Indian Journal of Biotechnology Vol. 18, July 2019, pp 260-268 Mathematically optimized production, purification and characterization of penicillin G acylase from soil bacterial isolates AA17A and AA17B Abhishek Ajamani 1 Rajesh Kumar 1 , Prachi Bhargava 2 and Siddharth Vats 2 * 1 University Institute of Engineering and Technology, Kurukshetra University, Kurukshetra 136 119; 2 Faculty of Biotechnology, IBST, Shri Ramswaroop Memorial University, Lucknow 225 003 Received 31 January 2019 ; revised 27 March 2019 ; accepted 5 April 2019 This research article deals with production of industrial enzyme penicillin G acylase from soil bacterial isolates namely AA17A and AA17B, which are selected from 80 soil samples. The strains were selected based on qualitative (turbidity) and quantitative (HPLC) test for 6-aminopenicillanic acid (6APA) production. The enzyme was assayed for its activity and optimized for production of enzyme using design of experiments software (DOE) “Design Expert 8.0.7.1”. Optimization of enzyme production of four carbon sources (glucose, glycerol, sucrose and starch), four nitrogen sources (beef extract, tryptone, peptone and yeast extract), for temperature (25°C, 30°C, 35°C and 40°C), four pH (6, 7, 8, 9), four inoculum volumes (2.5 ml, 5.0 ml, 7.5 ml, 10.0 ml) and the phenyl acetic acid (PAA) level (0.15%, 0.17%, 0.185%, 0.2%). The penicillin acylase activity was enhanced to 1.2 fold under following optimized culture conditions: carbon source - glucose (8%), nitrogen source - beef extract (2%), pH 9.0, temperature 30ºC, phenyl acetic acid 0.185%, inoculum volume 5 ml. Approximately 1.22-fold purification from the initial culture broth was achieved during ammonium sulphate precipitation (70-80%) with a yield of 4.6% enzyme. The specific activity of the final partially purified enzyme was 13.73 IU/mg protein. Keywords: Penicillin acylase, mathematical modeling, design of experiments software Introduction Enzymes extracted from microorganisms are applicable in baking, brewing, alcohol production, cheese making, pharmaceuticals and bioethanol based energy sectors. Pharmacy sector is also not untouched by their applications for example enzyme like penicillin acylase amidase (penicillin amidohydrolase). Penicillin acylase was first reported from Penicillium chrysogenum Wisc. Q 176 1 . This enzyme splits benzylpenicillin into phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA) which is used to produce sem-synthetic β-lactam antibiotics. Penicillin acylase is produced by yeast, bacteria and mold. This is a heterodimeric periplasmic protein consisting of a small „a‟ subunit and a large „b‟ subunit, which are formed by processing of a precursor protein. The catalytic nucleophile, a serine, is located at the N-terminus, which is a hallmark of the family of N-terminal nucleophile (Ntn) hydrolases, a class of enzymes which share a common fold around the active site and contain a catalytic serine, cysteine or threonine at the N-terminal position 2 . About 7,500 tons of 6-APA is produced annually and 10-30 tons of immobilized penicillin acylase is consumed. Based on their substrate specificity, penicillin acylases were originally divided into three groups: penicillin G acylases (PGAs), penicillin V acylases (PVAs) and ampicillin acylases. At present, however, ampicillin acylases are classified as α-amino acid ester hydrolases. PGAs preferentially hydrolyze penicillin G and have wide substrate specificity. They were found produced by many bacteria, i.e. Alcaligenes faecalis, Arthrobacter viscosus, Bacillus megaterium, Proteus rettgeri, Pseudomonas melanogenum, Bovista plumbea, Escherichia coli, Kluyvera citrophila, and Providencia rettgeri. Penicillin acylases are of great importance to pharmaceutical industry for their application in the production of semi-synthetic β-lactam antibiotics via the key intermediates, 6-aminopenicillanic acid (6-APA) and 7-amino-3- deacetoxycephalosporanic acid (7-ADCA). In this work, we have isolated penicillin G acylase producing bacteria from soil. Further, the enzyme was for its activity and the production was optimized for enzyme using design of experiments software. Partial purification of enzyme was done followed by enzyme —————— *Author for correspondence: vatssidd@gmail.com Tel: 9140951132