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.

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 Stabilization of Penicillin G Acylase from Escherichia coli: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

2004 ◽  
Vol 70 (2) ◽  
pp. 1249-1251 ◽  
Author(s):  
Olga Abian ◽  
Valeria Grazú ◽  
Juan Hermoso ◽  
Ramón González ◽  
José Luis García ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Penicillin Acylase ◽  
Site Directed Mutagenesis ◽  
Covalent Attachment ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Protein Surface ◽  
Improved Stability ◽  
Multipoint Covalent Attachment

ABSTRACT Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4°C, pH 5 at 60°C, pH 7 at 55°C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

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 The use of immobilized penicillin acylase for estimation of penicillin G.

1993 ◽  
Vol 40 (1) ◽  
pp. 93-94
Author(s):  
A Marciniak-Rusek ◽  
L Paśś-Dziegielewska
Keyword(s):  
Penicillin Acylase ◽  
Penicillin G ◽  
Immobilized Penicillin Acylase
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