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

Preparation method and application of penicillium genetically engineering bacterium

A technology of genetically engineered bacteria and Penicillium, applied in the field of genetic engineering, can solve problems such as non-expression of genes

Active Publication Date: 2014-02-12
ANHUI LEVEKING BIOTECH CO LTD
View PDF2 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of heterologous glyceraldehyde-3-phosphate dehydrogenase promoters to construct expression vectors is very common, the gene on the vector is not expressed in some fungal receptors

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
  • Preparation method and application of penicillium genetically engineering bacterium
  • Preparation method and application of penicillium genetically engineering bacterium
  • Preparation method and application of penicillium genetically engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Construction of overexpression vector

[0065] See figure 1 , the detailed steps are as follows:

[0066] (1) utilize restriction endonuclease Sac I, Kpn I to hygromycin resistance expression cassette from plasmid PV2 + The fragments were excised by the upper enzyme, and the fragments were recovered by gel, and cloned into the corresponding restriction site of the pCAMBIA2300 plasmid to obtain the recombinant plasmid pCHAMBIA2302 with hygromycin resistance; the hygromycin expression cassette can also be derived from any other DNA containing this sequence or synthesized directly. In addition to pCAMBIA2300, plasmids used to construct PEL gene overexpression vectors can also be used to express plasmids in filamentous fungi, such as pCAMBIA1300, pCAMBIA3300 and other pCAMBIA series vectors and pHB vectors.

[0067] (2) Take 0.2 g of Penicillium extensa mycelium and grind it with liquid nitrogen, add 1 mL of high-salt and low-pH extract, bathe in 65 ° C for...

Embodiment 2

[0074] Embodiment 2: the acquisition of Penicillium genetically engineered bacteria

[0075] The detailed steps are as follows:

[0076] (1) Pick isolated and purified wild-type Penicillium and inoculate it on a PDA plate, culture it at 28° C. for about 20 days, and wash the mature spores with sterile water.

[0077] (2) Inoculate the engineering Agrobacterium EHA105 containing the final vector pCHAMBIA2302::PgpdP-PEL-TtrpC in Example 1 in the LB liquid medium containing 100 μg / mL streptomycin and 100 μg / mL kanamycin at 28° C., 200 rpm Cultivate overnight, reactivate with MM medium containing 100 μg / mL streptomycin and 100 μg / mL kanamycin, and culture at 28°C, 220 rpm for 48 hours. Draw an appropriate amount of culture and centrifuge at 5000rpm to remove the supernatant, wash with IM medium, and finally dilute to OD with IM medium 600 =0.15, then cultured at 28°C and 220rpm for 6-8 hours until OD 600 = 0.5 to 0.6.

[0078] (3) The fresh spores obtained in step (1) are prep...

Embodiment 3

[0084] Embodiment 3: producing lipase ability comparison

[0085] The experiment randomly selected the Penicillium genetically engineered bacterium obtained in Example 2 to inoculate on the PDA medium, and after cultivating for 10 days, insert them into 50 mL of the seed medium, cultivate them on a shaker at 28° C. for 24 hours at 210 rpm, and then transfer them to 10% of the inoculum. Put into the fermentation medium (250mL Erlenmeyer flask with 30mL fermentation medium), ferment at 28°C and 210rpm for 48h; then centrifuge the fermentation broth, and take the supernatant to detect lipase activity.

[0086] Use the acid-base titration method to detect the lipase activity. The detection steps are: take 20 100mL Erlenmeyer flasks, add 4.0mL Gly-NaOH buffer solution of pH 9.4 and 5.0mL olive oil emulsion; Preheat the water bath at 36°C for 5 minutes. After filtering the enzyme solution, dilute it with 0.05mol / L Gly-NaOH buffer solution with pH9.4 to make the enzyme activity 4~5u...

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

No PUM Login to View More

Abstract

The invention discloses a preparation method and an application of a penicillium genetically engineering bacterium. The preparation method comprises the following steps: obtaining a hygromycin resistance expression cassette, cloning the hygromycin resistance expression cassette onto a target plasmid so as to obtain a hygromycin resistance recombinant plasmid; respectively cloning a penicillium lipase gene (PEL), a glyceraldehyde-3-phosphate dehydrogenase promoter PgpdP of penicillium expansum and a aspergillus nidulans tryptophan synthetase terminator TtrPC so as to obtain a PEL gene expression cassette driven by a strong promoter; cloning the PEL gene expression cassette to the hygromycin resistance recombinant plasmid, thus obtaining a hygromycin selection marker containing a PEL gene over-expression vector; and converting the over-expression vector into an engineering agrobacterium, and converting the PEL gene expression cassette to a penicillium strain by using an agrobacterium-mediated transformation method, thus obtaining the penicillium genetically engineering bacterium. The penicillium genetically engineering bacterium obtained by the method has strong lipase production capacity; the enzyme activity of the lipase prepared by the penicillium genetically engineering bacterium is 100%-150% higher than that of a starting strain penicillium wild fungus.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing a Penicillium genetically engineered bacterium, and an application of the Penicillium genetically engineered bacterium in preparing lipase. Background technique [0002] In recent years, lipase (Lipase EC 3.1.1.3) has made great progress in industrial application. Alkaline lipase has the advantages of high substrate hydrolysis efficiency, mild reaction, non-toxicity, and ability to hydrolyze fat under certain conditions. It has been widely used in the fields of detergent, papermaking, tanning, food, textile and light industry And it has become an important variety in the enzyme preparation market all over the world. Lipase can be extracted from animals and plants, and can also be produced by various microorganisms. Due to the variety of microbial lipases, wide sources, short cycle, wider pH, action temperature, and more specific types of subst...

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): C12N15/55C12N15/63C12N1/15C12N9/20C12R1/80
Inventor 张田王剑英唐克轩林智
Owner ANHUI LEVEKING BIOTECH CO LTD
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