CN104893990A - Aspergillus WL-Au and application of aspergillus WL-Au on preparation of gold nanoparticles - Google Patents

Aspergillus WL-Au and application of aspergillus WL-Au on preparation of gold nanoparticles Download PDF

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CN104893990A
CN104893990A CN201510358206.6A CN201510358206A CN104893990A CN 104893990 A CN104893990 A CN 104893990A CN 201510358206 A CN201510358206 A CN 201510358206A CN 104893990 A CN104893990 A CN 104893990A
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aspergillus
bacterial strain
gold nano
strain
nano grain
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曲媛媛
厉舒祯
李会杰
沈文丽
马桥
张旭旺
王经伟
张照婧
刘紫嫣
周集体
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Dalian University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
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    • C12N1/145—Fungal isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide

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Abstract

The invention provides aspergillus WL-Au and an application of the aspergillus WL-Au on preparation of gold nanoparticles, and belongs to the field of biotechnology. The bacterial strain WL-Au is separated from a bioreactor of a laboratory, is sequenced and identified into aspergillus through a 26S rRNA (ribosomal robonucleic acid) gene sequence, is classified and named as Aspergillus sp., and is stored in a common microorganism center of the China Committee for Culture Collection of Microorganisms; and the storage number is CGMCC No. 10648. The bacterial strain grows for 108 hours in an LB medium containing 0.05g/L of tetracycline and 0.05g/L of oxytetracycline and then is stable; and in the stable period, the width of hypha is about 1 micrometer, and the spore size is 0.25-0.65*1.1-2.5 micrometers. The wet hypha of 0.2g of the bacterial strain is mixed with HAuCl4 solution of which the final concentration is 0.5-5.0 mmol/L, the mixture is incubated for 15 hours under the condition of 150/min, and Au3+ in solution can be reduced into the gold nanoparticles in different shapes.

Description

One strain aspergillus tubigensis WL-Au and preparing the application in gold nano grain
Technical field
The present invention relates to a strain aspergillus tubigensis WL-Au and preparing the application in gold nano grain, belong to biological technical field.
Background technology
Gold is a kind of well-known precious metal material, and because it has multiple excellent properties, gold is applied to numerous areas, as done lagging material at automobile manufacturing field gold, some high-end CD products makes reflecting layer etc. with gold.Compared to the gold copper-base alloy of bulk, gold nano-material has unique performance, and the stability of its height and unique photoelectricity, light thermal property, be used widely in fields such as information storage, chemical sensitisation, medical imaging, medicament transport and biomarkers.
The gold nano-material particle size range of traditional physics and chemistry synthetic method synthesis at 1-100 nm, form of diverse.Although its related methods of synthesis is widely studied, still there is obvious shortcoming, as conventional physical synthetic method needs complicated instrument and energy consumption is high; Traditional chemical synthesis needs harsh synthesis condition, and the end-capping reagent used in synthesis and organic solvent are then unfavorable for the application of gold nano grain at biomedical sector, and cause negative impact to environment.Conventional physical and chemical process easily cause the nano particle of variform particle diameter to mix, and need following process process, as differential centrifugation etc., and the high and inefficiency of cost.Therefore, people more and more need clean, nontoxic, the environmental protection of exploitation, continuable gold nano-material synthesizes.
Microorganism is extensive in distributed in nature, growth and breeding is rapid, and easily separated cultivation, and nano materials process has the low and large-scale feature of easy expansion of gentleness, green low toxicity, cost relatively.Bacterium, fungi, actinomycetes and plant all have the ability of nano materials.Wherein, fungi can secrete a large amount of extracellular enzyme, polypeptides matter and the secondary metabolite etc. relevant to nano materials, and the nano material output of synthesis is high, be easily separated with fungi, can be used for the scale operation of nano material.In addition, the nano material synthesized by fungi also has good monodispersity.There are some researches show that at present fungi and meta-bolites thereof can reduce Au 3+/ Au +synthesis gold nano grain.As the researchs such as Shankar find, the outer extract of fungi born of the same parents can synthesize gold nano grain, pichia jadiniiwith yarrowia lipolyticatwo Yeasts all have the ability of well synthesis gold nano grain.The discoveries such as Liangwei Du penicilliumsp. cell leaches vacuole and reduces HAuCl outward 4synthesis gold nano grain only uses 1 min, and in full cell response born of the same parents, synthesis gold nano grain also only needs 8 h.
The aspergillus bacterium that this patent relates to is the important microbial resources of a class, it is one of saprophytic mushroom that distributed in nature is general, be widely used in traditional fermentation industry, modern fermentation industry and bio-engineering research, as for organic acid, zymin and antibiotic production, bio-transformation and genetics research etc., and have not been reported so far about the research of aspergillus tubigensis synthetic metals nano material.
Summary of the invention
The object of the present invention is to provide a strain aspergillus tubigensis, and prepare the method for gold nano grain, this bacterial strain has broad application prospects in biosynthesizing field of nanometer material technology.
The present invention relates to a strain aspergillus tubigensis, Classification And Nomenclature is aspergillussp., described bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and it is registered on the books and is numbered CGMCC No. 10648, and preservation date is on 04 09th, 2015; The accession number of 26S rRNA gene order in GenBank database of bacterial strain is KR090574.
This strains separation in this laboratory biological reactor, utilize 26S rRNA sequential analysis show bacterial strain for Eurotium ( aspergillus).This strain growth liquid nutrient medium used is LB substratum, and solid medium used is add 2% (W/V) agar in corresponding liquid nutrient medium.LB substratum consists of: NaCl 10 g/L, peptone 10 g/L, yeast leaching powder 5 g/L, tsiklomitsin 0.05 g/L, terramycin 0.05 g/L, pH 7.0.All in 115 DEG C of moist heat sterilization 20 min before all substratum use.
Bacterial strain switching and activation act are add 5 mL inoculation liquid in 100 mL LB liquid nutrient mediums, and in 30 DEG C, under 150 r/min conditions, shaking culture 108 h is to stationary phase.
This bacterial strain has the ability preparing gold nano grain: 0.2 g wet mycelium is joined HAuCl 4final concentration is in the reaction system of 0.5 ~ 5.0 mmol/L, and in 30 DEG C, 150 r/min conditions hatch 15 h altogether, can generate gold nano grain that is spherical, the different shape such as sexangle, trilateral.
The invention has the beneficial effects as follows: strains separation, in the bio-reactor in laboratory early stage, grows 108 h and reaches stationary phase in LB liquid nutrient medium.This bacterial strain can catalysis HAuCl 4the gold nano grain of synthesis different shape, particle diameter.This bacterial strain extends the application of fungi in nano material green syt, for the biology preparation of gold nano-material provides a kind of new Microbial resources, in biosynthesizing field of nanometer material technology, has potential using value.
Accompanying drawing explanation
Fig. 1 is bacterial strain 26S rRNA gene order phylogenetic tree.
Fig. 2 is the stereoscan photograph of bacterial strain.
Fig. 3 is strain growth curve.
Fig. 4 is the full wavelength scanner figure of different time points synthesis gold nano grain.
Fig. 5 is different HAuCl 4concentration is on the impact of synthesis gold nano grain.
Fig. 6 is the transmission electron microscope photo of gold nano grain.
Embodiment
embodiment 1:the screening of Aspergillus strain of the present invention
This bacterial strain screening stage enrichment medium used is improvement Martin substratum.Screening process is: by pedotheque natural air drying, with 10% ( w/V) inoculum size joins 100 mL and improve in Martin liquid nutrient mediums, at 150 r/min, under 30 DEG C of conditions, domestication is cultivated.After 3 days, nutrient solution is staticly settled, discard supernatant liquor, then add 100 mL improvement Martin substratum, continue domestication and cultivate.Continuous domestication about 30 days, take out the earth sample after domestication, adopt the method for dilution spread, mud mixture is coated on the culture dish containing LB solid medium, be placed in 30 DEG C of constant incubators and cultivate 72 h, choose single bacterium colony from culture dish and cultivate in LB liquid nutrient medium, repeat dull and stereotyped coating and choose the process of single bacterium colony, final screening obtains a strain bacterium, biomaterial called after WL-Au.
Improvement Martin substratum consists of: glucose 1 g/L, (NH 4) 2sO 41 g/L, MgSO 40.5 g/L, K 2hPO 41 g/L, tsiklomitsin 0.05 g/L, terramycin 0.05 g/L.
LB substratum consists of: LB substratum consists of: NaCl 10 g/L, peptone 10 g/L, yeast leaching powder 5 g/L, tsiklomitsin 0.05 g/L, terramycin 0.05 g/L, pH 7.0.
embodiment 2:bacterial strain (WL-Au) molecular biology identification
Precious biotechnology (Dalian) company limited is sent to identify bacterial strain (WL-Au), obtain this bacterial strain 26S rRNA gene order, inputted NCBI, by carrying out BLAST similarity analysis with the nucleic acid database sequence in GenBank, find bacterial strain and the aspergillus tubigensis to have delivered ( aspergillus protuberuswith aspergillus sydowii) similarity reaches more than 99%, therefore bacterial strain (WL-Au) for Eurotium ( aspergillus), its phylogenetic tree is as shown in Figure 1.
Bacterial strain aspergillussp. 26S rRNA gene order is:
GTAACGGCGAGTGAAGCGGCAAGAGCTCAAATTTGAAATCTGGCCCCTCCGGGGTCCGAGTTGTAATTTGCAGAGGATGCTTCGGGTGCGGCCCCTGTCTAAGTGCCCTGGAACGGGCCGTCAGAGAGGGTGAGAATCCCGTCTTGGGCAGGGTGCCCGTGCCCGTGTGAAGCTCCTTCGACGAGTCGAGTTGTTTGGGAATGCAGCTCAAAATGGGTGGTAAATTTCATCTAAAGCTAAATACCGGCCGGAGACCGATAGCGCACAAGTAGAGTGATCGAAAGATGAAAAGCACTTTGAAAAGAGAGTTAAACAGCACGTGAAATTGTTGAAAGGGAAGCGCTTGCAACCAGACTCGGCCTCGGGGTTCAGCCAGCATTCGTGCTGGTGTACTTCCCCGGGGCCGGGCCAGCGTCGGTTTGGGCGGCCGGTCAAAGGCCCCAGGAATGTATCGTCCTCCGGGACGTCTTATAGCCTGGGGTGCAATGCGGCCAGCCTGGACCGAGGAACGCGCTTCGGCACGGACGCTGGCGTAATGGTCGCAAACGAC
embodiment 3:the cultivation of bacterial strain (WL-Au)
In 250 mL Erlenmeyer flasks, add 100 mL LB liquid nutrient mediums, add 5 mL inoculation liquid, in 150 r/min, 30 DEG C of condition shaking culture.Adopt dry weight method, get bacterium liquid 100 mL every 12 h, filter paper filtering, mycelium is transferred in clean load weighted in advance glass culture dish, dries to constant weight for 105 DEG C, record of weighing after being down to room temperature, analytical data obtains the growth characteristics of bacterial strain (WL-Au), as shown in Figure 3.Result shows, bacterial strain (WL-Au) 0 ~ 48 h is in the adaptive phase; After 48 h, the speed of growth is accelerated gradually, enters logarithmic phase, reaches maximum growth amount during 108 h, and 108-144 h is in stationary phase.
embodiment 4:bacterial strain (WL-Au) morphological specificity
Get the bacterium liquid that 2 mL grow to late log phase, in centrifugal 10 min of 12000 r/min, abandoning supernatant.Wash thalline 2 times with 50 mmol/L phosphate buffered saline buffers (PBS), the resuspended thalline of 1.5 mL 5% glutaraldehyde solution, is placed in 4 DEG C of refrigerators and hatches 2 h, then 12000 r/min, 4 DEG C of pelleted by centrifugation 15 min, abandoning supernatant.Hanged thalline with 1.5 mL 30%, 50%, 75%, 90%, 95% ethanolic solns successively, centrifugal condition is the same, abandoning supernatant, adds 1.5 mL 100% ethanol, saves backup.Sample conductive resin sticks in sample table, after vacuum ion sputtering spraying plating platinum film, scanning electronic microscope (SEM) is observed, and as shown in Figure 2, in figure, a is mycelia, b is spore for the mycelia of bacterial strain (WL-Au) and spore shape.
embodiment 5:the process monitoring of bacterial strain (WL-Au) synthesis of nano gold
Get grow to late log phase wet mycelium 0.2 g in 5 mL centrifuge tubes, add 1960 μ L ultrapure waters and 40 μ L HAuCl 4solution, makes HAuCl 4final concentration is the reaction system of 1 mmol/L, produces 6 same sample with aforesaid method.Sample is placed in 150 r/min, 30 DEG C of condition lucifuges are hatched, at different time point (0 h, 3h, 6 h, 9 h, 12 h, 15 h) take out 2 mL samples, ultrasonication 30 min, get gained solution 1 mL, fully mix with 16% NaOH solution of same volume, react 1 h, centrifugal 5 min of 3000 r/min, get supernatant liquor.Ultraviolet-visible spectrophotometer is used to carry out full wavelength scanner, sensing range 400-700nm, scanning interval 1nm, as shown in Figure 4.Result shows, occurs obvious gold nano grain characteristic peak after reacting 3 h at about 545nm, and peak intensity increases gradually along with the prolongation in reaction times, and when reaction 15 h, gold nano grain characteristic peak intensity reaches the highest.
embodiment 6:different HAuCl 4concentration is on the impact of synthesis gold nano grain
According to embodiment 5, get wet mycelium 0.2 g growing to late log phase, add ultrapure water and HAuCl 4mother liquor, makes HAuCl 4final concentration be respectively 0.5,1.0,2.0,5.0 mmol/L, in 30 DEG C, oscillation incubation 15 h in 150 r/min shaking tables.By solution ultrasonication 30 min reacted completely, get gained solution 1 mL, fully mix, react 1 h with 16% NaOH solution of same volume, centrifugal 5 min of 3000 r/min, get supernatant liquor.As shown in Figure 5, result shows HAuCl to corresponding ultraviolet-visible full wavelength scanner 4when concentration is 0.5 mmol/L, due to Au in reaction system 3+concentration is too little, and phenomenon is not obvious; Work as HAuCl 4when concentration reaches 1.0 ~ 2.0 mmol/L, the characteristic peak intensity of gold nano grain is along with Au in solution 3+the increase of concentration and increasing; Work as HAuCl 4when concentration is 5.0 mmol/L, due to Au 3+to the restraining effect participating in the reductase enzyme reacted, the characteristic peak of gold nano grain reduces.
embodiment 7:the transmission electron microscope of the nanometer gold that bacterial strain HJ synthesizes characterizes
Suspension containing gold nano grain is dripped on copper mesh, room temperature nature volatile dry, uses transmission electron microscope to observe the pattern of nano particle in 200 kv conditions, as shown in Figure 6, the gold nano grain generated presents variform, as spherical, trilateral, hexagon etc.
SEQUENCE LISTING
<110> Dalian University of Technology
<120> mono-strain aspergillus tubigensis WL-Au and preparing the application in gold nano grain
<210> 1
<211> 550
<212> DNA
<213> aspergillus tubigensis (Aspergillus sp.)
<400> 1
GTAACGGCGAGTGAAGCGGCAAGAGCTCAAATTTGAAATCTGGCCCCTCC50
GGGGTCCGAGTTGTAATTTGCAGAGGATGCTTCGGGTGCGGCCCCTGTCT100
AAGTGCCCTGGAACGGGCCGTCAGAGAGGGTGAGAATCCCGTCTTGGGCA150
GGGTGCCCGTGCCCGTGTGAAGCTCCTTCGACGAGTCGAGTTGTTTGGGA200
ATGCAGCTCAAAATGGGTGGTAAATTTCATCTAAAGCTAAATACCGGCCG250
GAGACCGATAGCGCACAAGTAGAGTGATCGAAAGATGAAAAGCACTTTGA300
AAAGAGAGTTAAACAGCACGTGAAATTGTTGAAAGGGAAGCGCTTGCAAC350
CAGACTCGGCCTCGGGGTTCAGCCAGCATTCGTGCTGGTGTACTTCCCCG400
GGGCCGGGCCAGCGTCGGTTTGGGCGGCCGGTCAAAGGCCCCAGGAATGT450
ATCGTCCTCCGGGACGTCTTATAGCCTGGGGTGCAATGCGGCCAGCCTGG500
ACCGAGGAACGCGCTTCGGCACGGACGCTGGCGTAATGGTCGCAAACGAC550

Claims (3)

1. a strain aspergillus tubigensis, is characterized in that: described aspergillus tubigensis ( aspergillussp.WL-Au) be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 04 09th, 2015, register on the books and be numbered CGMCC No. 10648.
2. a strain aspergillus tubigensis according to claim 1, is characterized in that: described strain growth adopts LB substratum, in 30 DEG C, under 150 r/min conditions, shaking culture 108 h is to stationary phase, thalline is spherical in white, and mycelia is wide about 1 μm, spore size 0.25 ~ 0.65 × 1.1 ~ 2.5 μm; Described LB substratum consists of: NaCl 10 g/L, peptone 10 g/L, yeast leaching powder 5 g/L, tsiklomitsin 0.05 g/L, terramycin 0.05 g/L, pH 7.0.
3. a strain aspergillus tubigensis according to claim 2 is preparing the application in gold nano grain, it is characterized in that: by the HAuCl of described mycelia 0.2 g and final concentration 0.5-5.0 mmol/L 4solution mixes, and in 30 DEG C, under 150 r/min conditions, lucifuge hatches 15 h, by Au in solution 3+be reduced to spherical, trilateral or hexagonal gold nano grain.
CN201510358206.6A 2015-06-26 2015-06-26 Aspergillus WL-Au and application of aspergillus WL-Au on preparation of gold nanoparticles Pending CN104893990A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117089465A (en) * 2023-08-22 2023-11-21 陕西省微生物研究所 Aspergillus wart and application thereof
CN117089465B (en) * 2023-08-22 2024-05-03 陕西省微生物研究所 Aspergillus wart and application thereof

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