WO2023058037A1

WO2023058037A1 - Biomolecule mixture for biogenic synthesis of metal nanoparticles

Info

Publication number: WO2023058037A1
Application number: PCT/IN2021/051188
Authority: WO
Inventors: Kiran D. PAWAR; Megha Prakash Desai
Original assignee: Shivaji University
Current assignee: Shivaji University
Priority date: 2021-10-05
Filing date: 2021-12-18
Publication date: 2023-04-13
Anticipated expiration: 2024-04-05

Abstract

The present invention relates to a method for preparation of biomolecule mixture in dried/freeze-dried form using bacterial strains selected from the group comprising Lysinibacillus xylanilyticus, Lysinibacillus sp, Lysinibacillus pakistanensis and Lysinibacillus macroides for simple and rapid biogenic synthesis of metal nanoparticles namely gold, silver, palladium and platinum nanoparticles. The method of preparation includes growing the bacterial strain, harvesting the biomass, centrifugation, washing, re- suspension, and incubation, collection of homogenate, centrifugation, collection of biomolecule mixture, precipitation, incubation, centrifugation, dissolution, dialysis, and freeze drying biomolecule mixture and the use of biomolecules mixtures for simple and rapid biogenic synthesis of gold, silver, palladium and platinum nanoparticles at broad range of reaction parameters such as reaction volume, concentration of biomolecule mixture and metal precursor, pH; incubation and reaction time.

Description

BIOMOLECULE MIXTURE FOR BIOGENIC SYNTHESIS OF METAL NANOPARTICLES

FIELD OF THE INVENTION

[0001] The present invention relates to the nano-biotechnology, and particularly to the preparation of bacterial biomolecule mixture in dried/freeze -dried form, and to the use of this biomolecule mixture for the biogenic synthesis of precious metal nanoparticles such as gold, silver, palladium and platinum nanoparticles.

BACKGROUND OF THE INVENTION

[0002] Nanotechnology, an emerging and rapidly evolving field of science combines the knowledge from physics, chemistry, biology and other related branches of science. Nanotechnology based products and services have numerous applications in many fields of science and human life. In nanotechnology, synthesis of precious metal nanoparticles has great importance and therefore, is highly prioritized area of research and development in the recent decades. The properties of metal nanoparticles such as high dispersity with large surface area, good solubility, excellent catalytic activities, novel photonics and optoelectronic properties make them an excellent nanomaterial for wide range of applications.

[0003] Although, various physical and chemical methods are mostly employed to synthesize metal nanoparticles, the use of hazardous and expensive chemicals greatly limits their biomedical applications, particularly in clinical fields. Moreover, physical and chemical methods are complicated, outdated, inefficient and produce hazardous toxic wastes that are harmful, not only to the environment but also to human health. In addition, these existing methods for producing metal nanoparticles are lengthy, energy intensive, require specialized instruments to complete the production process and, therefore, reduce the commercial feasibility of producing metal nanoparticles on large scale. In the light of these risks associated, development of reliable, nontoxic, and eco-friendly methods for synthesis of metal nanoparticles is of utmost importance to expand their applications. One of the widely accepted and best recognized alternative to physical and chemical methods is to use biogenic methods that employ plants, their extract and microorganisms such as bacteria and fungi to synthesize metal nanoparticles. Biogenic methods are more acceptable, environment friendly “green” route as they are less energy intensive. In addition, nanoparticles produced by biogenic methods are far superior, in several ways, to those particles produced by chemical methods.

[0004] Among the biogenic methods, the use of plant species largely depends upon phytochemical potential and presence of biomolecule that can act as reducing and capping agents. Not all plant species exhibit similar phytochemical potential, and therefore cannot be employed to synthesize metal nanoparticles. Significant research efforts are put into screening the large numbers of plant species to select the potentially useful plant. In addition, using plants and their extract to produce large quantities of precious metal nanoparticles may not be always feasible as it requires large quantities of plants or their parts and may raise ecological and environmental concerns in case plant species selected and being used is rare and endangered.

[0005] Due to these short comings associated with the use of plants and their extract, biological approaches using bacteria become obvious choice and viable alternative to physical and chemical methods. Use of bacteria for biogenic synthesis is also preferred for the fact that the majority of them can grow in and inhabit ambient conditions of varying temperature, pH, and pressure. The metal nanoparticles generated by these processes show higher catalytic reactivity, greater specific surface area, excellent biocompatibility, no cytotoxicity and ease of surface modification.

[0006] To make the best possible use of bacteria mediated nanoparticles in wide range of application, one need to have potentially capable bacterium/bacteria that can synthesize metal nanoparticles. To have such potential bacteria, significant research efforts have to put into isolation, screening, identification, optimization of synthesis, separation and purification of metal nanoparticles and maintenance of the bacterial culture.

[0007] Mechanistically, bacteria mediated synthesis of metal nanoparticles is complex, mostly extracellular phenomena that involves many proteins, enzymes and other biomolecules. The exact mechanism involved, the identities of proteins, enzymes and other biomolecules are still not clear. In this regard, developing the method for preparation of bacterial biomolecule mixture for synthesis of precious metal nanoparticles would definitely save research efforts and time required for isolation, screening, identification, optimisation of synthesis, separation and purification of metal nanoparticles and maintenance of the bacterial culture.

[0008] There is various literature published, specifically related to the potential of some bacteria for synthesis of gold, silver and palladium nanoparticles such as (Lloyd J R et al., 1998. Enzymatic recovery of elemental palladium by using sulfate -reducing bacteria. Applied and Environmental Microbiology 64(11): 4607-4609; Yong P et al., 2002. Bioreduction and biocrystallization of palladium by Desulfovibrio desulfuricans NCIMB 8307. Biotechnology and Bioengineering 80 (4): 369-379; Windt W D et al., 2005. Bioreductive deposition of palladium (0) nanoparticles on Shewanella oneidensis with catalytic activity towards reductive dechlorination of polychlorinated biphenyls. 2005. Environmental Microbiology 7(3): 314-325; Gericke M and Pinches A. 2006. Microbial Production of Gold Nanoparticle. Gold Bulletin. 39(1): 22-28; Pollmann K et al., 2006. Manufacturing and characterization of Pd nanoparticles formed on immobilized bacterial cells. Letters in Applied Microbiology 43:39-45; Lengke M F et al., 2006. Synthesis of platinum nanoparticles by reaction of filamentous cyanobacteria with platinum (IV)-chloride complex. Langmuir. 22: 7318-7323; Merroun, et al., 2006. Spectroscopic characterization of gold nanoparticles formed by cells and S -layer protein of Bacillus sphaericus JG-A12. Materials Science and Engineering C. 27(1): 188-192; Shahverdi A R et al., 2007. Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: A novel biological approach. Process Biochemistry 42: 919-923; Shahverdi A R et al., 2007. Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomedicine 3(2): 168-171; Konishi Y et al., 2007. Bioreductive deposition of platinum nanoparticles on the bacterium Shewanella algae. Journal of Biotechnology 128: 648-653; Husseiny M I et al., 2007. Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa. Spectrochimica Acta Part A. 67: 1003-1006; He S et al., 2007. Biosynthesis of gold nanoparticles using the bacteria Rhodopseudomonas capsulate. Materials Letters 61: 3984-3987; Du L et al., 2007. Biosynthesis of gold nanoparticles assisted by Escherichia coli DH5a and its application on direct electrochemistry of haemoglobin. Electrochemistry Communications. 9: 1165-1170; Bunge M et al., 2010. Formation of palladium (0) nanoparticles at microbial surfaces. Biotechnology and Bioengineering 107(2): 206-215; Badri Narayanan K et al., 2010. Biological synthesis of metal nanoparticles by microbes Advances in Colloid and Interface Science 156: 1-13; Sharma S et al., 2010. Synthesis of crystalline Ag nanoparticles (AgNPs) from microorganisms. Materials Sciences and Applications. 1: 1-7; Deplanche K et al., 2010. Involvement of hydrogenases in the formation of highly catalytic Pd (0) nanoparticles by bioreduction of Pd(II) using Escherichia coli mutant strains. Microbiology 156: 2630-2640; Natarajan K et al., 2010. Microbial production of silver nanoparticles. Digest Journal of Nanomaterials and Biostructures. 5( 1): 135-140; Balagurunathan et al. 2011. Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10. Indian Journal of Biochemistry and Biophysics. 48:331-335; Corte S D et al., 2011. Gold nanoparticle formation using Shewanella oneidensis'. a fast biosorption and slow reduction process. J Chem Technol Biotechnol. 86: 547-553; Hennebel T et al., 2011. Palladium nanoparticles produced by fermentatively cultivated bacteria as catalyst for diatrizoate removal with biogenic hydrogen. Appl Microbiol Biotechnol.91: 1435-1445; Korbekandi H et al., 2012. Optimization of biological synthesis of silver nanoparticles using Lactobacillus casei subsp. Casei. J. Chem Technol Biotechnol. 2012: 87: 932-937; Srivastava S K and Constant! M. 2012. Room temperature biogenic synthesis of multiple nanoparticles (Ag, Pd, Fe, Rh, Ni, Ru, Pt, Co, and Li) by Pseudomonas aeruginosa SMI. J Nanopart Res. 14:831-841; Prakasham S R et al., 2012. Characterization of silver nanoparticles synthesized by using marine strain Streptomyces albidoflavus . J. Microbiol. Biotechnol. 22(5): 614-621; Correa-Llanten D N et al., 2013. Gold nanoparticles synthesized by Geobacillus sp. strain ID 17 a thermophilic bacterium straind from Deception Island, Antarctica. Microbial Cell Factories 12:75; Yates M D et al., 2013. Extracellular palladium nanoparticle production using Geobacter sulfurreducens . ACS Sustainable Chem. Eng. 1: 1165-1171; Martins M et al., 2013. Palladium recovery as nanoparticles by an anaerobic bacterial community. J Chem Technol Biotechnol 88: 2039-2045; Ghorbani H R 2013. Biosynthesis of Silver Nanoparticles by Escherichia coli. Asian Journal of Chemistry. 25 (3): 1247-1249; Gaidhani S V et al., 2014. Bio-reduction of hexachloroplatinic acid to platinum nanoparticles employing Acinetobacter calcoaceticus . Process Biochemistry 49: 2313-2319; Singh P K and Kundu S. 2014. Biosynthesis of Gold Nanoparticles Using Bacteria., Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.84(2): 331-336; Chauhan R et al., 2015. Biosynthesis of silver and zinc oxide nanoparticles using Pichia fermentans JA2 and their antimicrobial property. Appl. Nanosci. 5:63-71; Omajali J B et al., 2015. Characterization of intracellular palladium nanoparticles synthesized by Desulfovibrio desulfuricans and Bacillus benzeovorans. J Nanopart Res. 17:264; Abo-State M A M and Partila A M. 2015. Microbial production of silver nanoparticles by Pseudomonas aeruginosa Cell Free Extract. J. Eco. Heal. Env. 3(3): 91-98; Capeness M J et al., 2015. Nickel and platinum group metal nanoparticles production by Desulfovibrio alaskensis G20. New Biotechnology. 32(60):727-731; Rajeshkumar S. 2016. Anticancer activity of eco-friendly gold nanoparticles against lung and liver cancer cells. Journal of Genetic Engineering and Biotechnology 14:195-202; Kumar A and Ghosh A. 2016. Biosynthesis and characterization of silver nanoparticles with bacterial strain from gangetic- Alluvial Soil. International Journal of Biotechnology and Biochemistry 12 (2): 95-102; Nadaf N Y and Kanase S S. 2016. Biosynthesis of gold nanoparticles by Bacillus marisflavi and its potential in catalytic dye degradation. Arabian Journal of Chemistry. Article in press; Li J et al.,

2016. Biosynthesis of gold nanoparticles by the extreme bacterium Deinococcus radiodurans and an evaluation of their antibacterial properties. International Journal of Nanomedicine 11: 5931-5944; Ghorbani H R. 2017. Biosynthesis of nanosilver particles using extract of Salmonella typhirium. Arabian Journal of Chemistry 10: 1699-1702; Wadhwania S A et al., 2017. Biosynthesis of gold and selenium nanoparticles by purified protein from Acinetobacter sp. SW 30. Enzyme and Microbial Technology 111: 81-86; Dakhil A S 2017. Biosynthesis of silver nanoparticle (AgNPs) using Lactobacillus and their effects on oxidative stress biomarkers in rats. Journal of King Saud University- Science. 29: 462-467; Srinath B S et al., 2017. Eco-friendly synthesis of gold nanoparticles by gold mine bacteria Brevibacillus formosus and their antibacterial and biocompatible studies. IOSR Journal of Pharmacy 7(8): 53-60; Shivaji S et al.,

2017. Extracellular synthesis of antibacterial silver nanoparticles using psychrophilic bacteria. Process Biochemistry 46: 1800-1807; Buszewski B et al., 2018. Antimicrobial activity of biosilver nanoparticles produced by a novel Streptacidiphilus durhamensis strain. Journal of Microbiology, Immunology and Infection 51 (1): 45-54.).

[0009] The patent literature related to bacterial synthesis of metallic nanoparticles mostly consist use of bacterial biomass, cell free extract, culture suspension or contacting the bacteria with metal salts. The US patent 8,455,226 B2 discloses the method for producing a composition comprising colloidal nanoparticles of metals including silver, gold, zinc, mercury, copper, palladium, platinum, or bismuth on bacterial membrane by contacting a metal or metal compound with probiotic Lactobacillus fermentum strains.

[0010] The above-mentioned literature related to synthesis of metal nanoparticles, using bacteria involves lengthy methods, specific for only one or two metal nanoparticle synthesis and require post synthesis purifications of nanoparticles for further applications. In addition, there is limit to which bacterial suspension, biomass and extract can be stored and may sometime lose synthesis potential if stored for longer period.

[0011] For solving aforementioned problems, there is a strong need to have commercially viable method for synthesis of biomolecule mixture and a biogenic synthesis of gold, silver, palladium and platinum nanoparticles using the biomolecule mixture for various applications.

OBJECTIVE OF THE INVENTION

[0012] The objective of the present invention is to isolate bacterial strains to prepare freeze-dried biomolecule mixtures.

[0013] Another objective of the present invention is to develop novel, simple, reliable methodology to prepare freeze-dried biomolecule mixtures using isolated bacterial strain.

[0014] Yet another objective of the present invention is to use freeze dried biomolecule mixture for biogenic synthesis of gold, silver, palladium and platinum nanoparticle.

SUMMARY OF THE INVENTION

[0015] Accordingly, the present invention provides a method for preparing a freeze-dried biomolecule mixture from bacterial strains for biogenic synthesis of metal nanoparticles comprises: a) growing the bacterial strain in Luria Bertoni broth medium for 24-72 h; b) harvesting bacterial cell biomass by centrifugation and washing thrice with water; c) suspending cell biomass in water and incubating at 50-90° C for 1 -4 h; d) centrifuging the homogenate and collecting clear supernatant containing biomolecule mixture; e) precipitating biomolecule mixture with ammonium sulphate and incubating at 4° C for overnight; f) centrifuging precipitated biomolecule mixture and re-suspending in phosphate buffer; g) dialyzing re-suspended biomolecule mixture in phosphate buffer using semi -permeable membrane; h) concentrating dialyzed biomolecule mixture in sucrose; and i) freeze-drying biomolecule mixture.

[0016] Accordingly, the present invention provides a method for biogenic synthesis of gold nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding chloroauric acid (HAuCh) in the range 1-5 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 40-100° C; d) incubating the reaction mixture of step c) for 10-60 min; and e) obtaining the gold nanoparticles.

[0017] Accordingly, the present invention provides a method for biogenic synthesis of silver nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 2.5-25 mg/mL reaction volume; b) adding silver nitrate (AgNCL) in the range 0.5-2 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 30-70° C; d) incubating the reaction mixture of step c) for 15 min-24 h; and e) obtaining the silver nanoparticles. [0018] Accordingly, the present invention provides a method for biogenic synthesis of palladium nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding PdCh in the range 0.5-3 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 4-7 and at the temperature range 50-100° C; d) incubating the reaction mixture of step c) for 15 min-8 h; and e) obtaining the palladium nanoparticles.

[0019] Accordingly, the present invention provides a method for biogenic synthesis of platinum nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding Potassium tetra chloropalatinate (K^PtCLj) in the range 1-5 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 30-70° C; d) incubating the reaction mixture of step c) for 15 min-2 h; and e) obtaining the platinum nanoparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] With the above and other related objectives in view, the present invention consists in the details of different methods and parts thereof as will be more fully understood from the following description, when read in conjunction with the accompanying drawings in which:

Fig.1 shows flow chart of method for preparation of freeze-dried biomolecule mixture

Fig.2 shows digital photograph image of freeze-dried biomolecule mixture

Fig.3 shows LC MS/MS mass fingerprint of biomolecule mixture Fig.4 shows transmission electron microscopy (TEM) image of AuNPs synthesized using biomolecule mixture

Fig.5 shows energy dispersive spectrum (EDS) pattern of AuNPs showing with dominant peaks for Au atoms

Fig.6 shows UV-Vis. absorption spectra of AuNPs synthesized using biomolecule mixture in the range comprising 5 to 50 mg/mL

Fig.7 shows colour of reactions of AuNPs synthesis using biomolecule mixture in the range comprising 5 to 50 mg/mL

Fig.8 shows UV-Vis. absorption spectra of AuNPs synthesized using HAuCL precursor in the range comprising 1 to 5 mM

Fig. 9 shows colour of reactions of AuNPs synthesis using HAuCL precursor in the range comprising 1 to 5 mM

Fig.10 shows UV-Vis. absorption spectra of AuNPs synthesized at reaction pH in the range comprising 5 to 9

Fig.11 shows colour of reactions of AuNPs synthesis at reaction pH in the range 5 to 9

Fig.12 shows UV-Vis. absorption spectra of AuNPs synthesized using incubation temperature in the range comprising 40 to 100°C

Fig.13 shows colour of reactions of AuNPs synthesis at incubation temperature in the range comprising 40 to 100°C

Fig.14 shows UV-Vis. absorption spectra of AuNPs synthesized using reaction time in the range comprising 10 to 60 min.

Fig.15 shows transmission electron microscopy (TEM) image of AgNPs synthesized using biomolecule mixture Fig.16 shows energy dispersive spectrum (EDS) pattern of AgNPs showing with dominant peaks for Ag atoms

Fig.17 shows UV-Vis. absorption spectra of AgNPs synthesized using biomolecule mixture in the range comprising 2.5 to 25 mg/mL

Fig.18 shows colour of reactions of AgNPs synthesis using biomolecule mixture in the range comprising 2.5 to 25 mg/mE

Fig.19 shows UV-Vis. absorption spectra of AgNPs synthesized using AgNCh precursor in the range comprising 0.5 to 2 mM

Fig.20 shows colour of reactions of AgNPs synthesis using AgNCh precursor in the range comprising 0.5 to 2 mM

Fig.21 shows UV-Vis. absorption spectra of AgNPs synthesized at reaction pH in the range comprising 5 to 9

Fig.22 shows colour of reactions of AgNPs synthesis at reaction pH in the range comprising 5 to 9.

Fig.23 shows UV-Vis. absorption spectra of AgNPs synthesized using incubation temperature range comprising 30 to 70°C

Fig. 24 shows colour of reactions of AgNPs synthesis using incubation temperature in the range comprising 30 to 70°C

Fig.25 shows UV-Vis. absorption spectra of AgNPs synthesized using reaction time in the range comprising 15 min. to 24 h

Fig.26 shows transmission electron microscopy (TEM) image of PdNPs synthesized using biomolecule mixture

Fig.27 shows energy dispersive spectrum (EDS) pattern of PdNPs showing with dominant peaks for Pd atoms Fig.28 shows UV Vis. absorption spectra of PdNPs synthesized using biomolecule mixture in the range comprising 5-50 mg/mL

Fig.29 shows colour of reactions of PdNPs synthesis using biomolecule mixture in the range comprising 5-50 mg/mL

Fig.30 shows UV-Vis. absorption spectra of PdNPs synthesized using PdCh precursor in the range comprising 0.5 to 3 mM

Fig.31 shows colour of reactions of PdNPs synthesis using PdCh precursor in the range comprising 0.5 to 3 mM

Fig.32 shows UV-Vis. absorption spectra of PdNPs synthesized at reaction pH comprising 4 to 7

Fig.33 shows colour of reactions of PdNPs synthesis at reaction pH in the range comprising 4 to 7

Fig.34 shows UV-Vis. absorption spectra of PdNPs synthesized using incubation temperature in the range comprising 50 to 100°C

Fig.35 shows colour of reactions of PdNPs synthesis using incubation temperature in the range comprising 50 to 100°C

Fig.36 shows UV-Vis. absorption spectra of PdNPs synthesized using reaction time range comprising 15 min. to 8 h

Fig.37 shows transmission electron microscopy (TEM) image of PtNPs synthesized using biomolecule mixture

Fig.38 shows energy dispersive spectrum (EDS) pattern of PtNPs showing with dominant peaks for Pt atoms

Fig.39 shows UV-Vis. absorption spectra of PtNPs synthesized using biomolecule mixture in the range comprising 5 to 50 mg/mL Fig.40 shows colour of reactions of PtNPs synthesis using biomolecule mixture in the range comprising 5 to 50 mg/mL

Fig.41 shows UV-Vis. absorption spectra of PtNPs synthesized using K2PtC14 precursor in the range comprising 1 to 5 mM

Fig.42 shows colour of reactions of PtNPs synthesis using K2PtC14 precursor in the range comprising 1 to 5 mM

Fig.43 shows UV-Vis. absorption spectra of PtNPs synthesized at reaction pH comprising 5 to 9

Fig. 44 shows colour of reactions of PtNPs synthesis at reaction pH in the range comprising 5 to 9

Fig.45 shows UV-Vis. absorption spectra of PtNPs synthesized using incubation temperature range comprising 30 to 70°C

Fig.46 shows UV-Vis. absorption spectra of PtNPs synthesized using incubation temperature range comprising 30 to 70°C

Fig.47 shows colour of reactions of PtNPs synthesis using reaction time in the range comprising 1 to 8 h

DETAILED DESCRIPTION

[0021] Accordingly, the present invention relates to a method for preparation of biomolecule mixture in dried/freeze-dried form using bacterial strains selected from the group comprising Lysinibacillus xylanilyticus, Lysinibacillus sp, Lysinibacillus pakistanensis and Lysinibacillus macroides for simple and rapid biogenic synthesis of metal nanoparticles namely gold, silver, palladium and platinum nanoparticles. The method of preparation includes growing the bacterial strain, harvesting the biomass, centrifugation, washing, re- suspension, and incubation, collection of homogenate, centrifugation, collection of biomolecule mixture, precipitation, incubation, centrifugation, dissolution, dialysis, and freeze drying biomolecule mixture and the use of biomolecules mixtures for simple and rapid biogenic synthesis of gold, silver, palladium and platinum nanoparticles at broad range of reaction parameters such as reaction volume, concentration of biomolecule mixture and metal precursor, pH; incubation and reaction time.

[0022] The present invention is described below in details and the disclosures of various patents and publications that are referenced and cited in their entities are hereby incorporated in order to more fully describe this invention and the state of the art to which this invention pertains.

[0023] In the present invention, following terms and definitions are used for interpretation of the claims and specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter belongs.

[0024] As used herein, a term “freeze-dried” refers to dehydrated at low temperature process which involves freezing the biomolecule mixture, lowering pressure and then removing the ice by sublimation.

[0025] As used herein, the term “mass fingerprint” refers to LC MS/MS based mass spectrum giving a characteristic profile indicating complex series of molecular masses, each of which corresponding to that of biomolecule of biomolecule mixture.

[0026] As used herein, the term “precious metal nanoparticle” refers to nanoparticles of gold and/or silver and/or platinum and/or palladium. [0027] As used herein, the term “bacterial strain” refers to a single bacterial species capable of biosynthesizing precious metal nanoparticles.

[0028] As used herein, the term "biomolecule mixture’ encompasses, for example, any biological chemical or combination of chemicals of biological origin prepared using methodology depicted in figure 1.

[0029] Accordingly, the present invention provides a method for preparing a freeze-dried biomolecule mixture from bacterial strains for biogenic synthesis of metal nanoparticles comprises: a) growing the bacterial strain in Luria Bertoni broth medium for 24-72 h; b) harvesting bacterial cell biomass by centrifugation and washing thrice with water; c) suspending cell biomass in water and incubating at 50-90° C for 1 -4 h; d) centrifuging the homogenate and collecting clear supernatant containing biomolecule mixture; e) precipitating biomolecule mixture with ammonium sulphate and incubating at 4° C for overnight; f) centrifuging precipitated biomolecule mixture and re-suspending in phosphate buffer; g) dialyzing re-suspended biomolecule mixture in phosphate buffer using semi-permeable membrane; h) concentrating dialyzed biomolecule mixture in sucrose; and i) freeze-drying biomolecule mixture.

[0030] According to one other embodiment of the present invention, the biomolecule mixture can be prepared using any of the four bacterial strains selected from the group comprising Lysinibacillus xylanilyticus, Lysinibacillus sp, Lysinibacillus pakistanensis and Lysinibacillus macroides.

[0031] The fig. 1 shows the flow chart for the preparation of freeze-dried biomolecule mixture. The method comprises, growing the bacterial strain in Luria Bertoni (LB) broth medium at 37°C for 24-72 h; harvesting the bacterial cell biomass by centrifugation at 10,000-15000 rpm for 10- 20 min. and washing it thrice with sterile distilled water; suspending cell pellet in sterile distilled water and incubating at 60-80° C for 2-4 h. The further steps in the methods can include: centrifuging the homogenate/suspension at 10,000-15000 rpm for 10-15 min. to collect clear supernatant containing biomolecule mixture; precipitating biomolecule mixture with ammonium sulphate (60-70%) and incubating at 4°C overnight; centrifuging precipitated biomolecule mixture at 10,000-15000 rpm for 10-15 min and dissolving in phosphate buffer (0.1 M, pH 7); then dialyzing re-suspended biomolecule mixture in phosphate buffer (0.1M, pH 7) using 10-15 kD semipermeable membrane; and freeze drying biomolecule mixture. Fig.2 shows digital photograph image of prepared freeze-dried biomolecule mixture and Fig.3 shows LC MS/MS mass fingerprint of prepared biomolecule mixture.

[0032] According to one other embodiment of present invention, the prepared freeze-dried biomolecule mixture comprises one or more of the biomolecules corresponding to one or more LC MS/MS mass ions identified in Table 1 as shown below, more preferably the prepared freeze- dried biomolecule mixture comprises at least twenty biomolecules corresponding to at least twenty LC MS/MS mass ions identified in Table 1, more preferably the prepared freeze-dried biomolecule mixture comprises at least hundred biomolecules corresponding to at least hundred LC MS/MS mass ions identified in Table 1, more preferably the prepared freeze-dried biomolecule mixture comprises at least two hundred biomolecules corresponding to at least two hundred LC MS/MS mass ions identified in Table 1, more preferably the prepared freeze-dried biomolecule mixture comprises at least three hundred biomolecules corresponding to at least three hundred LC MS/MS mass ions identified in Table 1, most preferably the prepared freeze- dried biomolecule mixture comprises all the biomolecules corresponding to all the LC MS/MS mass ions identified in Table 1.

Table 1 Identities of Molecular masses of biomolecules in freeze dried biomolecule mixture

No m/z No m/z No m/z No m/z

I 102.09 51 234.91 101 315.19 151 383.4 104.11 52 235.13 102 316.21 152 385.29 105.03 53 237.15 103 316.32 153 388.25 107.05 54 239.24 104 317.14 154 390.28 111.12 55 240.16 105 317.24 155 395.24 113.13 56 242.21 106 319.12 156 395.3 119.09 57 243.23 107 321.24 157 402.26 120.08 58 245.11 108 321.31 158 404.3 121.03 59 249.18 109 323.22 159 405.26 122.1 60 251.16 110 323.26 160 406.33 131.05 61 255.12 111 324.22 161 407.17 132.08 62 256.26 112 325.2 162 411.28 133.03 63 261.08 113 325.23 163 413.27 135.08 64 265.18 114 327.2 164 415.21 136.08 65 265.25 115 332.88 165 416.87 139.11 66 267.16 116 336.32 166 417.21 144.08 67 267.27 117 337.27 167 417.25 146.06 68 268.15 118 338.34 168 418.22 149.02 69 269.25 119 339.16 169 419.28 149.06 70 271.26 120 339.29 170 422.33 153.05 71 273.11 121 340.36 171 425.25 158.03 72 273.18 122 341.27 172 425.26 159.09 73 274.27 123 346.29 173 426.83 161.06 74 277.18 124 347.85 174 429.09 161.1 75 277.21 125 349.23 175 432.28 163.11 76 279.16 126 351.25 176 432.33 165.09 77 280.15 127 353.27 177 436.34 167.03 78 281.25 128 354.41 178 437.29 179.1 79 282.28 129 355.07 179 441.33 181.09 80 283.26 130 355.3 180 445.82 183.08 81 284.29 131 356.24 181 447.27 183.1 82 288.29 132 358.37 182 450.36 186.08 83 295.19 133 360.32 183 452.28 188.07 84 297.24 134 361.23 184 453.34 199.17 85 297.28 135 363.15 185 460.27 202.18 86 299.29 136 363.25 186 460.27 203.11 87 301.14 137 365.27 187 463.3 209.12 88 301.14 138 366.37 188 465.82 209.15 89 301.21 139 367.27 189 466.23 214.09 90 302.2 140 369.09 190 471.22 219.17 91 302.3 141 369.3 191 474.28 42 220.12 92 307.08 142 371.1 192 475.24

43 223.13 93 309.2 143 371.31 193 476.3

44 224.09 94 309.24 144 375.18 194 483.2

45 225.15 95 310.24 145 375.25 195 484.38

46 225.2 96 310.31 146 379.28 196 485.35

47 227.13 97 311.19 147 381.26 197 488.36

48 228.05 98 312.08 148 381.3 198 491.37

49 228.2 99 313.24 149 382.44 199 491.42

50 230.25 100 315.16 150 383.25 200 502.37

Table 1 (continued) Identities of Molecular masses of biomolecules in freeze dried biomolecule mixture

No m/z No m/z No m/z

201 502.82 251 609.6 301 736.54

202 505.69 252 613.18 302 737.71

203 507.37 253 615.49 303 742.74

204 520.33 254 617.43 304 746.34

205 521.38 255 617.75 305 749.51

206 522.6 256 618.14 306 752.51 531.86 257 620.43 307 767.35 532.38 258 622.78 308 775.71 533.42 259 624.78 309 776.57 534.37 260 628.74 310 783.57 535.79 261 631.45 311 786.7 536.16 262 631.78 312 802.72 536.69 263 633.77 313 808.68 543.4 264 634.45 314 813.71 546.4 265 636.56 315 820.6 546.78 266 639.45 316 824.69 550.63 267 641.51 317 832.24 551.39 268 648.64 318 835.24 551.5 269 651.14 319 844.68 557.22 270 653.96 320 851.71 562.56 271 654.6 321 864.62 564.36 272 655.94 322 873.68 565.41 273 656.62 323 884.67 565.56 274 661.46 324 909.71 566.43 275 663.45 325 918.74 567.55 276 664.46 326 922.67 567.89 277 664.59 327 926.74 568.45 278 666.74 328 928.75 573.41 279 675.68 329 933.66 574.43 280 677.73 330 935.49 575.50 281 677.95 331 942.74 576.41 282 681.8 332 944.65 576.44 283 683.47 333 946.77 577.12 284 684.2 334 950.62 579.29 285 687.2 335 951.8 579.35 286 688.72 336 955.64 580.12 287 693.75 337 956.86 581.57 288 697.49 338 965.5 584.78 289 704.74 339 970.79 587.42 290 705.99 340 982.65 590.42 291 707.96 341 1026.6 590.75 292 708.49 342 1042.6 593.16 293 708.51 343 1064.6 595.33 294 719.5 344 1102.6 595.92 295 722.52 345 1124.6 596.17 296 728 346 1244.5 247 597.90 297 729.98

248 600.47 298 730.45

249 601.52 299 731.83

250 609.44 300 732.54

Isolation of bacterial strains

[0033] According to one embodiment of the present invention, the bacterial strains of the present invention were derived from bacterial community enriched from water and soil samples collected from waste water disposal site of foundry industrial area of Kolhapur, India and hot water springs at Rajavadi, Tural, India and selected for the ability to grow in presence of high iron content and synthesize precious metal nanoparticles.

[0034] Briefly, collected samples were inoculated in 10 mL of modified nutrient medium supplemented with 1 mM ferric quinate; then agitated on a shaker at 120 rpm, 37° C for 2 days. After 2 days, 1 mL of the culture was sub-cultured to 9 mL of fresh modified nutrient medium with same (1 mM) concentration of ferric quinate. One millilitre from remaining 9 mL culture was serially diluted up to 10’⁸ dilutions, and 20-100 pL of each dilution was spread onto nutrient agar (NA) medium containing ImM ferric quinnate. After 24 hours of incubation at 37°C, bacterial colonies with different morphological characters were manually picked and transferred to freshly prepared medium. These procedures of sampling, sub-cultivation, spreading and streaking were repeated and continued for two weeks. The bacterial colonies with distinct morphologies were made into pure culture by repeated streaking, screened and four bacterial strains were selected for the ability to synthesize precious metal nanoparticles such as gold, silver, palladium and platinum nanoparticles.

[0035] The selected four bacterial strains were identified by molecular identification using 16S rRNA gene amplification, sequencing and analysis. Based on 16S rRNA gene sequencing and analysis, the four strains of present invention were identified as Lysinibacillus xylanilyticus, Lysinibacillus sp, Lysinibacillus pakistanensis and Lysinibacillus macroides. [0036] The GeneBank accession numbers for the 16S rRNA gene sequences of Lysinibacillus xylanilyticus strain is MT102374 (disclosed herein as SEQ IDs NO: 1). Strains Lysinibacillus xylanilyticus was deposited with the international depositary authority at National Centre for Microbial Resource, National Centre for Cell Science, Pune, India on 4th October, 2019 as “Lysinibacillus xylanilyticus (KDP-SUK-M8)” and was assigned accession numbers MCC0176. The said deposit was made under the terms of the Budapest Treaty. Maintenance of a viable culture is assured for 20 years from the date of deposit. All restrictions on the availability to the public of the deposited microorganism will be irrevocably removed upon the granting of a patent for this application.

SEQ ID NO: 1

TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACAGAGAAGG AGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTACCT TATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCTATTTCACTT CATGGTGAAATACTGAAAGACGGTCTCGGCTGTCGCTATAAGATGGGCCCGCGGCGC ATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGA GAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTG AAGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTA ACTGGCTGTACCTTGACGGTACCTTATTAGAAAGCCACGGCTAACTACGTGCCAGCA GCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGC GCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATT GGAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTG AAATGCGTAGAGATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAAC TGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCC ACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGC TAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGA ATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGA AGAACCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATGGTTTTCCCTTC GGGGACAACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGC ACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCA TCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAACGGTTG CCAACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCT GCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT GAATACGTTCCCGG

[0037] The GeneBank accession numbers for the 16S rRNA gene sequences of Lysinibacillus sp is MT102373 (disclosed herein as SEQ IDs NO: 2). Strains Lysinibacillus sp was deposited with the international depositary authority at National Centre for Microbial Resource, National Centre for Cell Science, Pune, India on 4th October, 2019 as “Lysinibacillus sp (KDP-SUK-M5)” and was assigned accession numbers MCC0182. The said deposit was made under the terms of the Budapest Treaty. Maintenance of a viable culture is assured for 20 years from the date of deposit. All restrictions on the availability to the public of the deposited microorganism will be irrevocably removed upon the granting of a patent for this application.

SEQ ID NO: 2

TGTTACGACTTCACCCCAATCATCTATCCCACCTTCGGCGGCTGGCTCCAAAAGGTTA CCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGG CCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTC ATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGACTTTATCGGATTAGCTC CCTCTCGCGAGTTGGCAACCGTTTGTATCGTCCATTGTAGCACGTGTGTAGCCCAGGT CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCA GTCACCTTAGAGTGCCCAACTAAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTT GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT CACCGTTGCCCCCGAAGGGGAAACTATATCTCTACAGTGGTCAACGGGATGTCAAGA CCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGG GCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCT TAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCG TTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCT CAGCGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCAAATCTCT ACGCATTTCACCGCTACACTTGGAATTCCACTTTCCTCTTCTGCACTCAAGTCCCCCA GTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAAGGACC GCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTAC CGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTAATAAGGTACCGTCAAGGTACA GCCAGTTACTACTGTACTTGTTCTTCCCTTACAACAGAGTTTTACGATCCGAAAACCT TCTTCACTCACGCGGCGTTGCTCCATCAGGCTTTCGCCCATTGTGGAAGATTCCCTAC TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTC TCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATG CGCCGCGGGCCCATCCTATAGCGACAGCCGAGACCGTCTTTCAGTATGTCACCATGA GGTGACATAGATTATTCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAAACTATAG GGTAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCTAACGTCAAAGGAGCAAGCTC CTTCTCTGTTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGA

[0038] The GeneBank accession numbers for the 16S rRNA gene sequences of Lysinibacillus pakistanensis is MT102370 (disclosed herein as SEQ IDs NO: 3). Strains Lysinibacillus pakistanensis was deposited with the international depositary authority at National Centre for Microbial Resource, National Centre for Cell Science, Pune, India on 4th October, 2019 as “Lysinibacillus pakistanensis (KDP-SUK-M9)” and was assigned accession numbers MCC0185. The said deposit was made under the terms of the Budapest Treaty. Maintenance of a viable culture is assured for 20 years from the date of deposit. All restrictions on the availability to the public of the deposited microorganism will be irrevocably removed upon the granting of a patent for this application.

SEQ ID NO: 3

CAATCATCTATCCCACCTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGG GTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGC AGCCTACAATCCGAACTGAGAACGACTTTATCGGATTAGCTCCCTCTCGCGAGTTGG CAACCGTTTGTATCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGC CCAACTAAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCA ACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGTTGCCCCCGA AGGGGAAACTATATCTCTACAGTGGTCAACGGGATGTCAAGACCTGGTAAGGTTCTT CGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCC TTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGC AGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACT ACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAG ACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCAAATCTCTACGCATTTCACCGCTA CACTTGGAATTCCACTTTCCTCTTCTGCACTCAAGTCCCCCAGTTTCCAATGACCCTC CACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAAGGACCGCCTGCGCGCGCTTTA CGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG TAGTTAGCCGTGGCTTTCTAATAAGGTACCGTCAAGGTACAGCCAGTTACTACTGTA CTTGTTCTTCCCTTACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGC GTTGCTCCATCAGGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGG AGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGC ATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGCCCATC CTATAGCGACAGCGAGATGCCGTCTTTCAGTCTTTCACCATGAAGTAAAAGAGATTA TTCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAAACTATAGGGTAGGTTGCCCAC GTGTTACTCACCCGTCCGCCGCTAACGTCAAAGGAGCAAGCTCCTTTTCTGTTCGCTC GACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCA

[0039] The GeneBank accession numbers for the 16S rRNA gene sequences of Lysinibacillus macroides strain is MT102369 (disclosed herein as SEQ IDs NO: 4). Strains Lysinibacillus macroides was deposited with the international depositary authority at National Centre for Microbial Resource, National Centre for Cell Science, Pune, India on 4th October, 2019 as “Lysinibacillus macroides (KDP-SUK-M4)” and was assigned accession numbers MCC0186. The said deposit was made under the terms of the Budapest Treaty. Maintenance of a viable culture is assured for 20 years from the date of deposit. All restrictions on the availability to the public of the deposited microorganism will be irrevocably removed upon the granting of a patent for this application.

SEQ ID NO: 4

CAATCATCTATCCCACCTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGG

GTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC

ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGC

AGCCTACAATCCGAACTGAGAACGACTTTATCGGATTAGCTCCCTCTCGCGAGTTGG

CAACCGTTTGTATCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA

TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGC

CCAACTAAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCA

ACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGTTGCCCCCGA

AGGGGAAACTATATCTCTACAGTGGTCAACGGGATGTCAAGACCTGGTAAGGTTCTT

CGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCC

TTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGC

AGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACT

ACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAG

ACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCAAATCTCTACGCATTTCACCGCTA

CACTTGGAATTCCACTTTCCTCTTCTGCACTCAAGTCCCCCAGTTTCCAATGACCCTC

CACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAAGGACCGCCTGCGCGCGCTTTA

CGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG

TAGTTAGCCGTGGCTTTCTAATAAGGTACCGTCAAGGTACAGCCAGTTACTACTGTA

CTTGTTCTTCCCTTACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGC

GTTGCTCCATCAGGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGG

AGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGC

ATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGCCCATC

CTATAGCGACAGCGAGATGCCGTCTTTCAGTCTTTCGCCATGAAGTAAAAGAGATTA

TTCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAAACTATAGGGTAGGTTGCCCAC

GTGTTACTCACCCGTCCGCCGCTAACGTCAAAGGAGCAAGCTCCTTTTCTGTTCGCTC

GACTTGCATGTATTAGGCACGC. [0040] According to one embodiment of the present invention, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus xylanilyticus . Preferably, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 1.

[0041] According to one embodiment of the present invention the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus sp. Preferably, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 2.

[0042] According to one embodiment of the present invention the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus pakistanensis . Preferably, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 3

[0043] According to one embodiment of the present invention the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus macroides. Preferably, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 4.

[0044] In one other embodiment of present invention, the prepared freeze-dried biomolecule mixture is used for biogenic synthesis of precious metal nanoparticles such as gold, silver, palladium and platinum.

[0045] Accordingly, the present invention provides a method for biogenic synthesis of gold nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding chloroauric acid (HAuCU) in the range 1-5 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 40-100° C; d) incubating the reaction mixture of step c) for 10-60 min; and e) obtaining the gold nanoparticles.

[0046] In one other embodiments of the present invention, the formation of gold nanoparticles is monitored by change of colour from pale yellow to pinkish ruby red by using spectrophotometer. Typically, gold nanoparticles have mean diameter in the range 5-50 nm. Fig.4 shows transmission electron microscopy (TEM) image of AuNPs synthesized using biomolecule mixture. Fig.5 shows energy dispersive spectrum (EDS) pattern of AuNPs showing dominant peaks for Au atoms. Fig.6 shows UV-Vis. absorption spectra of AuNPs synthesized using biomolecule mixture in the range comprising 5 to 50 mg/mL. Fig.7 shows colour of reactions of AuNPs synthesis using biomolecule mixture in the range comprising 5 to 50 mg/mL. Fig.8 shows UV-Vis. absorption spectra of AuNPs synthesized using HALICU precursor in the range comprising 1 to 5 mM. Fig. 9 shows colour of reactions of AuNPs synthesis using HAuCL precursor in the range comprising 1 to 5 mM.Fig.lO shows UV-Vis. absorption spectra of AuNPs synthesized at reaction pH in the range comprising 5 to 9. Fig.11 shows colour of reactions of AuNPs synthesis at reaction pH in the range 5 to 9. Fig.12 shows UV-Vis. absorption spectra of AuNPs synthesized using incubation temperature in the range comprising 40 to 100°C. Fig. 13 shows colour of reactions of AuNPs synthesis at incubation temperature in the range comprising 40 to 100°C. Fig.14 shows UV-Vis. absorption spectra of AuNPs synthesized using reaction time in the range comprising 10 to 60 min.

[0047] Accordingly, the present invention provides a method for biogenic synthesis of silver nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 2.5-25 mg/mL reaction volume; b) adding silver nitrate (AgNCb) in the range 0.5-2 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 30-70° C; d) incubating the reaction mixture of step c) for 15 min-24 h; and e) obtaining the silver nanoparticles.

[0048] In one other embodiments of the present invention, the formation of silver nanoparticles is monitored by change of colour from pale yellow to brown and using spectrophotometer. Typically, silver nanoparticles have mean diameter in the range 10-80 nm. Fig.15 shows transmission electron microscopy (TEM) image of AgNPs synthesized using biomolecule mixture. Fig.16 shows energy dispersive spectrum (EDS) pattern of AgNPs showing dominant peaks for Ag atoms. Fig.17 shows UV-Vis. absorption spectra of AgNPs synthesized using biomolecule mixture in the range comprising 2.5 to 25 mg/mL. Fig.18 shows colour of reactions of AgNPs synthesis using biomolecule mixture in the range comprising 2.5 to 25 mg/mE. Fig.19 shows UV-Vis. absorption spectra of AgNPs synthesized using AgNCh precursor in the range comprising 0.5 to 2 mM. Fig.20 shows colour of reactions of AgNPs synthesis using AgNCh precursor in the range comprising 0.5 to 2 mM. Fig.21 shows UV-Vis. absorption spectra of AgNPs synthesized at reaction pH in the range comprising 5 to 9. Fig.22 shows colour of reactions of AgNPs synthesis at reaction pH in the range comprising 5 to 9. Fig.23 shows UV- Vis. absorption spectra of AgNPs synthesized using incubation temperature range comprising 30 to 70°C. Fig. 24 shows colour of reactions of AgNPs synthesis using incubation temperature in the range comprising 30 to 70°C. Fig.25 shows UV-Vis. absorption spectra of AgNPs synthesized using reaction time in the range comprising 15 min to 24 h.

[0049] Accordingly, the present invention provides a method for biogenic synthesis of palladium nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mE reaction volume; b) adding PdCh in the range 0.5-3 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 4-7 and at the temperature range 50-100° C; d) incubating the reaction mixture of step c) for 15 min-8 h; and e) obtaining the palladium nanoparticles.

[0050] In one other embodiments of the present invention the formation of palladium nanoparticles can be monitored by change of colour from pale yellow to black by using spectrophotometer. Typically, palladium nanoparticles have mean diameter in the range 2-40 nm. Fig.26 shows transmission electron microscopy (TEM) image of PdNPs synthesized using biomolecule mixture. Fig.27 shows energy dispersive spectrum (EDS) pattern of PdNPs showing with dominant peaks for Pd atoms. Fig.28 shows UV Vis. absorption spectra of PdNPs synthesized using biomolecule mixture in the range comprising 5-50 mg/mL. Fig.29 shows colour of reactions of PdNPs synthesis using biomolecule mixture in the range comprising 5-50 mg/mL. Fig.30 shows UV-Vis. absorption spectra of PdNPs synthesized using PdCh precursor in the range comprising 0.5 to 3 mM. Fig.31 shows colour of reactions of PdNPs synthesis using PdCh precursor in the range comprising 0.5 to 3 mM. Fig.32 shows UV-Vis. absorption spectra of PdNPs synthesized at reaction pH comprising 4 to 7. Fig.33 shows colour of reactions of PdNPs synthesis at reaction pH in the range comprising 4 to 7. Fig.34 shows UV-Vis. absorption spectra of PdNPs synthesized using incubation temperature in the range comprising 50 to 100°C. Fig.35 shows colour of reactions of PdNPs synthesis using incubation temperature in the range comprising 50 to 100°C. Fig.36 shows UV-Vis. absorption spectra of PdNPs synthesized using reaction time range comprising 15 min to 8 h.

[0051] Accordingly, the present invention provides a method for biogenic synthesis of platinum nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding Potassium tetra chloropalatinate (K^PtCLj) in the range 1-5 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 30-70° C; d) incubating the reaction mixture of step c) for 15 min-2 h; and e) obtaining the platinum nanoparticles.

[0052] In one other embodiments of the present invention the formation of platinum nanoparticles can be monitored by change of colour from pale yellow to black by using spectrophotometer. Typically, platinum nanoparticles have mean diameter in the range 1-20 nm. Fig.37 shows transmission electron microscopy (TEM) image of PtNPs synthesized using biomolecule mixture. Fig.38 shows energy dispersive spectrum (EDS) pattern of PtNPs showing with dominant peaks for Pt atoms. Fig. 39 shows UV-Vis. absorption spectra of PtNPs synthesized using biomolecule mixture in the range comprising 5 to 50 mg/mL. Fig.40 shows colour of reactions of PtNPs synthesis using biomolecule mixture in the range comprising 5 to 50 mg/mL. Fig. 41 shows UV-Vis. absorption spectra of PtNPs synthesized using K2PtCL precursor in the range comprising 1 to 5 mM. Fig. 42 shows colour of reactions of PtNPs synthesis using K2PtC14 precursor in the range comprising 1 to 5 mM. Fig. 43 shows UV-Vis. absorption spectra of PtNPs synthesized at reaction pH comprising 5 to 9. Fig. 44 shows colour of reactions of PtNPs synthesis at reaction pH in the range comprising 5 to 9. Fig. 45 shows UV-Vis. absorption spectra of PtNPs synthesized using incubation temperature range comprising 30 to 70°C. Fig. 46 shows UV-Vis. absorption spectra of PtNPs synthesized using incubation temperature range comprising 30 to 70°C. Fig. 47 shows colour of reactions of PtNPs synthesis using reaction time in the range comprising 1 to 8 h.

[0053] The main advantage of the present invention is, it provides a method for preparation of biomolecule mixture from bacteria which have good commercial value as freeze dried biomolecule mixture can commercially be distributed which consequently will save much of research efforts and time of those interested in using biogenic metal nanoparticles for various applications. The present invention isolates the bacterial strains that can be used for preparation of freeze-dried biomolecule mixture capable of biosynthesizing precious metal nanoparticles. The method for preparing commercially viable, bacterial biomolecule mixture in freeze dried form from four bacterial strains/species for biogenic synthesis of gold, silver, palladium and platinum nanoparticles is not found in literature thus the present invention provides a solution to prior art problems.

EXAMPLES:

[0054] Examplel: Isolation of bacterial strains: collected samples were inoculated in 10 mL of modified nutrient medium supplemented with 1 mM ferric quinate; then agitated on a shaker at 120 rpm, 37° C for 2 days. After 2 days, 1 mL of the culture was sub-cultured to 9 mL of fresh modified nutrient medium with same (1 mM) concentration of ferric quinate. One millilitre from remaining 9 mL culture was serially diluted up to 10’⁸ dilutions, and 20-100 pL of each dilution was spread onto nutrient agar (NA) medium containing ImM ferric quinnate. After 24 hours of incubation at 37°C, bacterial colonies with different morphological characters were manually picked and transferred to freshly prepared medium. These procedures of sampling, subcultivation, spreading and streaking were repeated and continued for two weeks. The bacterial colonies with distinct morphologies were made into pure culture by repeated streaking, screened and four bacterial strains were selected for the ability to synthesize precious metal nanoparticles such as gold, silver, palladium and platinum nanoparticles.

[0055] Example 2: Preparation of biomolecule mixture: the biomolecule mixture is prepared using any of the four bacterial strains selected from the group comprising Lysinibacillus xylanilyticus, Lysinibacillus sp, Lysinibacillus pakistanensis and Lysinibacillus macrolides and the method comprises: growing the bacterial strain in Luria Bertoni (LB) broth medium at 37°C for 24-72 h; harvesting the bacterial cell biomass by centrifugation at 10,000-15000 rpm for 10- 20 min and washing it thrice with sterile distilled water; suspending cell pellet in sterile distilled water and incubating at 60-80° C for 2-4 h. In one other aspect, the further steps in the methods can include: centrifuging the homogenate/suspension at 10,000-15000 rpm for 10-15 min to collect clear supernatant containing biomolecule mixture; precipitating biomolecule mixture with ammonium sulphate (60-70%) and incubating at 4°C overnight; centrifuging precipitated biomolecule mixture at 10,000-15000 rpm for 10-15 min and dissolving in phosphate buffer (0.1 M, pH 7); then dialyzing re-suspended biomolecule mixture in phosphate buffer (0.1M, pH 7) using 10-15 kD semipermeable membrane; and freeze drying biomolecule mixture.

[0056] Example 3: Biogenic synthesis of gold nanoparticles: a biogenic synthesis of gold nanoparticles which comprises the method; 5-50 ml reaction comprising 5-50 mg biomolecule mixture; 1-5 mM chloroauric acid (HAuCL); 5-9 pH; incubation at 40-100°C for 10-60 min. The formation of gold nanoparticles is monitored by change of colour from pale yellow to pinkish ruby red by using spectrophotometer. Typically, gold nanoparticles have mean diameter in the range 5- 50 nm.

[0057] Example 4: Biogenic synthesis of silver nanoparticles: a biogenic synthesis of gold nanoparticles which comprises the method; 5-50 mL reaction comprising 2.5-25 mg biomolecule mixture; 0.5-2 mM silver nitrate (AgNO3); 5-9 pH; incubation at 30-70°C for 15 min.-24 h. The formation of silver nanoparticles can be monitored by change of colour from pale yellow to brown and using spectrophotometer. Typically, silver nanoparticles have mean diameter in the range 10 - 80 nm [0058] Example 5: Biogenic synthesis of palladium nanoparticles: a biogenic synthesis of gold nanoparticles which comprises the method; 5-50 mL reaction comprising 5-50 mg biomolecule mixture; 0.5-3.0 mM Palladium chloride (PdCh); 4-7 pH; incubation at 50-100°C for 15min-8h. The formation of palladium nanoparticles can be monitored by change of colour from pale yellow to black by using spectrophotometer. Typically, palladium nanoparticles have mean diameter in the range 2- 40 nm.

[0059] Example 6: Biogenic synthesis of platinum nanoparticles: a biogenic synthesis of gold nanoparticles which comprises the method; 5-50 mL reaction comprising 5-50 mg biomolecule mixture; 1-5 mM Potassium Tetra Chloro Platinate (K^PtCLj); 5-9 pH; incubation at 30-70° C for 15min-2h. The formation of platinum nanoparticles can be monitored by change of colour from pale yellow to black by using spectrophotometer. Typically, platinum nanoparticles have mean diameter in the range 1-20 nm.

[0060] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure with specific examples. Thus, it is intended that the present disclosure covers the modifications and variations of this disclosure that come within the scope of any claims and their equivalents. Freeze- dried biomolecule mixture and the biogenic synthesis of nanoparticles of the present disclosure as defined by claims and/or as described in foregoing nonlimiting examples and description can be prepared using various combination and permutation, the various modification in the above processes and examples may be applied as to obtained the desired products and which are known to person skilled in the art.

Claims

WE CLAIM:

1. A method for preparing a freeze-dried biomolecule mixture from bacterial strains for biogenic synthesis of metal nanoparticles comprises: a) growing the bacterial strain in Luria Bertoni broth medium for 24-72 h; b) harvesting bacterial cell biomass by centrifugation and washing thrice with water; c) suspending cell biomass in water and incubating at 50-90° C for 1 -4 h; d) centrifuging the homogenate and collecting clear supernatant containing biomolecule mixture; e) precipitating biomolecule mixture with ammonium sulphate and incubating at 4° C for overnight; f) centrifuging precipitated biomolecule mixture and re-suspending in phosphate buffer; g) dialyzing re-suspended biomolecule mixture in phosphate buffer using semi-permeable membrane; h) concentrating dialyzed biomolecule mixture in sucrose; and i) freeze-drying biomolecule mixture.

2. The method as claimed in claim 1 wherein in step a) the bacterial strain is selected from group comprising Lysinibacillus xylanilyticus, Lysinibacillus sp, Lysinibacillus pakistanensis and Lysinibacillus macroides.

3. The method as claimed in claim 1 wherein in step a) the bacterial strain is selected from group comprising of the genus Lysinibacillus or more particularly the one or more stain(s) is selected from the group consisting of one or more of Lysinibacillus xylanilyticus (GeneBank Acc. No- MT102374), Lysinibacillus sp (GeneBank Acc. No- MT102373), Lysinibacillus pakistanensis (GeneBank Acc. No- MT102370) and Lysinibacillus macroides (GeneBank Acc. No- MT102369).

4. The method as claimed in claim 1 wherein in step a) the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus xylanilyticus preferably, the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 1.

5. The method as claimed in claim 1 wherein in step a) the bacterial strain f has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus sp. preferably, the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 2.

6. The method as claimed in claim 1 wherein in step a) the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus pakistanensis preferably, the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 3

7. The method as claimed in claim 1 wherein in step a) the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Lysinibacillus macrolides preferably, the bacterial strain has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 4.

8. The method as claimed in claim 1 wherein freeze-dried biomolecule mixture comprises one or more of the biomolecules corresponding to one or more LC MS/MS mass ions identified in Table 1.

9. The method as claimed in claim 1 wherein the freeze-dried biomolecule mixture comprises at least twenty biomolecules corresponding to at least twenty LC MS/MS mass ions identified in Table 1.

10. The method as claimed in claim 1 wherein the freeze-dried biomolecule mixture comprises at least hundred biomolecules corresponding to at least hundred LC MS/MS mass ions identified in Table 1.

11. The method as claimed in claim 1 wherein the freeze-dried biomolecule mixture comprises at least two hundred biomolecules corresponding to at least two hundred LC MS/MS mass ions identified in Table 1.

12. The method as claimed in claim 1 wherein the freeze-dried biomolecule mixture comprises at least three hundred biomolecules corresponding to at least three hundred LC MS/MS mass ions identified in Table 1.

13. The method as claimed in claim 1 wherein the freeze-dried biomolecule mixture comprises all the biomolecules corresponding to all the LC MS/MS mass ions identified in Table 1.

14. A method for biogenic synthesis of gold nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding chloroauric acid (HAuCL) in the range 1-5 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 40-100° C; d) incubating the reaction mixture of step c) for 10-60 min; and e) obtaining the gold nanoparticles.

15. A method for biogenic synthesis of silver nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 2.5-25 mg/mL reaction volume; b) adding silver nitrate (AgNCL) in the range 0.5-2 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 30-70° C; d) incubating the reaction mixture of step c) for 15 min-24 h; and e) obtaining the silver nanoparticles.

16. A method for biogenic synthesis of palladium nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding PdCh in the range 0.5-3 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 4-7 and at the temperature range 50-100° C; d) incubating the reaction mixture of step c) for 15 min-8 h; and e) obtaining the palladium nanoparticles.

17. A method for biogenic synthesis of platinum nanoparticles using freeze-dried biomolecule mixture comprises: a) taking freeze dried biomolecule mixture in the range 5-50 mg/mL reaction volume; b) adding Potassium tetra chloropalatinate (K^PtCLj) in the range 1-5 mM in the step a) reaction mixture; c) carrying the reaction in the pH range 5-9 and at the temperature range 30-70° C; d) incubating the reaction mixture of step c) for 15 min-2 h; and e) obtaining the platinum nanoparticles.