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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.


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Journal ArticleDOI
TL;DR: Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed, and the reaction performed under these conditions allowed us to produce (2 S,2′ S)‐ N‐2′‐hydroxy‐2‐phenyl)‐ 2‐hydroxyphenylacetamide with a diasteromeric excess higher than 98%.
Abstract: Immobilization of penicillin G acylase on glyoxyl agarose and its further hydrophilization by physicochemical modification with ionic polymers has made it possible to perform the direct condensation between (+/-)-2-hydroxy-2-phenylethylamine [(+/-)-1] and different acyl donors in the presence of high concentrations of organic cosolvent (up to 90%) in the reaction medium. Using 50 mM phenyl acetic acid and these drastic reaction conditions, too harsh for any other PGA preparation, we have achieved an almost quantitative transformation (more than 99%) of 10 mM (+/-)-1 into the corresponding amide. Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed. Thus, using phenylacetic acid (2), the enantioselectivity was almost negligible (1.3 favoring the S isomer), whereas using S-mandelic acid [(S)-4], the E factor reached a value of 21 (also favoring the S isomer). By using R-mandelic acid [(R)-4], we observed a different enantioselectivity (E was 3.6 favoring the R). At 4 degrees C, the E value reached a value higher than 100 when (S)-4 was used as the acyl donor. The reaction performed under these conditions allowed us to produce (2S,2'S)-N-2'-hydroxy-2'-phenyl)-2-hydroxyphenylacetamide [(2S,2'S)-7] with a diasteromeric excess higher than 98%.

15 citations

Journal ArticleDOI
TL;DR: It is shown that the alpha- and beta-subunits of the enzyme can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids.
Abstract: Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the α- and β-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of α-subunit linked to a 54-amino-acid endopeptide and β-subunit was lower than with the subunits alone.

15 citations

Journal ArticleDOI
TL;DR: This immobilization method for penicillin acylase prepared by glutaraldehyde crosslinking on hydrogenated porous polyacrylonitrile fibers was superior to other methods in specific activity and immobilization yield.

15 citations

Journal ArticleDOI
TL;DR: The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications to create a hybrid enzyme which was catalytically active.
Abstract: A cross-species penicillin G amidase (PGA) gene (pac) coding for an α-peptide and a linker peptide fromK. citrophila ATCC 21285 and a β-peptide fromE. coli ATCC 11105 has been constructed and cloned inE. coli. The naturally occurring PGA specific processing pathway led to the formation of a hybrid enzyme which was catalytically active. In comparison with the two wild-type enzymes the hybrid PGA was found to have higherk cat values for the three tested substrates benzylpenicillin, ampicillin and 6-nitro-3-phenylacetamido-benzoic acid (NIPAB).K m was between the values of the wild-type enzymes or close to that ofK. citrophila. The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications.

15 citations

Journal ArticleDOI
TL;DR: The mechanism without binding of the nucleophile N (6-APA i n Eq. 2, or an amino-acid derivative in peptide semisynthesis), before the deacylation of the acylenzyme E-A, is the most frequently used mechanism in the literature in this field.
Abstract: where an activated substrate must be used, and the enzyme carries out a group transfer reaction. The yield of the desired product in this case is often much larger than the yield for the equilibrium-controlled process.'-' This requires that the catalyst be removed when the maximum is reached, and this is easily performed with immobilized enzymes. The detailed reaction mechanism for the kinetically controlled process' must be known for' a rational analysis of the factors controlling yield. Two different mechanisms have been proposed here. They are given in TABLE I . The mechanism without binding of the nucleophile N (6-APA i n Eq. 2, or an amino-acid derivative in peptide semisynthesis), before the deacylation of the acylenzyme E-A, is the most frequently used mechanism in the literature in this field. The other mechanism where the nucleophile N is bound to the acylenzyme before the deacylation may be inferred from the sequence specificity of the hydrolases and the principle of microscopic reversibility. The two mechanisms can be distinguished by measuring the initial rates of formation of AN (oaN) and A (uA) as functions of nucleophile concentration. The expressions for this and the yield at the kinetically controlled maximum are given in TABLE I.' Once the mechanism has been established, the yield-controlling factors can be rationally analyzed to evaluate the biotechnological potential of such processes. Some data pertinent to the kinetically controlled, enzyme-catalyzed synthesis of semisynthetic penicillins and peptides are presented here. Free and immobilized penicillin amidase (EC 3.5.1.1 1) from E. coli, the substrates and 6-APA used to prepare semisynthetic penicillins were gifts from Dr. Sauber (Hoechst AG) and Dr. Kramer (Rohm), respectively. Phenyl-acetyl-glycine was

15 citations

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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20234
20222
20183
20175
20165
20153