Rational design of biocatalysts based on covalent immobilization of acylase enzymes

• Published in: Enzyme and microbial technology Vol. 171; p. 110323
• Main Authors: Castillo, Patricio, Cutiño-Avila, Bessy V., González-Bacerio, Jorge, Chávez Planes, María de los Ángeles, Díaz Brito, Joaquín, Guisán Seijas, José Manuel, del Monte-Martínez, Alberto
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.12.2023
• Subjects: Covalent immobilization; Enzymatic bioconversion; Protein immobilization; Rational design of immobilized derivatives; Site directed mutagenesis; pACY1; ImmDer. FC; Site directed mutagenesis; eMQ; Rational design of immobilized derivatives; Covalent immobilization; mMQ; BSA; cCP; Enzymatic bioconversion; pMQ; U; tMQ; E.C; ε-Lys; Protein immobilization; HPLC-MS; NTerm; RDID; PGA; wtPGA; diff. IG; FCP; Conv; MutPGA; RI; diff. IG prot; SIR; DA; FC
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2023.110323

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst’s total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes. •The RDID strategy allows optimizing the covalent immobilization of acylase enzymes.•RDID predicts biocatalysts’ optimal immobilization pH and configuration probability.•RDID predicts biocatalysts’ operational stability and catalytic competence.•RDID complements protein engineering approaches: mutagenesis and fusion proteins.•RDID is a new strategy to allow enzymes to be used in industrial processes.