BIOMAT Database Logo BIOMAT Database Logo

Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus. 2013

Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Faculty of Life and Environmental Sciences, University of Tsukuba.

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
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
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
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
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005819 Genetic Markers A phenotypically recognizable genetic trait which can be used to identify a genetic locus, a linkage group, or a recombination event. Chromosome Markers,DNA Markers,Markers, DNA,Markers, Genetic,Genetic Marker,Marker, Genetic,Chromosome Marker,DNA Marker,Marker, Chromosome,Marker, DNA,Markers, Chromosome
D006358 Hot Temperature Presence of warmth or heat or a temperature notably higher than an accustomed norm. Heat,Hot Temperatures,Temperature, Hot,Temperatures, Hot
D006921 Hygromycin B Aminoglycoside produced by Streptomyces hygroscopicus. It is used as an anthelmintic against swine infections by large roundworms, nodular worms, and whipworms.
D000900 Anti-Bacterial Agents Substances that inhibit the growth or reproduction of BACTERIA. Anti-Bacterial Agent,Anti-Bacterial Compound,Anti-Mycobacterial Agent,Antibacterial Agent,Antibiotics,Antimycobacterial Agent,Bacteriocidal Agent,Bacteriocide,Anti-Bacterial Compounds,Anti-Mycobacterial Agents,Antibacterial Agents,Antibiotic,Antimycobacterial Agents,Bacteriocidal Agents,Bacteriocides,Agent, Anti-Bacterial,Agent, Anti-Mycobacterial,Agent, Antibacterial,Agent, Antimycobacterial,Agent, Bacteriocidal,Agents, Anti-Bacterial,Agents, Anti-Mycobacterial,Agents, Antibacterial,Agents, Antimycobacterial,Agents, Bacteriocidal,Anti Bacterial Agent,Anti Bacterial Agents,Anti Bacterial Compound,Anti Bacterial Compounds,Anti Mycobacterial Agent,Anti Mycobacterial Agents,Compound, Anti-Bacterial,Compounds, Anti-Bacterial

Related Publications

Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.
January 1987, European journal of biochemistry,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.
February 1986, Nucleic acids research,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Molecular cloning and expression in streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.
November 1983, Biochemical and biophysical research communications,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.
January 1987, European journal of biochemistry,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Enzymatic modification of hygromycin B in Streptomyces hygroscopicus.
April 1982, The Journal of antibiotics,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
An O-phosphotransferase catalyzes phosphorylation of hygromycin A in the antibiotic-producing organism Streptomyces hygroscopicus.
October 2008, Antimicrobial agents and chemotherapy,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Biochemical basis of resistance to hygromycin B in Streptomyces hygroscopicus--the producing organism.
June 1985, Journal of general microbiology,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus.
August 2005, Journal of bioscience and bioengineering,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
Pentalenolactone I and hygromycin A, immunosuppressants produced by Streptomyces filipinensis and Streptomyces hygroscopicus.
May 2001, Bioscience, biotechnology, and biochemistry,
Naohisa Sugimoto, and Yasuaki Takakura, and Kentaro Shiraki, and Shinya Honda, and Naoki Takaya, and Takayuki Hoshino, and Akira Nakamura
[Morphological and biochemical characteristics of Streptomyces hygroscopicus mutants induced by streptomycin and hygromycin B].
November 1980, Antibiotiki,
Copied contents to your clipboard!