caa a22 4500 605505934 CHVBK 20210128100624.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s00253-015-6522-4 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6522-4 Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures [Elektronische Daten] [Jyumpei Kobayashi, Megumi Furukawa, Takashi Ohshiro, Hirokazu Suzuki] 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 (CATA138T). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CATA138T 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 CATA138S and CATA138V, 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. CATA138T conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CATA138T may be useful as a genetic marker in Geobacillus spp. Notably, CATA138T generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes. Springer-Verlag Berlin Heidelberg, 2015 Geobacillus kaustophilus nationallicence Chloramphenicol resistance nationallicence Chloramphenicol acetyltransferase nationallicence Thermoadaptation-directed evolution nationallicence Error-prone thermophile nationallicence Kobayashi Jyumpei Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 680-8552, Tottori, Japan aut Furukawa Megumi Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, 812-8581, Fukuoka, Japan aut Ohshiro Takashi Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 680-8552, Tottori, Japan aut Suzuki Hirokazu Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 680-8552, Tottori, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/13(2015-07-01), 5563-5572 0175-7598 99:13<5563 2015 99 253 https://doi.org/10.1007/s00253-015-6522-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6522-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kobayashi Jyumpei Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 680-8552, Tottori, Japan aut NATIONALLICENCE 700 1- Furukawa Megumi Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, 812-8581, Fukuoka, Japan aut NATIONALLICENCE 700 1- Ohshiro Takashi Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 680-8552, Tottori, Japan aut NATIONALLICENCE 700 1- Suzuki Hirokazu Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 680-8552, Tottori, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/13(2015-07-01), 5563-5572 0175-7598 99:13<5563 2015 99 253