Increasing synthetic performance of penicillin G acylase from Bacillus megaterium by site-directed mutagenesis

2007 ◽  
Vol 74 (5) ◽  
pp. 1023-1030 ◽  
Author(s):  
Jingang Wang ◽  
Qing Zhang ◽  
He Huang ◽  
Zhongyi Yuan ◽  
Dafu Ding ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Site Directed Mutagenesis ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Directed Mutagenesis

scholarly journals Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers

2006 ◽  
Vol 73 (1) ◽  
pp. 312-319 ◽  
Author(s):  
Tamara Montes ◽  
Valeria Grazú ◽  
Fernando López-Gallego ◽  
Juan A. Hermoso ◽  
Jose L. García ◽  
...  
Keyword(s):  
Specific Activity ◽  
Enzyme Stability ◽  
Enzyme Engineering ◽  
Enzyme Inactivation ◽  
Site Directed Mutagenesis ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Enzyme Adsorption ◽  
Reversible Immobilization

ABSTRACT A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.

Inoculum Studies in Production of Penicillin G Acylase by Bacillus megaterium ATCC 14945

2002 ◽  
pp. 679-686 ◽  
Author(s):  
Laura M. Pinotti ◽  
Rosineide G. Silva ◽  
Roberto C. Giordano ◽  
Raquel L. C. Giordano
Keyword(s):  
Bacillus Megaterium ◽  
Penicillin G Acylase ◽  
Penicillin G

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis

1991 ◽  
Vol 17 (2) ◽  
pp. 99-108 ◽  
Author(s):  
Joo Hyun Kang ◽  
Young Hwang ◽  
Ook Joon Yoo
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Bacillus Megaterium ◽  
Penicillin G Acylase ◽  
Penicillin G

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.

Study of Different Media for Production of Penicillin G Acylase from Bacillus megaterium ATCC 14945

2000 ◽  
Vol 84-86 (1-9) ◽  
pp. 655-664 ◽  
Author(s):  
Laura M. Pinotti ◽  
Astrea F. S. Silva ◽  
Rosineide G. Silva ◽  
Raquel L. C. Giordano
Keyword(s):  
Bacillus Megaterium ◽  
Penicillin G Acylase ◽  
Penicillin G

High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant

2000 ◽  
Vol 89 (1) ◽  
pp. 152-157 ◽  
Author(s):  
W. Panbangred ◽  
K. Weeradechapon ◽  
S. Udomvaraphant ◽  
K. Fujiyama ◽  
V. Meevootisom
Keyword(s):  
Bacillus Megaterium ◽  
High Expression ◽  
Penicillin G Acylase ◽  
Penicillin G

Study of Different Media for Production of Penicillin G Acylase from Bacillus megaterium ATCC 14945

2000 ◽  
pp. 655-663
Author(s):  
Laura M. Pinotti ◽  
Astrea F. S. Silva ◽  
Rosineide G. Silva ◽  
Raquel L. C. Giordano
Keyword(s):  
Bacillus Megaterium ◽  
Penicillin G Acylase ◽  
Penicillin G

scholarly journals Using a medium of free amino acids to produce penicillin g acylase in fed-batch cultivations of Bacillus megaterium ATCC 14945

2006 ◽  
Vol 23 (1) ◽  
pp. 37-43 ◽  
Author(s):  
R. G. Silva ◽  
V. R. Souza ◽  
E. R. Nucci ◽  
L. M. Pinotti ◽  
A. J. G. Cruz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Megaterium ◽  
Free Amino Acids ◽  
Free Amino ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Fed Batch

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.

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