Penicillin amidase

About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.

IS2-mediated re-arrangement of the promoter sequence suppresses metabolic burden of the recombinant plasmid

Abstract: Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase (‘penicillin G acylase’) bears the 3′ end region of IS2 element. The IS2 sequence replaces the −35 region of promoter ofpga and extends up to TAGTAT box at position −10 of the promoter region. It therefore forms a hybrid promoter ofpga ppgaHT. A natural promoterppgaWT was not detected on any recombinant plasmid isolated from recombinant strains ofEscherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity ofppgaWT was 5× higher than that ofppgaHT. The same nucleotide “G” localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place thepga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with highpga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.

Purification of Penicillin G Amidase using Quaternary Ammonium Salts and effect on the activity of the immobilised enzymes

Abstract: This paper describes the purification of Penicillin G Amidase (EC 3.5.1.11) using quaternary ammonium salts with the aim of increasing the activity of immobilised enzymes prepared from the purified solutions. Two different quaternary ammonium salts were tested with different solutions of the enzyme. It was concluded that the quaternary ammonium salts used selectively precipitated the non-enzymatic protein leaving in solution practically all the enzyme resulting in a high yield of purification. Optimal conditions for purification using the two types of quaternary ammonium salts were determined. Immobilisation studies were performed from various purified enzyme solutions, using different amounts of a quaternary ammonium salt. The immobilised enzymes so obtained showed a much higher activity than the immobilised enzyme obtained from non-purified enzyme solutions.

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Abstract: The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg−1) as compared to PCSIR-102 (77 IU mg−1) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K m value) which may show the superiority of the latter in terms of better enzyme properties.

Method for producing cephalexin

Abstract: The invention relates to a method for producing cephalexin with the aid of a penicillin amidase that is immobilized on a pearl-shaped, cross-linked and hydrophilic copolymer that binds to ligands having nucleophilic groups.