Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures

Applied Microbiology and Biotechnology
Jyumpei KobayashiHirokazu Suzuki

Abstract

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance ...Continue Reading

References

May 1, 1989·Journal of General Microbiology·L J Wu, N E Welker
Feb 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·H LiaoR Hageman
Jun 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·A G LeslieW V Shaw
May 25, 2002·Journal of Bacteriology·Olafur FridjonssonRalf Mattes
Jan 11, 2005·The Journal of Biological Chemistry·Stan J J BrounsJohn van der Oost
Jun 9, 2005·Journal of Molecular Biology·D Allan DrummondFrances H Arnold
Jul 12, 2005·Current Opinion in Structural Biology·Jesse D BloomFrances H Arnold
Mar 6, 2010·Current Protein & Peptide Science·Nikolaos E Labrou
Feb 2, 2012·Protein Science : a Publication of the Protein Society·Tapan BiswasOleg V Tsodikov
Jul 21, 2012·Journal of Microbiology and Biotechnology·Hirokazu Suzuki, Ken-ichi Yoshida
Aug 14, 2012·Applied and Environmental Microbiology·Hirokazu SuzukiKen-ichi Yoshida
Jun 25, 2013·Applied and Environmental Microbiology·Hirokazu SuzukiToshihisa Ohshima
Sep 3, 2013·Journal of Biotechnology·Sameh Ben MabroukSamir Bejar
Nov 10, 2013·Bioscience, Biotechnology, and Biochemistry·Hirokazu SuzukiToshihisa Ohshima
Aug 6, 2014·Journal of Biotechnology·Dawn Elizabeth StephensKugen Permaul
Oct 19, 2014·Applied and Environmental Microbiology·Hirokazu SuzukiKatsumi Doi

❮ Previous
Next ❯

Citations

Sep 4, 2015·Bioscience, Biotechnology, and Biochemistry·Keisuke WadaHirokazu Suzuki
Jun 23, 2016·ACS Synthetic Biology·Benjamin ReeveTom Ellis
Nov 26, 2019·Protein Science : a Publication of the Protein Society·Ashley AlcalaMisty L Kuhn
Oct 13, 2018·Applied Microbiology and Biotechnology·Hirokazu Suzuki
Sep 29, 2018·Molecular Biotechnology·Angelica Jimenez-Rosales, Miriam V Flores-Merino
Jan 20, 2018·The Journal of Antibiotics·Hirokazu SuzukiTakashi Ohshiro
Nov 9, 2019·Extremophiles : Life Under Extreme Conditions·Ryota KurashikiHirokazu Suzuki
Apr 13, 2021·Frontiers in Microbiology·Hirokazu SuzukiTakashi Ohshiro

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.