Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

Abstract

A tag of three lysines alternating with three glycines was added to the C-terminal end of the ␤ chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…Perhaps the most popular domain is the poly-His tag, with high affinity for metal chelates (Figure 5) (e.g. for purification by IMAC) (Porath et al, 1975, Porath and Olin, 1983, Porath, 1992 but the range of these affinity peptides is huge and still growing: domains of affinity for cellulose, chitin binding domain, peptide tags, among 9 others (Arroyo et al, 2011, Bello-Gil et al, 2014, Bergeron et al, 2009, Bolivar and Nidetzky, 2012a, b, Cassimjee et al, 2011, Chern and Chao, 2005, Daunert et al, 2007, Kondo and Teshima, 1995, Kowsari et al, 2014, Kweon et al, 2005, Linder and Teeri, 1997, Martinez et al, 2000, Mateo et al, 2001b, Moldes et al, 2004a, Moldes et al, 2004b, Scaramozzino et al, 2005, Shpigel et al, 1999, Vishwanath et al, 1995, Wang et al, 2013a, Wiesbauer et al, 2011, Zhao et al, 2013. Table 1 shows a summary of the main domains used for this purpose while Table 2 shows some specific examples of uses of these domains.…”

“…Terreni and coworkers have shown that the introduction of poly-Lys tags on the enzyme penicillin G acylase may permit the immobilization of the tagged protein even at pH 7 on glyoxyl supports (Scaramozzino et al, 2005). Although the objective of this tag was to get an oriented immobilization, this means that under these conditions only poly-Lys tagged proteins and multimeric proteins having the terminal amino groups on the same plane will be immobilized on the support, enabling a significant purification via a simple immobilization protocol (Figure 15).…”

“…For example, using glyoxyl supports, this may be performed by adding poly-Lys tags or by adding new Lys on rich-Lys areas of proteins, this can be a simple way to get the one pot immobilization, orientation, purification and, at least in the second case, the hyper-stabilization of enzymes (Abian et al, 2004, Cecchini et al, 2007, Cecchini et al, 2012, Scaramozzino et al, 2005, Serra et al, 2009, Serra et al, 2013, Temporini et al, 2010. (Mateo et al, 2005).…”

Coupling of Site‐Directed Mutagenesis and Immobilization for the Rational Design of More Efficient Biocatalysts: The Case of Immobilized 3G3K PGA from E.coli

“…Immobilization of 80 IU/g of the commercial wt PGA on glyoxyl agarose gave a 58 % yield. For the 3G3K PGA, because of the poor stability of this enzyme, [32] the immobilization was performed at 4°C and pH 9.5. The immobilization on glyoxyl agarose under these conditions was remarkable: in fact, 51 % of the offered activity was bound with a 30 % yield.…”

“…[30] Recently, a new mutant of PGA, characterized by a basic and flexible tag of three lysines alternating with three glycines introduced at the C-terminus of the β-chain (on the side-face of the protein [31] ), has been described. [32] Preliminary results demonstrated that this mutant (3G3K PGA), compared with the wild-type acylase, is characterized by a higher vs/vh 1 ratio in the kcNa of 7-aminocephalosporanic acid (7-ACA) [32] and maintains this property after immobilization. We hypothesized that the flexibility of the tag and the increased number of lysines probably induce the preferential and oriented immobilization of this PGA, leading the β-lactam substrate to freely access the active site of the immobilized enzyme.…”