caa a22 4500 606213287 CHVBK 20210128101046.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s10295-015-1690-x doi (NATIONALLICENCE)springer-10.1007/s10295-015-1690-x Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases [Elektronische Daten] [Priyabrata Panigrahi, Deepak Chand, Ruchira Mukherji, Sureshkumar Ramasamy, C. Suresh] Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in β-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme. Society for Industrial Microbiology and Biotechnology, 2015 Thermostability nationallicence Penicillin acylase nationallicence Consensus approach nationallicence Alkalistable nationallicence Ion pairs nationallicence β-lactam antibiotics nationallicence Disulfide bridge nationallicence Panigrahi Priyabrata Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut Chand Deepak Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut Mukherji Ruchira Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut Ramasamy Sureshkumar Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut Suresh C. Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/11(2015-11-01), 1493-1506 1367-5435 42:11<1493 2015 42 10295 https://doi.org/10.1007/s10295-015-1690-x 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/s10295-015-1690-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Panigrahi Priyabrata Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut NATIONALLICENCE 700 1- Chand Deepak Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut NATIONALLICENCE 700 1- Mukherji Ruchira Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut NATIONALLICENCE 700 1- Ramasamy Sureshkumar Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut NATIONALLICENCE 700 1- Suresh C. Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008, Pune, India aut NATIONALLICENCE 773 0- Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/11(2015-11-01), 1493-1506 1367-5435 42:11<1493 2015 42 10295