately at a temperature of 121°C for 20 minutes at a pressure of 15 psi.
10) The method as claimed in claim 1, wherein the centrifugation in step (b) is carried out in a centrifuge at 8000 rpm at 4°C.
11) The method as claimed in claim 1, wherein the extraction and purification of siderophore in step (e) is carried out by using ethyl acetate and evaporating the collected ethyl acetate in a rotary evaporator to obtain the purified siderophore.
12) The method as claimed in claim 1, wherein the mycobacterial exochelin siderophore acts as a drug or in combination of drugs and as a drug delivery vehicle for anti-tuberculosis activity against multiple drug resistant Mycobacterium tuberculosis in-vitro.
13) The method as claimed in claim 1, wherein the mycobacterial exochelin siderophore prevents proliferation of and kills cancer cells in vitro
Dated this 10th Day of March, 2021