Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis. 2020

Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

UI MeSH Term Description Entries
D007218 Industrial Microbiology The study, utilization, and manipulation of those microorganisms capable of economically producing desirable substances or changes in substances, and the control of undesirable microorganisms. Microbiology, Industrial
D007700 Kinetics The rate dynamics in chemical or physical systems.
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D004220 Disulfides Chemical groups containing the covalent disulfide bonds -S-S-. The sulfur atoms can be bound to inorganic or organic moieties. Disulfide
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D005968 Glutamate Decarboxylase A pyridoxal-phosphate protein that catalyzes the alpha-decarboxylation of L-glutamic acid to form gamma-aminobutyric acid and carbon dioxide. The enzyme is found in bacteria and in invertebrate and vertebrate nervous systems. It is the rate-limiting enzyme in determining GAMMA-AMINOBUTYRIC ACID levels in normal nervous tissues. The brain enzyme also acts on L-cysteate, L-cysteine sulfinate, and L-aspartate. EC 4.1.1.15. Glutamate Carboxy-Lyase,Glutamic Acid Decarboxylase,Acid Decarboxylase, Glutamic,Carboxy-Lyase, Glutamate,Decarboxylase, Glutamate,Decarboxylase, Glutamic Acid,Glutamate Carboxy Lyase
D013696 Temperature The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms. Temperatures
D013816 Thermodynamics A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed) Thermodynamic
D016297 Mutagenesis, Site-Directed Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion. Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses

Related Publications

Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
April 2019, Sheng wu gong cheng xue bao = Chinese journal of biotechnology,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
May 2008, Bioscience, biotechnology, and biochemistry,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
July 1997, Bioscience, biotechnology, and biochemistry,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
March 2018, World journal of microbiology & biotechnology,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
September 2018, Biochemical and biophysical research communications,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
January 2014, Enzyme and microbial technology,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
January 2013, Bioscience, biotechnology, and biochemistry,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
February 2014, Protein expression and purification,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
November 2013, Journal of industrial microbiology & biotechnology,
Yujiao Hua, and Changjiang Lyu, and Chunyan Liu, and Hongpeng Wang, and Sheng Hu, and Weirui Zhao, and Jiaqi Mei, and Jun Huang, and Lehe Mei
September 2006, Biotechnology letters,
Copied contents to your clipboard!