Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

Abstract

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH₂ group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.

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

“…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).…”

Impact of polybasic alcohols on biocompatibility and selectivity of Penicillin G Acylase for kinetically controlled synthesis

“…Protein engineering or directed evolution studies have been used to understand the structure-function relationship and to improve PGA properties such as activity, substrate specificity and stability [7][8][9][10][11][12][13][14][15][16].…”

“…Immobilization methods have also been pursued in the recent past to improve the synthetic performance of PGA [7,36] In this study, we wanted to see if we could improve the performance of PGA for CEX synthesis by medium engineering using co-solvents and optimizing pH conditions. The non-toxic effect of a co-solvent on the function of an enzyme in a specific reaction medium has been studied.…”

“…Polybasic alchols for synthesis by PGA 7 Ethylene glycol and glycerol were purchased from Beijing Yili Fine Chemical Co., Ltd. Polyethylene glycol 400 (PEG400), n-butanol and DMF (N,N-dimethylformamide) were purchased from Sinopharm Medicine Holding Co., Ltd.…”

Impact of polybasic alcohols on biocompatibility and selectivity of Penicillin G Acylase for kinetically controlled synthesis

“…Polybasic alchols for synthesis by PGA 5 superfamily [6]. Protein engineering or directed evolution studies have been used to understand the structure-function relationship and to improve PGA properties such as activity, substrate specificity and stability [7][8][9][10][11][12][13][14][15][16].…”