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

Recombinant plasmid for constitutive expression of aspergillus niger surface display lipase and application of recombinant plasmid

A technology of constitutive expression and surface display, applied in the direction of recombinant DNA technology, enzymes, hydrolytic enzymes, etc., to achieve the effect of strong operational stability and reduced production costs

Active Publication Date: 2016-12-07
SOUTH CHINA UNIV OF TECH
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when lipase is displayed on the surface of Aspergillus niger mycelium, an inducible promoter is used to induce enzyme production at a lower inducing sugar concentration, and the Aspergillus niger cells displaying lipase need to grow well and maintain the shape of the mycelium itself , There is a natural contradiction in maintaining a high bacterial enzyme activity

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Acquisition of Escherichia coli strains carrying recombinant plasmid pGpdA-CALB-CwpA

[0030] The recombinant plasmid pGpdA‐CALB‐CwpA was transformed on the inducible Aspergillus niger expression vector pCALB‐C. pCALB-C is derived from Aspergillus.Nidulans (Aspergillus nidulans) filamentous fungal dominant selection marker AMDS (gene encoding acetamidase), Aspergillus. Enzyme promoter), Aspergillus oryzae (A. The element, which constitutes the basic skeleton, fuses Candida antarctica lipase B (CALB, GI:515791, 1029bp) with a FLAG tag at the C-terminus and Aspergillusniger endogenous GPI cell wall anchoring protein CwpA (GI:51860184), and connects to Between the promoter Pgla and the terminator TagdA, the plasmid pCALB-C based on CwpA-anchored lipase displayed on the cell surface of Aspergillus niger was obtained. (Pan Z‐Y, Yang Z‐M, Pan L, Zheng S‐P, Han S‐Y, Lin Y. Displaying Candida antarctica lipase Bon the cell surface of Aspergillus niger as a potentia...

Embodiment 2

[0032] Example 2: Construction, cultivation and identification of Aspergillus niger engineering bacteria displaying high-activity lipase

[0033] The -80°C glycerol tube of Aspergillus niger SH‐1 was quickly thawed in a 37°C water bath, and 50 μl of the thawed bacterial liquid was inoculated into a 250ml Erlenmeyer flask containing 50ml of Aspergillus niger CD culture base. Incubate at 34°C for 3‐4 days. Shake the flask regularly in the morning and evening during the cultivation until a large number of small mycelium balls grow in the flask. Transfer the grown Aspergillus niger liquid CD medium into a 100ml sterile centrifuge tube, centrifuge at 8000rpm for 5min, discard the supernatant, add an appropriate amount of sterile saline, and repeatedly blow and inhale until the OD is 2.0 to obtain a mycelial suspension. Inoculate 200 μl of mycelia suspension into a 500 ml Erlenmeyer shaker flask filled with 100 ml of YPD medium. The conditions of 35° C. and 250 rpm were cultivated...

Embodiment 3

[0038] Example 3 Aspergillus niger mycelium surface display CALB lipase activity detection

[0039] Absorbance method can be used to measure the activity of lipase catalyzed by whole cells of Aspergillus niger showing CALB. Glucose fermentation medium after shake flask fermentation for 48 hours, using nitrophenol butyrate as substrate, under the conditions of 45°C and pH=8.0, hydrolyze p-nitrophenol butyrate substrate (C4 substrate), use enzyme Measure the absorbance value at a wavelength of 405nm with a standard instrument, and then calculate the enzyme activity. Enzyme activity unit definition: 1U is equal to the amount of enzyme needed to hydrolyze pNPB to generate 1 μmol p-nitrophenol per minute. The determination steps are as follows:

[0040] (1) Aspergillus niger AN‐GpdA‐CALB‐CwpA fermentation broth was centrifuged at 14000rpm for 2min, the supernatant was discarded, and an equal volume of Tris‐HCl buffer (pH8.0) was added to resuspend the bacteria, mixed well, and ce...

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 recombinant plasmid for constitutive expression of an aspergillus niger surface display lipase and application of the recombinant plasmid. An aspergillus niger surface display carrier pCALB-C serves as a departure plasmid, a constitutive promoter PgpdA substitutes for an inducible glucoamylase promoter Pgla, and accordingly, the recombinant plasmid pGpdA-CALB-CwpA is obtained. The recombinant plasmid transforms escherichia coli to obtain escherichia coli DH5alpha with the plasmid pGpdA-CALB-CwpA; the recombinant plasmid transforms an aspergillus niger protoplast to obtain an engineering bacterium AN-GpdA-CALB-CwpA displaying the lipase CALB on the surface of aspergillus niger; after fermented cultivation of the engineering bacterium, mycelia are collected centrifugally, refrigerated and dried to obtain an aspergillus niger surface display CALB whole-cell catalyst which can be used for efficient compounding of glyceryl phosphatidylcholine.

Description

technical field [0001] The present invention relates to a recombinant plasmid pGpdA-CALB-CwpA capable of constitutively and efficiently expressing CALB lipase in Aspergillus niger, especially the aspergillus niger mutant strain Aspergillus niger, and the surface display of Aspergillus niger obtained after the plasmid is transformed into aspergillus niger The immobilized lipase, and the Aspergillus niger display CALB whole-cell catalyst catalyzes the reaction system of ethanol and various lecithin (PC) to produce glycerol phosphatidylcholine (GPC). Background technique [0002] Lipase has shown better catalytic performance than other enzymes in esterification, hydrolysis, transesterification, and other types of reactions. However, the stability of free lipase to temperature and organic solvents is poor, the separation of reaction products is difficult, and it is difficult to adapt to the needs of different industrial production conditions. Using cell surface display technolo...

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
IPC IPC(8): C12N15/80C12N9/20C12P13/00C12R1/685
CPCC12N9/20C12N15/80C12P7/6481C12P13/001C12Y301/01003C12N2800/10
Inventor 林影韩双艳靳坤潘志友郑穗平梁书利
Owner SOUTH CHINA UNIV OF TECH
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