Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00790 (PGA)
 2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The technique of cassette and site-specific mutagenesis were used to study the role of residue No. 177 in penicillin G acylase (PGA, EC 3.5.1.11). Ser is conserved at residue No. 177 in all penicillin binding proteins. We got a series of mutants in which the amino acid at residue No. 177 was replaced by other amino acids through the site-specific and cassette mutagenesis, and we characterized the mutants by colony hybridization, NIPAB paper test and DNA sequence analysis. These mutants all show no activity of enzyme, even if the Ser residue was replaced by Thr, Gly and Ala respectively. The results show that Ser residue may be essential for substrate-binding or catalysis of PGA.
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PMID:[Effect of mutagenesis at Ser 177 residue in penicillin G acylase on activity of the enzyme]. 190 33

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.
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PMID:Immobilization-stabilization of penicillin G acylase from Escherichia coli. 209 28

Fermentation parameters for the production of penicillin G acylase by Escherichia coli NCIM 2400 have been evaluated. The bacterium produced the enzyme intracellularly when grown in nutrient broth containing PAA. PAA stimulated the enzyme synthesis by 8-10 fold and reduced the lag period. The optimum concentration of PAA for induction was 20 mM and addition of PAA prior to inoculation gave maximum production of PGA. Glucose, lactose, sorbitol, acetate and lactate even at 0.1% concentration catabolically repressed the enzyme formation. Peptone was the best utilised 'N' source for the enzyme production. Phosphate and yeast extract were found to be essential for both the growth and for enzyme biosynthesis. Temperature between 22-24 degrees C was optimum and under ideal condition E. coli NCIM 2400 produced 0.45-0.55 U/ml of penicillin G acylase.
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PMID:Biosynthesis of benzylpenicillin acylase by Escherichia coli NCIM-2400. 269 12

In many GL-7ACA acylases, the first Ser residue at the N-terminal of beta-subunit is the catalytic center. In order to investigate relationship between the N-terminal structure and catalytic activities, peptide replacement and site-directed mutagenesis were performed at the N-terminal of beta-subunit of GL-7ACA acylase C130. When the N-terminal 8 amino acid residues of C130 were replaced by the corresponding sequence of penicillin acylases PAC and PGA, respectively, the first mutant B8PAC lost the activity of the acylase, and the second mutant B8PGA had lower activity with the K(m) value increasing from 0.44x10(-3)mol.L(-1) to 0.55x10(-3) mol.L(-1), and the k(cat) decreasing from 4.92 s(-1) to 1.64 s(-1). Although the substitution of Trp (beta4) by Tyr did not change the K(m) value, the k (cat) decreased to 2.29 s(-1). When the Trp was substitued by Leu, both the K ( m ) and k ( cat ) values decreased. Compared with the wild type, mutations of Ser (beta3) to Met, Ala and Cys caused decrease of K(m) values by 52.27%, 43.18% and 38.64%, respectively. Mutation of Asn (beta2) to Gln caused the K ( m ) value being increased by 5-fold, and k ( cat ) decreased by 10-fold. These results suggested that the N-terminal amino acid residues of beta-subunit in GL-7ACA acylase C130 are important for enzyme function.
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PMID:Mutagenesis of N-terminal Amino Acid Residues in beta-subunit of Glutaryl-7-amino-cephalosporanic Acid Acylase C130. 1203 60

Site-directed mutagenesis and chemical modification were performed at Ser290 of the penicillin G acylase from E. coli ATCC11105. The Ser290 was substituted by Cys or Secys. Wild type and mutant proteins were purified, and the activities and kinetic constants of penicillin acylases for hydrolysis and synthesis were determined, respectively. Although their K(m) values were not changed, the k(cat) values of the thiol-PGA and seleno-PGA were decreased from 135s(-1) to 0.63s(-1) and 0.38s(-1) against NIPAB, and from 34.38s(-1) to 0.23s(-1) and 0.06s(-1) against penicillin G. Contrary to Choi's report(Choi K S (et al. J Bacteriology), 1992, 10 6270-6276), we found that hydrolysis activity was certainly kept in the mutant of penicillin acylase. In addition, the specific activities of synthesis were decreased by 5-fold and 20-fold, respectively.
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PMID:Site-directed Mutagenesis of the Active Center of Penicillin Acylase from E. coli ATCC 11105. 1211 70

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.
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PMID:Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model. 1211 22

The development of a new chromatographic reactor based on immobilized Candida rugosa lipase (CRL) is described. The chromatographic system has been used to evaluate the rate differences by which the product enantiomers of esterolytic reactions catalyzed by immobilized CRL are obtained. The method has been applied to a series of racemic 2-aryloxyalkanoic acids and isosteric analogous methyl esters and to some non-steroidal antiinflammatory drugs 2-arylpropanoic acids methyl esters in order to study the structure effects on reaction rate and enantioselectivity. Lipase from C. rugosa has been non-covalently and covalently immobilized on HPLC chromatographic silica supports to develop an immobilized enzyme reactor (IMER). The reactor was connected through a switching valve to an analytical reversed-phase column, which was used for the on-line determination of the hydrolysis rate by peak area integration. The enantiomeric excess of the hydrolytic reaction products was determined off-line on a CSP utilizing immobilized penicillin G acylase (PGA-CSP).
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PMID:Lipases for biocatalysis: development of a chromatographic bioreactor. 1289 62

The hydrolysis of penicillin G in the presence of an organic solvent, used with the purpose of extracting it from the culture medium, may greatly simplify the industrial preparation of 6-APA. However, under these conditions, PGA immobilized onto Eupergit displays very low stability (half-life of 5 h in butanone-saturated water) and a significant degree of inhibition by the organic solvent (30%). The negative effect of the organic solvent strongly depended on the type of solvent utilized: water saturated with butanone (around 28% v/v) had a much more pronounced negative effect than that of methylisobutyl ketone (MIBK) (solubility in water was only 2%). These problems were sorted out by using a new penicillin G acylase derivative designed to work in the presence of organic solvents (with each enzyme molecule surrounded by an hydrophilic artificial environment) and a suitable organic solvent (MIBK). Using such solvent, this derivative kept its activity unaltered for 1 week at 32 degrees C. Moreover, the enzyme activity was hardly inhibited by the presence of the organic solvent. In this way, the new enzyme derivative thus prepared enables simplification of the industrial hydrolysis of penicillin G.
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PMID:Improving the industrial production of 6-APA: enzymatic hydrolysis of penicillin G in the presence of organic solvents. 1465 34

The matter of this work was to evaluate possibilities of biospecific immobilization of synthetic mannan-penicillin G acylase neoglycoconjugate on Concanavalin A support. The conjugate containing 37% (w/w) of yeast mannan was prepared. Significant biospecific interaction of this neoglycoenzyme with Con A was confirmed by precipitation method. The biospecific sorption of conjugate was investigated using Concanavalin A-triazine bead celluloses MT-100 with different content of Con A (from 1.4 to 9.8 mgCon A/gwet support). The results obtained under optimal conditions were compared with those from covalent immobilization of PGA. The sorbent capacity was observed higher for covalent binding of enzyme. On the other hand, the biospecifically immobilized neoglycoenzyme retained a greater amount of initial activity. The maximum amount of 6.6mgimmobilizedneoglycoenzyme/gwet Con A-sorbent (18.1 U/g) was achieved. The amount as well as activity of immobilized mannan-penicillin G acylase was increased by its two multiple layering on surface of sorbent (10.1mg, respectively, 23.5 U/gwet sorbent). Determined storage and operational (using flow calorimetric method) stabilities of biospecifically immobilized enzyme, were similar, possibly somewhat higher that those of covalent bound penicillin G acylase.
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PMID:Biospecific immobilization of mannan-penicillin G acylase neoglycoenzyme on Concanavalin A-bead cellulose. 1509 1

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%.
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PMID:Enantioselective synthesis of phenylacetamides in the presence of high organic cosolvent concentrations catalyzed by stabilized penicillin G acylase. Effect of the acyl donor. 1517 9


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