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

Methionine lyase, as well as encoding gene and biosynthesis method thereof

一种甲硫氨酸、编码基因的技术，应用在甲硫氨酸裂解酶领域，能够解决制约真核微生物发酵微生物功能、原核基因难高效表达等问题

Inactive Publication Date: 2015-09-23
HUBEI UNIV OF TECH
View PDF1 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the problem of codon preference, prokaryotic genes are difficult to express efficiently in eukaryotic fermenting microorganisms, which restricts the improvement of the functions of eukaryotic microorganisms, especially some food-related fermenting microorganisms

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
  • Methionine lyase, as well as encoding gene and biosynthesis method thereof
  • Methionine lyase, as well as encoding gene and biosynthesis method thereof
  • Methionine lyase, as well as encoding gene and biosynthesis method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of methionine lyase gene STR3 and construction of expression vector

[0046] (1) Based on the conservativeness of the amino acid sequence of methionine lyase and the degeneracy of codons, the conservative sequence of the methionine lyase gene in Nigrosporium truffles was cloned homologously, and 5 of the conservative sequence was amplified by RACE technology. At the'and 3'ends, NCBI Blast was performed after the sequence was spliced, and it was found that the amino acid level identity with the homologous protein in the model strain was 78%. Its nucleotide sequence is shown in SEQ ID NO.1, and its deduced amino acid sequence is shown in SEQ ID NO.2.

[0047] (2) Extraction of expression plasmid pYES2

[0048] Take 1 test tube containing 10mL LB (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, ampicillin 100mg / L) medium, and inoculate E.coli Top10 containing pYES2 plasmid into the test tube Incubate overnight at 37°C and 180 rpm, and extract the plasmid accor...

Embodiment 2

[0063] Example 2 Expression and purification of methionine degrading enzyme

[0064] (1) Preparation of yeast competent cells

[0065] Activate the preserved Saccharomyces cerevisiae INVSc1 on YPD solid medium, transfer three times, inoculate it into YPD liquid medium, 30℃, 200rpm, shake culture to OD 600 =1.0, collect the cells, wash the cells with pre-cooled ultrapure water, and resuspend them in 200 μL of 1M sorbitol solution to prepare competent cells of Saccharomyces cerevisiae.

[0066] YPD medium formula: Yeast Extract 10g, Trypton 20g, autoclaved at 121°C for 20 minutes, and 2% (w / v) glucose was added.

[0067] (2) Transformation of Saccharomyces cerevisiae overexpression STR3 plasmid

[0068] Extract the pYES2-STR3 plasmid in E.Coli Top10, transfer the extracted plasmid into Saccharomyces cerevisiae competent, mix gently, add to the pre-cooled 0.2cm electrode cup, tap gently on the ultraclean table to mix Flow to the bottom of the electrode cup and ice bath for 5 minutes. Pla...

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 methionine lyase, as well as an encoding gene and a biosynthesis method thereof. A gene for encoding the methionine lyase of which the sequence is as shown in SEQ ID No.1 is separated from tuber melanosporum. The invention further provides an efficient biosynthesis method for the methionine lyase. The efficient biosynthesis method comprises the following steps: (1) cloning the gene, of which the sequence is as shown in SEQ ID.1, for encoding the methionine lyase in a yeast expression vector to construct a recombinant yeast expression vector; (2) converting the recombinant yeast expression vector in brewer's yeast to obtain an expression strain; (3) inducing the expression strain to express the methionine lyase, collecting a thallus after induced expression, and purifying the expressed recombinant methionine lyase. The purity of the recombinant methionine lyase prepared according to the biosynthesis method is 90% or higher, and the methionine degrading efficiency can reach 0.53 + / - 0.0030 [mu]M MTL.h<-1>.mg protein<-1>.

Description

Technical field [0001] The present invention relates to a methionine lyase, in particular to a methionine lyase derived from fungi and its encoding gene. The invention further relates to a biosynthetic method for obtaining a methionine lyase, belonging to methionine Lyase and its biosynthesis field. Background technique [0002] Methionine lyase has a wide range of industrial and medical uses. In the industrial field, methionine lyase uses methionine as a substrate to directly catalyze the synthesis of methyl mercaptan and its derivatives dimethyl sulfide, dimethyl disulfide and dimethyl trisulfide, which contain sulfur. Compounds are important food flavor additives, mainly used in the preparation of corn, tomato, potato, dairy products, pineapple and orange fruity and green flavors (Martinez-Cuesta Mdel, C., Pelaez, C., Requena, T. ,2013.Methionine metabolism:major pathways and enzymes involved and strategies for control and diversification of volatile sulfur compounds in chee...

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): C12N15/60C12N15/81C12N9/88C12N1/19C12R1/865
CPCC12N9/88C12N15/81C12Y404/01011G01N27/447G01N30/50G01N30/80
Inventor 汤亚杰贾开志徐阳华张权李红梅
Owner HUBEI 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