Workflow for evaluating enzyme immobilization and performance for continuous flow manufacturing

Luteran, Emily Michelle; Aloisi, Marc Robert; Akerley, Wendi Lynn; Gilbertson, Robert David

doi:10.1016/j.crbiot.2025.100327

Workflow for evaluating enzyme immobilization and performance for continuous flow manufacturing

Journal Article · Sat Aug 16 00:00:00 EDT 2025 · Current Research in Biotechnology

DOI:https://doi.org/10.1016/j.crbiot.2025.100327· OSTI ID:2584683

^[1]; ^[1]; Akerley, Wendi Lynn ^[1]; ^[1]

Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Enzymes have shown promise in various industries due to their functional specificity, catalytic efficiency, and environmental sustainability. These biological catalysts can be a pivotal component of manufacturing pipelines like continuous flow chemistry. For this, there exists a need to robustly immobilize enzymes on solid supports and assess the effects of the solid supports on catalytic performance and stability. Here, we use an industrially relevant model enzyme, C. ensiformis (Jack bean) urease, to demonstrate immobilization and assess performance in the context of continuous flow manufacturing. Various immobilization strategies were screened focusing on immobilization efficiency, protocol simplicity, and urease biocatalyst kinetics. Based on this, CDI-agarose and NHS-agarose resins were identified as the best-performing immobilization strategies for urease. CDI-agarose-urease and NHS-agarose-urease were then scaled up and applied to a large-scale continuous flow reactor to evaluate product yields, operational stability, and long-term stability. These experiments identified differences in stability and performance depending on the immobilization method tested. This highlights the importance of screening immobilization methods and subsequent enzyme performance for each candidate biocatalyst used in manufacturing to promote optimal performance and stability. As such, this work provides a framework for evaluating enzyme biocatalyst immobilization approaches to improve performance and enable transition into industrial processes.

View Accepted Manuscript (DOE)

Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: USDOE Laboratory Directed Research and Development (LDRD) Program

Grant/Contract Number:: 89233218CNA000001

OSTI ID:: 2584683

Report Number(s):: LA-UR--25-24559; 10.1016/j.crbiot.2025.100327

Journal Information:: Current Research in Biotechnology, Journal Name: Current Research in Biotechnology Vol. 10; ISSN 2590-2628

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
Biocatalysis
Biomanufacturing
Continuous flow chemistry
Immobilization
Jack bean urease
Kinetics
Stability

Workflow for evaluating enzyme immobilization and performance for continuous flow manufacturing

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