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

Application of phenylpyruvate decarboxylase mutant M538A in biological fermentation production of phenethyl alcohol

A technology of phenylpyruvate decarboxylase and biological fermentation, which is applied in the field of enzyme engineering and genetic engineering, can solve the problems of low catalytic efficiency and low substrate specificity, and achieve the goal of improving catalytic efficiency, improving enzyme production ability, and improving enzyme activity Effect

Active Publication Date: 2019-10-15
HUBEI UNIV
View PDF4 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to modify certain genes involved in metabolism or biology called pyrolysis bacteria's gene product pigment precursor pathway system(PPR). By modifying specific parts of this process, they can improve their ability to produce various chemical compounds such as ethanols from other organisms without losing any useful properties like photosynthesis or cell division.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the performance or applicability of certain types of chemical compounds called phenylacetosides because they are difficult to manufacture industrially without generating unwanted side effects like harmful volatile organics during their preparation process. To address this issue, various methods were developed including gene manipulation techniques, molecular biology approaches, and synthetic chemistry tools.

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
  • Application of phenylpyruvate decarboxylase mutant M538A in biological fermentation production of phenethyl alcohol
  • Application of phenylpyruvate decarboxylase mutant M538A in biological fermentation production of phenethyl alcohol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Construction of recombinant plasmid pHY-P43-kivD:

[0022] An α-ketoacid decarboxylase kivD gene was cloned from Lactococcus lactis subsp.lactis (CICC6246) by using KivD-F and KivD-R. Using the Bacillus subtilis 168 genome as a template, the P43 promoter was amplified with P43-F and P43-R primers; the amylase terminator TamyL was amplified with TamyL-F and TamyL-R primers. P43-F and TamyL-R were used as primers to perform SOE-PCR on the three fragments of P43, kivD and TamyL to obtain the fusion fragment P43-kivD-TamyL. Using the plasmid pHY300PLK as a template and pHY-T5-F and pHY-T5-R as primers, the whole plasmid was amplified by PCR to obtain a linearized pHY300PLK vector. After the above amplified products were checked by electrophoresis, the PCR products were purified and recovered using a gel recovery kit. Using the ClonExpress II one-step cloning kit, the fusion fragment was fused with the linearized vector pHY300PLK to obtain the recombinant plasmid pHY-P43-k...

Embodiment 2

[0033] Preparation and enzyme activity determination of phenylpyruvate decarboxylase wild bacteria (WT):

[0034] The plasmid pHY-P43-kivD sequenced correctly in Example 1 was transformed into Bacillus licheniformis DW2. Select the transformant and inoculate it into LB medium after verifying that it is correct, culture it at 37°C for 14 hours; transfer it to the fermentation medium with an inoculum of 3%, culture it at 37°C for 24 hours, collect the bacteria by centrifugation, wash the bacteria twice with PBS, Finally, resuspend the cells with 1 ml of 50 mM potassium phosphate buffer (pH 6.8). Cells were disrupted using an ultrasonic breaker. Ultrasonic settings: 150W, 20kHz, working for 2s; off for 2s, a total of 8 minutes, centrifuged at 4°C to collect the supernatant, which was the crude enzyme solution.

[0035] The supernatant was tested for enzyme activity according to the following system, enzymatic reaction system (200μL): 50mM potassium phosphate buffer (pH 6.8), 1mM...

Embodiment 3

[0037] Preparation of Phenylpyruvate Decarboxylase Mutants

[0038] Using the constructed pHY-P43-kivD as a template, primers were designed for PCR amplification of the whole plasmid to obtain a linearized pHY300PLK vector with P43 promoter and amylase TamyL terminator. Perform site-directed mutation on the catalytic active site of kivD, and replace the mutation on the gene sequence by means of primers, specifically:

[0039] 1) Mutate the valine at position 461 of the catalytic domain of the phenylpyruvate decarboxylase molecule to isoleucine, split the base GTC encoding the amino acid at position 461 of kivD into two parts, primers V461I-AR and V461I -BF, using the plasmid in Example 1 as a template to amplify the upper and lower sections of the kivd mutant sequence; then use kivD-F and kivD-R as primers to perform SOE-PCR on the two sections to amplify the kivD mutant sequence V461I;

[0040] 2) Mutate the 538th methionine to alanine, split the base GCG encoding the 538th ...

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 belongs to the technical field of gene engineering and enzyme engineering, and discloses an application of a phenylpyruvate decarboxylase mutant M538A in the biological fermentation production of phenethyl alcohol. Through a site-directed mutagenesis way, 538th-site methionine which is from Lactococcus lactis subsp. Lactis of which the preservation number is CICC6246 and is near a phenylpyruvate decarboxylase KivD molecular catalytic structural domain is mutated into alanine. The enzyme activity of the phenylpyruvate decarboxylase is obviously improved, the problem that the existing phenylpyruvate decarboxylase has low catalytic efficiency for phenylpyruvic acid can be solved, the phenylpyruvate decarboxylase mutant M538A is used for the biological fermentation of phenethyl alcohol, a new thought is provided for the production of the phenethyl alcohol, and the phenylpyruvate decarboxylase mutant M538A is suitable for large-scale promotion.

Description

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

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
Owner HUBEI 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