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WO2023058037A1 - Mélange de biomolécules pour la synthèse biogène de nanoparticules métalliques - Google Patents

Mélange de biomolécules pour la synthèse biogène de nanoparticules métalliques Download PDF

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WO2023058037A1
WO2023058037A1 PCT/IN2021/051188 IN2021051188W WO2023058037A1 WO 2023058037 A1 WO2023058037 A1 WO 2023058037A1 IN 2021051188 W IN2021051188 W IN 2021051188W WO 2023058037 A1 WO2023058037 A1 WO 2023058037A1
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freeze
biomolecule
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lysinibacillus
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Kiran D. PAWAR
Megha Prakash Desai
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Shivaji University
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Shivaji University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F9/00—Making metallic powder or suspensions thereof
    • B22F9/16—Making metallic powder or suspensions thereof using chemical processes
    • B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
    • B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
    • C22C—ALLOYS
    • C22C1/00—Making non-ferrous alloys
    • C22C1/04—Making non-ferrous alloys by powder metallurgy
    • C22C1/0466—Alloys based on noble metals

Definitions

  • 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.
  • 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.
  • 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.
  • nanoparticles produced by biogenic methods are far superior, in several ways, to those particles produced by chemical methods.
  • 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.
  • 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.
  • the objective of the present invention is to isolate bacterial strains to prepare freeze-dried biomolecule mixtures.
  • Another objective of the present invention is to develop novel, simple, reliable methodology to prepare freeze-dried biomolecule mixtures using isolated bacterial strain.
  • Yet another objective of the present invention is to use freeze dried biomolecule mixture for biogenic synthesis of gold, silver, palladium and platinum nanoparticle.
  • HUACh chloroauric acid
  • AgNCL silver nitrate
  • K ⁇ PtCLj Potassium tetra chloropalatinate
  • 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
  • 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.
  • freeze-dried refers to dehydrated at low temperature process which involves freezing the biomolecule mixture, lowering pressure and then removing the ice by sublimation.
  • 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.
  • the term “precious metal nanoparticle” refers to nanoparticles of gold and/or silver and/or platinum and/or palladium.
  • the term “bacterial strain” refers to a single bacterial species capable of biosynthesizing precious metal nanoparticles.
  • biomolecule mixture encompasses, for example, any biological chemical or combination of chemicals of biological origin prepared using methodology depicted in figure 1.
  • 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.
  • 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.
  • Fig.2 shows digital photograph image of prepared freeze-dried biomolecule mixture
  • Fig.3 shows LC MS/MS mass fingerprint of prepared biomolecule mixture.
  • 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.
  • 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.
  • 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.
  • 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.
  • the GeneBank accession numbers for the 16S rRNA gene sequences of Lysinibacillus xylanilyticus strain is MT102374 (disclosed herein as SEQ IDs NO: 1).
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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
  • 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.
  • 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.
  • the prepared freeze-dried biomolecule mixture is used for biogenic synthesis of precious metal nanoparticles such as gold, silver, palladium and platinum.
  • HAuCU chloroauric acid
  • the formation of gold nanoparticles is monitored by change of colour from pale yellow to pinkish ruby red by using spectrophotometer.
  • 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.
  • AgNCb silver nitrate
  • the formation of silver nanoparticles is monitored by change of colour from pale yellow to brown and using spectrophotometer.
  • 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.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. 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.
  • the formation of palladium nanoparticles can be monitored by change of colour from pale yellow to black by using spectrophotometer.
  • 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.
  • K ⁇ PtCLj Potassium tetra chloropalatinate
  • platinum nanoparticles can be monitored by change of colour from pale yellow to black by using spectrophotometer.
  • 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.
  • 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.
  • 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.
  • NA nutrient agar
  • 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.
  • 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.
  • LB Luria Bertoni
  • 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.
  • 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.
  • 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.
  • silver nanoparticles have mean diameter in the range 10 - 80 nm
  • 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.
  • palladium nanoparticles have mean diameter in the range 2- 40 nm.
  • 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.
  • platinum nanoparticles have mean diameter in the range 1-20 nm.

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Abstract

La présente invention concerne un procédé de préparation d'un mélange de biomolécules sous forme séchée/lyophilisée utilisant des souches bactériennes choisies dans le groupe comprenant Lysinibacillus xylanilyticus, Lysinibacillus sp., Lysinibacillus pakistanensis et Lysinibacillus macroides pour la synthèse biogénique simple et rapide de nanoparticules métalliques, à savoir des nanoparticules d'or, d'argent, de palladium et de platine. Le procédé de préparation comprend les étapes suivantes : culture de la souche bactérienne, récolte de la biomasse, centrifugation, lavage, remise en suspension, et incubation, collecte d'un homogénat, centrifugation, collecte d'un mélange de biomolécules, précipitation, incubation, centrifugation, dissolution, dialyse, et la lyophilisation du mélange de biomolécules et l'utilisation de mélanges de biomolécules pour la synthèse biogénique simple et rapide de nanoparticules d'or, d'argent, de palladium et de platine suivant une large gamme de paramètres de réaction tels que le volume de réaction, la concentration du mélange de biomolécules et du précurseur métallique, le pH, l'incubation et le temps de réaction.
PCT/IN2021/051188 2021-10-05 2021-12-18 Mélange de biomolécules pour la synthèse biogène de nanoparticules métalliques Ceased WO2023058037A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119432655A (zh) * 2024-11-07 2025-02-14 沈阳农业大学 一株巴基斯坦赖氨酸芽胞杆菌Sneb2537与一种能够防治根结线虫病的制剂及其制备方法和应用

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP2035567A1 (fr) * 2006-07-05 2009-03-18 Janssen Pharmaceutica, N.V. Procédé de production de nanoparticules métalliques
WO2014029783A1 (fr) * 2012-08-20 2014-02-27 Chr. Hansen A/S Procédé pour la lyophilisation d'un concentré contenant des bactéries

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2035567A1 (fr) * 2006-07-05 2009-03-18 Janssen Pharmaceutica, N.V. Procédé de production de nanoparticules métalliques
WO2014029783A1 (fr) * 2012-08-20 2014-02-27 Chr. Hansen A/S Procédé pour la lyophilisation d'un concentré contenant des bactéries

Non-Patent Citations (2)

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Title
CARVALHO A S,ET AL: "Relevant factors for the preparation of freeze-dried lactic acid bacteria", INTERNATIONAL DAIRY JOURNAL, ELSEVIER APPLIED SCIENCE, BARKING,, GB, vol. 14, no. 10, 1 October 2004 (2004-10-01), GB , pages 835 - 847, XP002349908, ISSN: 0958-6946 *
DATABASE Nucleotide ANONYMOUS : "Lysinibacillus sp. strain Firmi-71 16S ribosomal RNA gene, partial seq -", XP093061248, retrieved from NCBI *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119432655A (zh) * 2024-11-07 2025-02-14 沈阳农业大学 一株巴基斯坦赖氨酸芽胞杆菌Sneb2537与一种能够防治根结线虫病的制剂及其制备方法和应用

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