Literature DB >> 17098917

Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers.

Tamara Montes1, Valeria Grazú, Fernando López-Gallego, Juan A Hermoso, Jose L García, Isabel Manso, Beatriz Galán, Ramón González, Roberto Fernández-Lafuente, José M Guisán.   

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.

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Year:  2006        PMID: 17098917      PMCID: PMC1797127          DOI: 10.1128/AEM.02107-06

Source DB:  PubMed          Journal:  Appl Environ Microbiol        ISSN: 0099-2240            Impact factor:   4.792


  34 in total

1.  Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment.

Authors:  Olga Abian; Valeria Grazú; Juan Hermoso; Ramón González; José Luis García; Roberto Fernández-Lafuente; José Manuel Guisán
Journal:  Appl Environ Microbiol       Date:  2004-02       Impact factor: 4.792

2.  Advances in enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA).

Authors:  A Parmar; H Kumar; S S Marwaha; J F Kennedy
Journal:  Biotechnol Adv       Date:  2000-07       Impact factor: 14.227

3.  Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis.

Authors:  Wei Zhang; Yuan Liu; Huabao Zheng; Sheng Yang; Weihong Jiang
Journal:  Appl Environ Microbiol       Date:  2005-09       Impact factor: 4.792

Review 4.  Engineered enzymes for improved organic synthesis.

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Journal:  Curr Opin Biotechnol       Date:  2003-08       Impact factor: 9.740

5.  Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli.

Authors:  Yali Xu; Ming-Yi Hsieh; Niju Narayanan; William A Anderson; Jeno M Scharer; Murray Moo-Young; C Perry Chou
Journal:  Biotechnol Prog       Date:  2005 Sep-Oct

6.  Fast horizontal electrophoresis. I. Isoelectric focusing and polyacrylamide gel electrophoresis using PhastSystem.

Authors:  I Olsson; U B Axiö-Fredriksson; M Degerman; B Olsson
Journal:  Electrophoresis       Date:  1988-01       Impact factor: 3.535

7.  Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes.

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8.  Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

Authors:  U K Laemmli
Journal:  Nature       Date:  1970-08-15       Impact factor: 49.962

9.  Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Authors:  J Mansfeld; G Vriend; B Van den Burg; V G Eijsink; R Ulbrich-Hofmann
Journal:  Biochemistry       Date:  1999-06-29       Impact factor: 3.162

10.  Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran.

Authors:  Manuel Fuentes; Benevides C C Pessela; Jorgette V Maquiese; Claudia Ortiz; Rosa L Segura; Jose M Palomo; Olga Abian; Rodrigo Torres; Cesar Mateo; Roberto Fernández-Lafuente; J M Guisán
Journal:  Biotechnol Prog       Date:  2004 Jul-Aug
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  3 in total

1.  High-throughput strategies for penicillin G acylase production in rE. coli fed-batch cultivations.

Authors:  Ana Maria Vélez; Adilson José da Silva; Antonio Carlos Luperni Horta; Cintia Regina Sargo; Gilson Campani; Gabriel Gonçalves Silva; Raquel de Lima Camargo Giordano; Teresa Cristina Zangirolami
Journal:  BMC Biotechnol       Date:  2014-01-21       Impact factor: 2.563

2.  Chromatographic Studies of Protein-Based Chiral Separations.

Authors:  Cong Bi; Xiwei Zheng; Shiden Azaria; Sandya Beeram; Zhao Li; David S Hage
Journal:  Separations       Date:  2016-09-05

3.  New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase.

Authors:  Davide A Cecchini; Immacolata Serra; Daniela Ubiali; Marco Terreni; Alessandra M Albertini
Journal:  BMC Biotechnol       Date:  2007-09-10       Impact factor: 2.563

  3 in total

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