Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction and application of ganoderma manganese peroxidase pichia pastoris gene engineering strain

A technology of ganoderma manganese peroxide and manganese peroxidase, applied in the field of genetic engineering and molecular biology, can solve the problems of poor broad spectrum, poor environmental adaptability, mineralization, etc., and achieve good activity effect

Inactive Publication Date: 2015-05-20
SHANGHAI JIAO TONG UNIV
View PDF4 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are the following main shortcomings in the prior art: one is that the decolorization is mostly achieved by the adsorption of the bacteria to the dye, and the dye cannot be further degraded and thoroughly mineralized; the other is the lack of efficient degradation strains; most single strains The broad-spectrum of the colony is poor, and it can only act on a few dye molecules with simple structures; and the decolorization rate cannot meet the actual wastewater treatment requirements
Third, the adaptability to the environment is not strong
The salt tolerance or alkali resistance of most strains is not strong, and the growth ability and decolorization and degradation ability of the bacteria decrease rapidly under high salt concentration

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of ganoderma manganese peroxidase pichia pastoris gene engineering strain
  • Construction and application of ganoderma manganese peroxidase pichia pastoris gene engineering strain
  • Construction and application of ganoderma manganese peroxidase pichia pastoris gene engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Expression of MnP in Pichia pastoris

[0040] 1) Obtaining the MnP gene Ganoderma lucidum strain (G. lucidum 50044), which is preserved by the Plant Biotechnology Research Center of Shanghai Jiaotong University. Ganoderma lucidum RNA extraction method refers to Tiangen (TIANGEN) RNA extraction kit.

[0041] Partial fragments of the Ganoderma manganese peroxidase gene were obtained by PCR sequencing, and the intermediate sequence, 5' upstream sequence and 3' downstream sequence of the obtained gene were sequenced on the Vector NTI Suite 8.0 software by RACE cloning, and the splicing results It was shown that a 1337bp fragment was obtained. Then, using the total RNA of Ganoderma lucidum hyphae as a template, primers were designed for RT-PCR amplification, and a specific band of about 1400bp was displayed on the 1.0% agarose gel electrophoresis. Sequencing results showed that the sequence was identical to the spliced ​​sequence, including an open reading frame (ORF) of 1...

Embodiment 2

[0050] Induced expression of MnP in Pichia pastoris

[0051] The constructed recombinant Pichia positive strain was inoculated in yeast glycerol medium BMGY (1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6.0, 1.34% yeast nitrogen source, 4×10 ‐5 % biotin, 1% glycerol) in shake flasks, the filling volume is 10%, cultured at 250r / min at 28°C for 16‐18h, until OD 600 =5, use a sterilized centrifuge tube at room temperature at 3000g for 5min, pour off the supernatant, collect the cell pellet, and use yeast methanol medium BMMY (1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH6. 0, 1.34% yeast nitrogen source, 4 x 10 ‐5 % biotin, 0.5% methanol) to resuspend the cells until OD600=1, add heme (final concentration 1mM) and manganese sulfate (final concentration 0.5mM) for induction. Methanol was added every 24h to a final concentration of 1%, and the fermentation samples on the 2nd, 3d, and 4th days were used for SDS-PAGE detection.

[0052] Th...

Embodiment 3

[0054] Decolorization of Azo Dyes by Crude Protein rGlMnP from Pichia pastoris

[0055] 1) Select 4 kinds of azo dyes Drimaren Blue CL‐BR (Drimaren Lilan CL‐BR), Drimaren Yellow X‐8GN (Drimaren Lihuang X‐8GN), Drimaren Red K‐4BL (Drimaren Red K‐ 4BL), Disperse Navy Blue HGL (disperse dark blue HGL) is used for the decolorization experiment of rGlMnP. (Four dyes were purchased from Shanghai Pingbo Trading Co., Ltd., No. 300, Xuanhua Road, Changning District, Shanghai)

[0056] 2) Use an Evolution 300UV-VIS spectrophotometer (Thermo Scientific) to scan the 150mg / L azo dye solution in the wavelength range of 200-800nm ​​to determine the absorption wavelength of various dyes.

[0057] 3) The decolorization reaction experiment was carried out in a 1mL reaction system. The main steps were: take 390μL of 100mmol / L malonate‐sodium malonate (pH 4.5), 1mmol / L MnSO 4 100μL, 400μL of 500mg / L dye solution, 100μL of crude protein containing MnP in a 2mL EP tube. The MnP enzyme solution i...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention provides construction and application of a pichia pastoris gene engineering strain in the field of molecular biology and gene engineering. A manganese peroxidase gene GlMnP is cloned from white rot fungus ganoderma, a pichia pastoris expression vector Pao815-GlMnP of an expression cassette containing the manganese peroxidase gene is constructed, the constructed pichia pastoris expression vector is used for converting pichia pastoris (P. pastoris) SMD1168H, and the pichia pastoris engineering strain of the expression cassette containing the manganese peroxidase gene with the expression vector highly copied and recombined by ampicillin resistance screening. The engineering strain is constructed and a biological enzyme is industrially produced in the invention to remove azo dyes in such systems as production, sewage and the like, for example, decoloration of textile industrial wastewater, printing and dyeing industrial wastewater and industrial wastewater related to production and application of dyes.

Description

technical field [0001] The invention relates to a technique in the fields of molecular biology and genetic engineering, in particular to the construction of a Pichia pastoris genetically engineered strain and the use of the strain in the production of manganese peroxidase. Background technique [0002] Manganese peroxidase (Manganese peroxida, MnP) is a class of glycoproteins, composed of a series of glycosylated isoenzymes containing a Fe(s)-porphyrin ring (IX) heme prosthetic group, the isoelectric point 4.2-4.5, the molecular weight of MnP produced by fungi is 38-62.5kDa, and the molecular weight of most purified enzymes is about 45kDa, and the enzyme activity center is composed of a heme group and a MnP 2+ composition, there are two Ca 2+ , whose molecule consists of ten long protein single chains and one short single chain. Its activity is not only dependent on H 2 o 2 , also depends on Mn 2+ The presence of , can decompose aromatic ring polymers under in vitro con...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N9/02C02F3/00
Inventor 周选围徐慧郭梦圆白晓慧
Owner SHANGHAI JIAO TONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com