Assessing the predictive capabilities of design heuristics and coarse‐grain simulation toward understanding and optimizing site‐specific covalent immobilization of β‐lactamase. Issue 6 (6th March 2022)

• Record Type: Journal Article
• Authors: Soltani, Mehran; Hunt, John Porter; Smith, Addison K.; Zhao, Emily Long; Knotts, Thomas A.; Bundy, Bradley C.
• Is Part Of: Biotechnology journal. Volume 17:Issue 6(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-03-06
• Subjects: cell‐free protein synthesis -- site‐specific covalent immobilization -- TEM‐1 β‐lactamase; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202100535
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: For industrial applications, covalent immobilization of enzymes provides minimum leakage, recoverability, reusability, and high stability. Yet, the suitability of a given site on the enzyme for immobilization remains a trial‐and‐error procedure. Here, we investigate the reliability of design heuristics and a coarse‐grain molecular simulation in predicting the optimum sites for covalent immobilization of TEM‐1 β‐lactamase. We utilized Escherichia coli ‐lysate‐based cell‐free protein synthesis (CFPS) to produce variants containing a site‐specific incorporated unnatural amino acid with a unique moiety to facilitate site directed covalent immobilization. To constrain the number of potential immobilization sites, we investigated the predictive capability of several design heuristics. The suitability of immobilization sites was determined by analyzing expression yields, specific activity, immobilization efficiency, and stability of variants. These experimental findings are compared with coarse‐grain simulation of TEM‐1 domain stability and thermal stability and analyzed for a priori predictive capabilities. This work demonstrates that the design heuristics successfully identify a subset of locations for experimental validation. Specifically, the nucleotide following amber stop codon and domain stability correlate well with the expression yield and specific activity of the variants, respectively. Our approach highlights the advantages of combining coarse‐grain simulation and high‐throughput experimentation using CFPS to identify optimal enzyme immobilization sites. Graphical Abstract and Lay Summary: In this work, we leverage cell‐free protein synthesis to develop a novel design heuristic for optimal site‐specific, covalent immobilization of TEM‐1. Importantly, this heuristic, which employs biochemical parameters and coarse‐grain simulation of enzyme structure, predicted the top three immobilization locations for extended‐spectrum TEM‐1 that preserve both enzyme stability and catalytic activity. We found domain the stability determined by a coarse‐grain simulation as a predictive of the impact of specific activity of different variants. We anticipate that this heuristic will assist investigators in creating highly active and cost‐effective immobilized enzyme biocatalysts.