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

Genetically engineered bacteria producing D-pantolactone hydrolase as well as construction method and application of genetically engineered bacteria

A technology of pantolactone and genetically engineered bacteria is applied in the field of construction of genetically engineered bacteria, which can solve the problems of low expression of D-pantolactone hydrolase and achieve the effect of efficient hydrolysis

Pending Publication Date: 2019-03-12
JIANGNAN UNIV
View PDF3 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention solves the problem of low expression of D-pantolactone hydrolase in the prior art

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
  • Genetically engineered bacteria producing D-pantolactone hydrolase as well as construction method and application of genetically engineered bacteria
  • Genetically engineered bacteria producing D-pantolactone hydrolase as well as construction method and application of genetically engineered bacteria
  • Genetically engineered bacteria producing D-pantolactone hydrolase as well as construction method and application of genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A genetically engineered bacterium producing D-pantolactone hydrolase, the construction method comprising the steps of:

[0038] (1) Construction of cloning vector pMD19-T-DL

[0039] The cDNA sequence of D-pantolactone hydrolase (GenBank: AY728018.1) was found on NCBI, and sent to Suzhou Jinweizhi Biological Co., Ltd. for optimized synthesis according to the codons preferred by yeast to obtain optimized D-pantolactone hydrolase Acid lactone hydrolase gene (DL), the DL sequence is shown in SEQ ID NO:1. Using the synthetic gene (DL) as a template, PCR amplification was carried out with the designed primers, and the results were as follows: figure 1 shown. The amplified product was connected with pMD19-T-Simple Vector, the cloning vector pMD19-T-DL was constructed and transformed into E.coli JM109 competent, the positive transformant was screened by ampicillin resistance plate, and the recombinant plasmid was extracted by alkaline cleavage method. For specific methods, ...

Embodiment 2

[0055] Embodiment 2: the enzyme production culture of genetically engineered bacteria

[0056] Preparation of seed solution: Take 200 μL of the preserved bacterial solution in YPD liquid medium, cultivate at 30°C, 200 r / min for 36 hours, and use it as the primary seed. In a 250mL Erlenmeyer flask containing 50mL of YPD liquid medium, inoculate primary seeds at a transfer rate of 4% (volume ratio), and cultivate them at 30°C and 200r / min for 36h as secondary seeds.

[0057] Shake flask enzyme production culture: medium components are sucrose 40g / L, peptone 20g / L, beef extract 15g / L, NaCl 6g / L, KCl 6g / L, CaCl 2 3g / L, MgSO 4 3g / L, 30℃, 200r / min fermentation time 85h

[0058] Determination of biomass: use the dry weight of the bacteria contained in the fermentation broth per unit volume (L) for a certain period of time to represent the growth of the bacteria, separate the fermentation broth at 10000r / min for 5min to obtain the bacteria, and wash with sterile deionized water fo...

Embodiment 3

[0061] Embodiment 3: the application of genetically engineered bacteria

[0062] (1) Preparation of immobilized enzyme

[0063] Put an appropriate amount of pretreated resin D380 into a 50mL Erlenmeyer flask, add a crude enzyme solution with a total enzyme activity of 12-80U per gram of resin, then place it in a shaker, and absorb it at 20-35°C at 100-200r / min for 2 ~6h, pre-cool at 4°C, then add glutaraldehyde to a final concentration of 0.1%~1%, crosslink for 0.5~3h, and finally wash repeatedly with deionized water to remove free enzyme. Or put an appropriate amount of pretreated resin D380 into a packed bed reactor such as Figure 6 As shown, use crude enzyme solution (total enzyme activity per gram of resin is 12-80U) for cyclic adsorption, 2-6h, pre-cool at 4°C, and then use 0.1%-1% glutaraldehyde for cyclic cross-linking for 0.5-3h, Finally, the free enzymes were removed by repeated washing with deionized water.

[0064] (2) Hydrolysis of DL-pantolactone by immobilize...

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 genetically engineered bacteria for producing D-pantolactone hydrolase. The construction method of the engineered bacteria comprises the following steps: transferring the encoding genes of the D-pantolactone hydrolase into Kluyveromyces lactis cells as a host to prepare the engineered bacteria; the engineered bacteria contain the nucleotide fragments of a recombinant vectorpZL505-DL, the DL sequence is as shown in SEQ ID NO: 1. By the genetically engineered bacteria, the problem in the prior art that the expression quantity of the D-pantolactone hydrolase is low is solved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a construction method and application of a genetically engineered bacterium producing D-pantolactone hydrolase. Background technique [0002] With the continuous deepening of theoretical research on molecular biology and the continuous innovation of molecular biological methods, genetic engineering technology has developed rapidly and achieved remarkable results. As an important content of genetic engineering technology, gene expression technology has penetrated into various fields of industry, agriculture and life science, and has been widely valued by people. According to the different hosts for exogenous gene expression, at present, the gene expression systems that have been developed include Escherichia coli expression system, mammalian expression system, yeast expression system and so on. Among them, the yeast expression system has the advantages of simple opera...

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): C12N1/19C12N15/55C12N15/81C12N9/18C12P41/00C12R1/645
CPCC12N9/18C12N15/815C12P41/001
Inventor 张梁辛瑜王开放王大伟顾正华
Owner JIANGNAN 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