Abstract
In biocatalytic processes, the use of free enzymes is often limited due to the lack of long-term stability and reusability. To counteract this, enzymes can be crystallized and then immobilized, generating cross-linked enzyme crystals (CLECs). As mechanical stability and activity of CLECs are crucial, different penicillin G acylases (PGAs) from Gram-positive organisms have proven to be promising candidates for industrial production of new semisynthetic antibiotics, which can be crystallized and cross-linked to characterize the resulting CLECs regarding their mechanical and catalytic properties. The greatest hardness and Young’s modulus determined by indentation with an atomic force microscope were observed for CLECs of Bacillus species FJAT-PGA CLECs (26 MPa/1450 MPa), followed by BmPGA (Priestia megaterium PGA, 23 MPa/1170 MPa) and BtPGA CLECs (Bacillus thermotolerans PGA, 11 MPa/614 MPa). In addition, FJAT- and BtPGA CLECs showed up to 20-fold higher volumetric activities compared to BmPGA CLECs. Correlation to structural characteristics indicated that a high solvent content and low number of cross-linking residues might lead to reduced stability. Furthermore, activity seems to be restricted by small water channels due to severe diffusion limitations. To the best of our knowledge, we show for the first time in this study that the entire process chain for the characterization of diverse industrially relevant enzymes can be performed at the microliter scale to discover the most important relationships and limitations.
Highlights
Penicillin G acylases (PGAs; EC 3.5.1.11) are key enzymes for the biotechnological production of β-lactam antibiotics in the pharmaceutical industry [1]
To guarantee a consistent morphology for similar mechanical behavior and a flat surface to perform the indentation with the cantilever tip, the crystal forms were adapted by varying the concentration of the different penicillin G acylases (PGAs) (5–10 mg/mL) as well as the concentration and pH (4.6–7.5) of the crystallization buffers
This study was designed for the production and characterization of cross-linked enzyme crystals (CLECs) at a small scale
Summary
Penicillin G acylases (PGAs; EC 3.5.1.11) are key enzymes for the biotechnological production of β-lactam antibiotics in the pharmaceutical industry [1]. PGAs are heterodimeric enzymes applied for the hydrolysis of natural penicillin G, yielding 6-aminopenicillanic acid (6-APA) as an important precursor for synthesis of semisynthetic β-lactam antibiotics such as ampicillin or amoxicillin. PGAs can catalyze the synthesis of these semisynthetic β-lactam antibiotics by condensation of activated acyl donors to β-lactam precursors such as 6-APA [1]. Due to increasing microbial resistance, new semisynthetic β-lactam antibiotics produced by PGA are developed to overcome microbial resistances [3]. PGAs from Gram-negative bacteria such as Escherichia coli (EcPGA) or Gram-positive bacteria such as Priestia megaterium (formerly known as Bacillus megaterium) (BmPGA) are mainly used [4,5].
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