scholarly journals Immobilization of alginate-PAC on Sepabeads EC-HA support

2011 ◽  
Vol 65 (4) ◽  
pp. 431-437 ◽  
Author(s):  
Milena Zuza ◽  
Nenad Milosavic ◽  
Zorica Knezevic-Jugovic
Keyword(s):  
Phenylacetic Acid ◽  
Immobilized Enzyme ◽  
Penicillin Acylase ◽  
Covalent Immobilization ◽  
Enzyme Inactivation ◽  
Penicillin G ◽  
Ph Profile ◽  
Covalently Linked ◽  
Hydrolysis Of ◽  
Derivatives Of

Penicillin acylase (PAC) is an important industrial enzyme for the production of many ?-lactam antibiotics. It is capable of catalyzing the hydrolysis of penicillin G (Pen G) to generate phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). In this paper, in order to prevent enzyme inactivation, an attempt of coupling enzyme modification and immobilization was presented. Chemical modification was promoted to introduce carbohydrate moiety into the PAC molecule, capable of being covalently linked to an amino support. This seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause a loss of activity. PAC molecules were modified by cross-linking with polyaldehyde derivatives of alginate in order to add them new and useful functions. Immobilization of alginate-PAC on Sepabeads EC-HA was used as a model system in order to demonstrate the potential of this strategy. Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA, were investigated. The immobilized enzyme was then characterized by evaluating the potential effects of immobilization on its thermal stability, temperature and pH profile in comparison with native non-modified PAC and modified non-immobilized PAC. The maximum amount of the alginate-PAC coupled on the dry support of 99 mg/g was satisfactory. Deactivation rate constants at 50 ?C for free PAC, alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA were 2,32; 50,65 and 1,68 h-1, respectively. Alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA had the same pH and temperature optimum as the native non-modified PAC.

scholarly journals Preparation and characterization of penicillin acylase immobilized on sepabeads EC-EP carrier

2007 ◽  
Vol 13 (4) ◽  
pp. 205-210 ◽  
Author(s):  
Milena Zuza ◽  
Slavica Siler-Marinkovic ◽  
Zorica Knezevic
Keyword(s):  
Thermal Stability ◽  
Immobilized Enzyme ◽  
Conformational Stability ◽  
Penicillin Acylase ◽  
Covalent Immobilization ◽  
Free Enzyme ◽  
Penicillin G ◽  
High Catalytic Activity ◽  
Decimal Reduction Time ◽  
Promising Solution

This paper reports the covalent immobilization of penicillin G acylase from E. coli on Sepabeads EC-EP, an epoxy-activated polymethacrylic carrier, and describes the properties of the immobilized enzyme. Due to its versatility to mediate hydrolysis of penicillins and semi-synthetic B-lactam antibiotics synthesis reactions, the selected enzyme belongs to a class of biocatalysts of great industrial interest. The immobilized enzyme was characterized in its pH and thermal stability and reaction kinetics. The immobilization of penicillin acylase resulted in a slightly different pH activity profile and temperature optima, indicating that the immobilization by this method imparted the structural and conformational stability to this enzyme. The immobilized enzyme also retained a high catalytic activity and showed the increased thermal stability compared with a free enzyme. By comparison of decimal reduction time values obtained at 50?C, it can be concluded that the immobilized enzyme was approximately 5-fold more stable than a free enzyme. The immobilization procedure developed is quite simple and easily reproduced, and provides a promising solution for the application of penicillin acylase for the purpose of 6-aminopenicillanic acid production.

scholarly journals Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

1993 ◽  
Vol 291 (3) ◽  
pp. 907-914 ◽  
Author(s):  
J Martín ◽  
J M Mancheño ◽  
R Arche
Keyword(s):  
Enzyme Assay ◽  
Ph Dependence ◽  
Penicillin Acylase ◽  
Enzyme Inactivation ◽  
Time Dependent ◽  
Penicillin G ◽  
Reversible Binding ◽  
Ph Increase ◽  
Kinetics Of ◽  
Covalent Activation

Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation. Moreover, enzyme re-activation is achieved by chromatographic removal of EEDQ, pH increase or displacement of the reagent with penicillin G. It was then concluded that PA inactivation is due to an equilibrium reaction. The kinetics of enzyme inactivation was analysed by fitting data to theoretical equations derived in accordance with this mechanism. Corrections for re-activation during the enzyme assay were a necessary introduction. The pH-dependence of the rate constant for EEDQ hydrolysis either alone or in the presence of enzyme was studied by u.v. spectroscopy. It turned out to be coincident with the pH-dependence of the forward and reverse rate constants for the inactivation process. It is suggested that previous protonation of the EEDQ molecule is required for these reactions to occur. The thermodynamic values associated with the overall reaction showed little change. Finally it is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction. The initial and rapid reversible binding is followed by a slow, time-dependent, non-covalent, reversible inactivating step. The expected behaviour in the case of enzyme modification by covalent activation of carboxy residues is also reviewed.

Immobilization of modified penicillin G acylase on Sepabeads carriers

2009 ◽  
Vol 63 (2) ◽  
Author(s):  
Milena Žuža ◽  
Nenad Milosavić ◽  
Zorica Knežević-Jugović
Keyword(s):  
Thermal Stability ◽  
Hydrolytic Activity ◽  
Covalent Immobilization ◽  
Stability Study ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Chemically Modified ◽  
Modified Enzyme ◽  
Derivatives Of ◽  
Thermal Stability Study

AbstractAn approach to stable covalent immobilization of chemically modified penicillin G acylase from Escherichia coli on Sepabeads® carriers with high retention of hydrolytic activity and thermal stability is presented. The two amino-activated polymethacrylate particulate polymers with different spacer lengths used in the study were Sepabeads® EC EA and Sepabeads® EC HA. The enzyme was first modified by cross-linking with polyaldehyde derivatives of starch in order to provide it with new useful functions. Such modified enzyme was then covalently immobilized on amino supports. The method seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause the loss of activity. Performances of these immobilized biocatalysts were compared with those obtained by the conventional method with respect to activity and thermal stability. The thermal stability study shows that starch-PGA immobilized on Sepabeads EC-EA was almost 4.5-fold more stable than the conventionally immobilized one and 7-fold more stable than free non-modified PGA. Similarly, starch-PGA immobilized on Sepabeads EC-HA was around 1.5- fold more stable than the conventionally immobilized one and almost 9.5-fold more stable than free non-modified enzyme.

scholarly journals Preparation of Sterically Demanding 2,2-Disubstituted-2-Hydroxy Acids by Enzymatic Hydrolysis

Catalysts ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 113 ◽  
Author(s):  
Andrea Pinto ◽  
Immacolata Serra ◽  
Diego Romano ◽  
Martina Contente ◽  
Francesco Molinari ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Phenylacetic Acid ◽  
General Structure ◽  
Hydroxy Acids ◽  
Pig Liver Esterase ◽  
Liver Esterase ◽  
Sterically Demanding ◽  
Optically Pure ◽  
Hydrolysis Of ◽  
Derivatives Of

Preparation of optically-pure derivatives of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid of general structure 2 was accomplished by enzymatic hydrolysis of the correspondent esters. A screening with commercial hydrolases using the methyl ester of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid (1a) showed that crude pig liver esterase (PLE) was the only preparation with catalytic activity. Low enantioselectivity was observed with substrates 1a–d, whereas PLE-catalysed hydrolysis of 1e proceeded with good enantioselectivity (E = 28), after optimization. Enhancement of the enantioselectivity was obtained by chemical re-esterification of enantiomerically enriched 2e, followed by sequential enzymatic hydrolysis with PLE. The preparation of optically-pure (S)-2e was validated on multi-milligram scale.

scholarly journals Stabilization of Lecitase Ultra® by Immobilization and Fixation of Bimolecular Aggregates. Release of Omega-3 Fatty Acids by Enzymatic Hydrolysis of Krill Oil

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1067
Author(s):  
Daniel Andrés-Sanz ◽  
Cristina Fresan ◽  
Gloria Fernández-Lorente ◽  
Javier Rocha-Martín ◽  
Jose M. Guisán
Keyword(s):  
Covalent Immobilization ◽  
Omega 3 Fatty Acids ◽  
Krill Oil ◽  
Omega 3 ◽  
Open Structures ◽  
Lecitase Ultra ◽  
Support Surfaces ◽  
Hydrophobic Supports ◽  
Hydrolysis Of ◽  
Derivatives Of

Lecitase Ultra® solutions are mainly composed of bimolecular aggregates of two open structures of the enzyme. The immobilization and fixation of these bimolecular aggregates onto support surfaces is here proposed as a novel protocol for the immobilization and stabilization of Lecitase. The resulting derivatives of Lecitase aggregates were much more stable than the diluted solutions of the enzyme. The most stable of them was obtained by covalent immobilization of the bimolecular aggregate: 300-fold more stable than the diluted enzyme and 75-fold more stable than open Lecitase adsorbed onto hydrophobic supports. The bimolecular aggregate that adsorbed onto polyethyleneimine-agarose exhibited the best combination of activity and stability for the hydrolysis of krill oil. Omega-3 acids are in the sn-2 position of the krill oil, but they are also released by a phospholipase A1 because of migration issues.

Repression of penicillin G acylase of Proteus rettgeri by tricarboxylic acid cycle intermediates

1982 ◽  
Vol 152 (1) ◽  
pp. 104-110
Author(s):  
G O Daumy ◽  
A S McColl ◽  
D Apostolakos
Keyword(s):  
Catabolite Repression ◽  
Phenylacetic Acid ◽  
Tricarboxylic Acid Cycle ◽  
Dicarboxylic Acids ◽  
Penicillin Acylase ◽  
Tricarboxylic Acid ◽  
Penicillin G ◽  
Acid Cycle ◽  
E Coli ◽  
Tricarboxylic Acid Cycle Intermediates

The regulation of the penicillin acylase in proteus rettgeri ATCC 31052 was compared with that of the enzyme in Escherichia coli ATCC 9637. Unlike the E. coli acylase, the P. rettgeri enzyme was not induced by phenylacetic acid, nor was it subject to catabolite repression by glucose. The P. rettgeri acylase appears to be expressed constitutively but is subject to repression by the C4-dicarboxylic acids of the tricarboxylic acid cycle, succinate, fumarate, and malate.

scholarly journals Penicillins and other acylamino compounds synthesized by the cell-bound penicillin acylase of Escherichia coli

1969 ◽  
Vol 115 (4) ◽  
pp. 747-756 ◽  
Author(s):  
M. Cole
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Phenylacetic Acid ◽  
Enzyme Reaction ◽  
Reaction Rates ◽  
Hydroxamic Acids ◽  
Penicillin Acylase ◽  
E Coli ◽  
Hydroxyphenylacetic Acid ◽  
Derivatives Of

1. The penicillin acylase of Eschericha coli N.C.I.B. 8743 is a reversible enzyme. Reaction rates for the two directions have been determined. 2. Measurements of the rates of enzymic synthesis of penicillins from 6-aminopenicillanic acid and various carboxylic acids revealed that p-hydroxyphenylacetic acid was the best substrate, followed by phenylacetic, 2-thienylacetic, substituted phenylacetic, 3-hexenoic and n-hexanoic acids. 3. The rate of synthesis of penicillin improved when amides or N-acylglycines were used; α-aminobenzylpenicillin and phenoxymethylpenicillin were only synthesized when using these more energy-rich compounds. 4. Phenyl-acetylglycine was the best substrate for the synthesis of benzylpenicillin compared with other derivatives of phenylacetic acid. 5. The enzyme was specific for acyl-l-amino acids, benzylpenicillin being synthesized from phenylacetyl-l-α-aminophenylacetic acid but not from phenylacetyl-d-α-aminophenylacetic acid. 6. α-Phenoxyethylpenicillin was synthesized from 6-aminopenicillanic acid and α-phenoxypropionylthioglycollic acid non-enzymically, but the rate was faster in the presence of the enzyme. 7. The E. coli acylase catalysed the acylation of hydroxylamine by acids or amides to give hydroxamic acids, the phenylacetyl group being the most suitable acyl group. The enzyme also catalysed other acyl-group transfers.

scholarly journals Immobilization of penicillin acylase from Escherichia coli on commercial sepabeads EC-EP carrier

2007 ◽  
pp. 173-182 ◽  
Author(s):  
Milena Zuza ◽  
Slavica Siler-Marinkovic ◽  
Zorica Knezevic
Keyword(s):  
Escherichia Coli ◽  
Conformational Stability ◽  
Maximum Yield ◽  
Penicillin Acylase ◽  
Covalent Immobilization ◽  
Penicillin G ◽  
Kinetic Properties ◽  
Enzyme Coupling ◽  
Immobilized Penicillin Acylase ◽  
Coupling Yield

This paper describes the covalent immobilization of penicillin G acylase from Escherichia coli on sepabeads EC-EP, an epoxy-activated polymethacrylic carrier and kinetic properties of the immobilized enzyme. The selected enzyme belongs to a class of biocatalysts whose industrial interest is due to their versatility to mediate hydrolysis of penicillins and semi-synthetic ?-lactam antibiotics synthesis reactions. About 2.7 mg of the pure enzyme was immobilized onto each gram of sepabeads with an enzyme coupling yield of 96.9%. However, it seems that the activity coupling yield is not correlated with the amount of enzyme bound and the maximum yield of 89.4% can be achieved working at low enzyme loading (0.14 mg g-1). Immobilization of the penicillin acylase resulted in slightly different pH activity profile and temperature optima, indicating that the immobilization by this method imparted structural and conformational stability of this enzyme. It appears that both free and immobilized penicillin acylase followed simple Michaelis-Menten kinetics, implying the same reaction mechanism in both systems.

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