Preparation of cross-linked enzyme aggregates of l-aminoacylase via co-aggregation with polyethyleneimine

l-Aminoacylase from Aspergillus melleus was co-aggregated with polyethyleneimine and subsequently cross-linked with glutaraldehyde to obtain aminoacylase–polyethyleneimine cross-linked enzyme aggregates (termed as AP-CLEA). Under the optimum conditions, AP-CLEA expressed 74.9% activity recovery and 81.2% aggregation yield. The said method of co-aggregation and cross-linking significantly improved the catalytic stability of l-aminoacylase with respect to temperature and storage. AP-CLEA were employed for enantioselective synthesis of three unnatural amino acids (namely: phenylglycine, homophenylalanine and 2-naphthylalanine) via chiral resolution of their ester-, amide- and N-acetyl derivatives. The enantioselectivity of AP-CLEA was the highest for hydrolysis of amino acid amides; was moderate for hydrolysis of N-acetyl amino acids and was the least for hydrolysis of amino acid esters. Furthermore, AP-CLEA were found to retain more than 92% of the initial activity after five consecutive batches of (RS)-homophenylalanine hydrolysis suggesting an adequate operational stability of the biocatalyst.

Graphical abstractScanning electron micrographs of AP-CLEA: (a) individual CLEA particle observed at lower magnification (1000×); (b) macroporous nature of CLEA observed at higher magnification (2500×).Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Cross-linked enzyme aggregates of l-aminoacylase were synthesized. ► Prior to cross-linking, the enzyme was co-aggregated with polyethyleneimine. ► Polyethyleneimine enhanced the cross-linking efficiency of l-aminoacylase. ► The cross-linked enzyme expressed 74.9% activity recovery and 81.2% aggregation yield. ► The cross-linking enhanced thermal-, storage- and operational stability of the enzyme.