Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability

Metabolic Rearrangements Causing Elevated Proline and Polyhydroxybutyrate Accumulation During the Osmotic Adaptation Response of Bacillus megaterium

“…Bacillus megaterium is a big rod-shaped Gram-positive soil bacterium that has been used industrially for decades and whose product portfolio is continuously growing. It includes enzymes such as αand β-amylases, penicillin G acylase, xylanase, hydrolases, glycosyltransferases, and cytochrome monooxygenases (Vary et al, 2007;Korneli et al, 2013;Lakowitz et al, 2017;Mayer et al, 2019). The bacterium displays several important biotechnological features such as its non-pathogenic character, broad substrate spectrum, high plasmid stability, and high secretion capacity for proteins.…”

Structure-Properties Correlation of Cross-Linked Penicillin G Acylase Crystals

“…However, the BmPGA CLECs show only slightly lower mechanical hardness, although BmPGA, with a melting point at 56.8 • C, is significantly less thermostable than FJAT-PGA (73.3 • C). In contrast, BtPGA shows intermediate thermostability (64.5 • C) but the lowest mechanical stability of the measured PGA CLECs [6]. This might be explained by a differing influence of intraand intermolecular interactions.…”

“…FJAT-and BtPGA, which were recently identified, as well as the already known BmPGA, were recombinantly produced and secreted with P. megaterium MS941 via plasmids pRBBm316, pRBBm311 and pALBm1, respectively [6,18,19]. For each, LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) supplemented with 10 mg/L tetracycline and 2.5 mM CaCl 2 (Bt-and BmPGA) was inoculated with an overnight culture and cultivated at 37 • C and 200 rpm in baffled flasks.…”

“…FJAT-and BmPGA were purified by cation and BtPGA by anion exchange chromatography from the cell-free supernatant followed by preparative gel permeation chromatography, as described previously [6]. Fractions with the respective PGAs were combined and concentrated using Amicon centrifugal filters MWCO 10,000 (Merck Millipore, Darmstadt, Germany).…”

“…FJAT-27231 (FJAT-PGA) were discovered. Compared to commonly used PGAs from P. megaterium and E. coli, they show significantly improved thermal stability and activity for the hydrolysis of penicillin G yielding 6-APA [6], making them promising candidates for use in a potential biocatalytic process to obtain precursor molecules for synthesis of semisynthetic antibiotics. Their enzymatic synthesis is usually preferred to chemical synthesis due to efficiency and selectivity of enzymes under mild reaction conditions without hazardous chemicals and solvents.…”