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Separation and optimization method and application of laccase-producing fungus strain

An optimization method and strain technology, applied in the field of microorganisms, to achieve good application prospects and potential, and good decolorization efficiency

Inactive Publication Date: 2016-07-06
DALIAN JIAOTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few reports on the rhizosphere microorganisms of Taxus chinensis. Hao Dacheng et al. screened taxane xyloside desugaring microorganisms from the rhizosphere of Taxus Mandia, and found that the bacterial community composition of Taxus rhizosphere in different regions of China has the characteristics of regional diversity; Ren Jiahong isolated bacteria capable of dissolving phosphorus from the rhizosphere soil of Taxus chinensis, and conducted a greenhouse pot experiment on them

Method used

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  • Separation and optimization method and application of laccase-producing fungus strain
  • Separation and optimization method and application of laccase-producing fungus strain
  • Separation and optimization method and application of laccase-producing fungus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 active green 19 degradation

[0047] 1.1 Experimental reagents

[0048] Reactive Green 19 (97%), CAS No.: 61931-49-5 (SigmaR9378)

[0049] 1.2 Medium

[0050] Sucrose 30g / L, peptone 8g / L, CuSO 4 1mmol / L, MgSO 4 2g / L, KH 2 PO 4 1.5g / L, pH6.0, FeSO 4 0.05g / L, set to 1L.

[0051] 1.3 Degradation experiment of reactive green 19

[0052]In this experiment, the degradation rate of microorganisms to high-concentration substrates was improved through uniform design optimization, and the degradation effect of H strain Myrothecium verrucaria on 30mg / L active green 19 within 7 days was determined.

[0053] Preparation of bacterial suspension: On an ultra-clean laboratory bench sterilized by ultraviolet light, 5% of the inoculum of the purified Verrucous verrucosa strain was added to 100 mL of culture medium, and cultured at 28° C. and 130 r / min for 4 days.

[0054] Degradation experiment of reactive green 19: Scan the dye aqueous solution at full wavelength (...

Embodiment 2

[0072] Example 2 Isolation and screening of laccase-producing strains

[0073] 2.1 Experimental materials

[0074] 2.1.1 Experimental reagents and materials

[0075] Screening strain source: Select the rhizosphere 5-10cm deep soil of Taxus chinensis in Dalian, Taxus var.

[0076] 2.1.2 Culture medium and solution preparation

[0077] (1) Enrichment medium: 30g sucrose, CuSO 4 ·5H 2 O0.5g, NaNO 3 2g, MgSO 4 ·7H 2 O0.5g, K 2 HPO 4 1.0g, FeSO 4 0.01g, 0.5g KCl, 1000mL distilled water, natural pH, sterilized at 115℃ for 15min;

[0078] (2) Strain purification and preservation medium (PDA medium): potato 20%, glucose 2%, KH 2 PO 4 3g, agar 2%, MgSO 4 -7H 2 O2g, VB 1 Micro-volume, set the volume to 1 L, with natural pH, sterilize at 121°C for 20 minutes, and add penicillin 50 μg / mL and streptomycin 100 μg / mL when the medium temperature drops to 70°C;

[0079] (3) Primary screening medium: PDA medium, 15g of agar, sterilized at 121°C for 20min. Add guaiacol solution ...

Embodiment 3

[0103] Example 3 Taxonomic identification of laccase-producing strains in yew rhizosphere soil

[0104] 3.1 Medium, solution and buffer

[0105] (1) 10×TBEBuffer (pH8.3): Tris108g, boric acid 55g, Na 2 EDTA·2H 2 O7.44g, dilute to 1L with deionized water, store at room temperature.

[0106] (2) Ethidium bromide EB (10 mg / mL): 1 g of ethidium bromide, 100 mL of deionized water, and store in a brown bottle at room temperature in the dark. The working concentration is 0.5 μg / mL.

[0107] (3) Agarose (1%): 100 mL of 1×TBE buffer, 1 g of agarose, 5 μL of 10 mg / mLEB.

[0108] (4) Proteinase K: The concentration of the mother solution is 20mg / L, and it is stored at -20°C.

[0109] 3.2 Molecular identification of strains

[0110] The genomic DNA of the four strains was extracted with Bao Biological Kit UniversalGenomicDNAExtractionKitVer5.0, and the extracted DNA was checked by agarose gel electrophoresis. The ITS of the four fungi were amplified using the universal primers ITS1...

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Abstract

The invention discloses a separation and optimization method and an application of a laccase-producing fungus strain. Through a color-changing reaction between guaiacol and laccase, a laccase-producing fungus is screened, and then according to morphologic observation and rDNA-ITS sequence analysis, myrothecium verrucaria is identified. The myrothecium verrucaria, as a patent preserved strain, is a strain highly producing laccase, and reaches 316 U / ml in enzyme activity of the produced laccase after optimization, which is higher than that of myrothecium verrucaria NF-05 by 45.08 U / ml. The enzyme activity and decolorizing rates on nine active dyes of the fungus strain are much higher than those of talaromyces, aspergillus and glomerella producing the laccase. Through a single factor experiment and uniform design, the optimum conditions of the myrothecium verrucaria producing the laccase are determined. Through HPLC, TLC and infrared spectrum, the myrothecium verrucaria is proved to have degradation on reactive green 19. The fungus separated from rhizosphere of taxus chinensis has excellent application prospect and potential in the fields of industrial wastewater treatment, green traffic, environment protection and sustainable development.

Description

technical field [0001] The invention belongs to the technical field of microbes, and relates to a separation and optimization method and application of laccase-producing fungal strains. Background technique [0002] Laccase is a copper-containing polyphenol oxidase that can catalyze the degradation of various organic compounds, for example, it has a high conversion efficiency for some anthraquinones and azo dyes. With the development of science and technology and the gradual deepening of the degree of cross-comprehension of various disciplines, the research on laccase has become more and more perfect, and the application technology in various fields has gradually matured. There are many types of laccase resources, which are abundant in nature. People have studied some animal and plant laccases, and some understanding of some microbial laccases, but little is known about the vast majority of microbial laccases, and the relevant resources need to be further developed. develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12R1/645C02F3/34
Inventor 郝大程宋思梦胡文丽
Owner DALIAN JIAOTONG UNIVERSITY
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