Method for increasing thermal stability of polymer protein and alcohol dehydrogenase with increased thermal stability

A thermostable, alcohol dehydrogenase technology, applied in the field of enzyme engineering, can solve problems such as low protein stability and inactivation, and achieve the effects of improving stability, avoiding cumbersome processes and broad market prospects.

Inactive Publication Date: 2018-10-23
WUHAN UNIV
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, despite the advantages of high selectivity and catalytic activity, proteins usually have low stability under non-p

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
  • Method for increasing thermal stability of polymer protein and alcohol dehydrogenase with increased thermal stability
  • Method for increasing thermal stability of polymer protein and alcohol dehydrogenase with increased thermal stability
  • Method for increasing thermal stability of polymer protein and alcohol dehydrogenase with increased thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] This embodiment is used to illustrate the above steps (1) by finding a protein with higher homology with alcohol dehydrogenase LnADH (SEQ ID NO.1) in the PDB database, and then using it as a template to perform homology modeling; by The protein structure was analyzed to find out the mutation site located at the N-terminal of the tetrameric protein LnADH that can form a disulfide bond on interface A. The specific method is as follows: by searching the protein database RCSB database, after blasting comparison, find the protein 4URE (PDB number, 49% homology) with high homology to the alcohol dehydrogenase, use the software Discovery Studio 4.0 to use 4URE as a template, Homology modeling of this alcohol dehydrogenase. Through the analysis of its structure, it is found that the N-terminal distance of the two subunits on the A interface is very close, only distance. Then several ways of inserting Cys at the N-terminus were devised: a. CysGlyGlySerGly amino acid sequence ...

Embodiment 2

[0046]This example is used to illustrate the above step (2) by using protein disulfide bond prediction software to find out the mutation site capable of forming a disulfide bond on the interface B of the tetrameric protein alcohol dehydrogenase LnADH. The specific method is: calculate and predict the possible disulfide bond introduction sites through the "site-directed mutagenesis" online program of the DSDBASE database (http: / / caps.ncbs.res.in / dsabase / / mainFrame.html). Through screening and exclusion, 6 groups of mutation sites with relatively high scores that can form disulfide bonds on interface B of the tetrameric protein were finally obtained, namely Val101Cys / Ala171Cys (V101C / A171C), Thr159Cys / Ala163Cys (T159C / A163C) , Thr159Cys / Leu167Cys (T159C / L167C), His162Cys (H162C), Ser155Cys / Asn170Cys (S155C / N170C), Thr100Cys / Glu174Cys (T100C / E174C).

Embodiment 3

[0048] This example is used to illustrate the above step (3) to construct a mutant that forms a disulfide bond on the contact interface A of the LnADH subunit of alcohol dehydrogenase and to verify its thermal stability. The specific method is as follows: design primers according to the GenBank accession number of alcohol dehydrogenase LnADH gene sequence (ERK72999.1), and obtain the alcohol dehydrogenase gene through PCR amplification. The test methods used include molecular cloning techniques such as PCR technology, DNA extraction, enzyme digestion, and enzyme linkage. Using Leifsonia aquatica ATCC 14665 genomic DNA as a template, the gene sequence of alcohol dehydrogenase LnADH was amplified, and this target gene was connected to the plasmid pET28aNdeI-HindIII site, and then transformed into Escherichia coli BL21 (DE 3 ) was expressed to determine its enzymatic activity. The specific process is as follows:

[0049] 1) The cultivation of alcohol dehydrogenase bacterial str...

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 method for increasing thermal stability of polymer protein and alcohol dehydrogenase with increased thermal stability, which belong to the technical field of enzyme engineering. The method for increasing the thermal stability of polymer protein is characterized in that a covalent bond mode is introduced into a contact interface of terminal N of polymer protein with adjacent N terminals (in a scope of 8 angstrom) and an internal subunit, and the subunits of polymer protein are linked to form a cyclized integral body. The sequences of alcohol dehydrogenase with increased thermal stability are respectively SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, compared with a wild type, a T5015 value of the alcohol dehydrogenase can be respectively increased by 5.5 DEG C, 6.0 DEGC and 18 DEG C. The method avoids the problems of large screening work amount and tedious processes in the prior art, and site-specific mutagenesis has pertinency by a strategy introduced through a disulfide bond between interfaces.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a method for improving the thermostability of multimeric proteins, and an alcohol dehydrogenase with improved thermostability obtained by the method. Background technique [0002] Enzymes are also regarded as functional proteins that can catalyze chemical reactions under very mild conditions and have high selectivity. Because of the green, economical and sustainable advantages, enzymes are favored in industrial applications (U.T. Bornscheuer et al. Nature, 2012, 485: 185-194). In addition, in the field of pharmacy, some proteins such as antibodies are widely used for disease treatment due to their very high selectivity (R.V.J.Chari et al. Angew. Chem. Int. Ed., 2014, 53: 3796-3827). However, despite the advantages of high selectivity and catalytic activity, proteins usually have low stability under non-physiological conditions, which easily leads to inacti...

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): C12N9/04C12N15/53
CPCC12N9/0006
Inventor 瞿旭东朱露马宏敏邓子新
Owner WUHAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap