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

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.

On-Line Monitoring of Biomass Concentration Based on a Capacitance Sensor: Assessing the Methodology for Different Bacteria and Yeast High Cell Density Fed-Batch Cultures

“…The second, third and forth clones, called E. coli "b", "c" and "e", produces fragments of the antigenic surface proteins PspA clade 1, 3 and 4 from Streptococcus pneumoniae (Carvalho et al, 2012;Sargo, 2011;Santos, 2012) and were kindly provided by Dr. Eliane Miyaji and Dr. Luciana Cerqueira Leite (Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil). The fifth clone, called E. coli "d", transformed with the plasmid pT101/D-TOPO with pac gene codifying for penicillin G acylase (PGA) production (Montes et al, 2007), was kindly donated by the Laboratório de Biocatálisis, ICP-CSIC-Madri, Spain.…”

Improved Stabilization of Genetically Modified Penicillin G Acylase in the Presence of Organic Cosolvents by Co‐ Immobilization of the Enzyme with Polyethyleneimine

“…In fact, using strategy 1, the titration of the enzyme derivatives with SPDP did not reveal a significant increase in the amount of primary amino groups ( Table 1), suggesting that PEI was not adsorbed on the enzyme surface This result agrees with the observation that PGA is not adsorbed on supports coated with PEI. [32] The failure of both treatments could be explained by the lack of polymer-enzyme interactions, since in this way, only those areas of the enzyme surface which are in close contact with the support can be protected by the polymer, while those areas of the protein oriented to the medium remain fully accessible to the organic solvent.…”

“…Therefore, this mutant PGA can be strongly adsorbed on PEI-coated supports. [32] In this manuscript, we have studied the possibilities of stabilizing this mutant PGA against the action of organic solvents by co-immobilizing on glyoxyl agarose the enzyme and polycationic polymers having primary amino groups. This support has been chosen because it has allowed one to stabilize many enzymes via multipoint covalent attachment [33] and has been specifically used to stabilize PGA.…”

“…[32] Recently, a new PGA mutant has been reported possessing 8 new Glu residues which have been introduced on the protein surface by changing Asn or Gln in different positions homogeneously distributed throughout the PGA surface. [32] When compared with the native enzyme, this mutant PGA presented very similar thermostability and activity/pH profiles while the isoelectric point decreased from 6.4 to 4.3. Therefore, this mutant PGA can be strongly adsorbed on PEI-coated supports.…”

A supervision and control tool based on artificial intelligence for high cell density cultivations

“…Five recombinant E. coli BL21(DE3) strains, modified with the following plasmids, were cultivated under the supervision of SUPERSYS_HCDC: a) pET37b+/PspA3 and PspA4Pro, kindly provided by Dr. Eliane Miyaji (Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil), and pET37b+/ PspA245, provided by Dr. Luciana Leite (Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil), all three clones expressing a fragment of the surface protein A of Streptococcus pneumoniae (Barazzone et al, 2011;Carvalho et al, 2012); b) pET28a/SpaA, producing a recombinant fragment of the SpaA protein from Erysipelothrix rhusiopathiae ; c) pT101/D-TOPO, kindly donated by Laboratório de Biocatálisis, ICP-CSIC-Madri, Spain, with pac gene codifying for penicillin G acylase (PGA) production (Montes et al, 2007).…”