fa3fb7479f1c54396a5e139ea17eb116 processes-12-02014.pdf f28ac3d0bab55f480def83ef8a6b1c45ffbfa3d2 processes-12-02014.pdf 5fd7ea0565685f6ea9c835c227e0eae6217868520f1aebb419c584a34c038357 processes-12-02014.pdf Title: Catalytic Potential-Guided Design of Multi-Enzymatic System for DHA Production from Glycerol Subject: The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni+2 agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations. Keywords: multi-enzymatic system; glycerol; sustainable bioprocess; dihydroxyacetone; cofactor regeneration; immobilized enzymes Author: Carolina Fernández-Pizarro, Lorena Wilson and Oscar Romero Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu Sep 19 09:58:18 2024 CEST ModDate: Thu Sep 19 10:01:01 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2668872 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AIQCCL+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 QCIEGT+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 NSRQAN+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 GBYEDF+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 YQFNMO+CMSY10 Type 1 Builtin yes yes yes 32 0 MDIDAP+VnURWPalladioL Type 1 Custom yes yes yes 37 0 BHUVWR+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 SQADDB+EURM10 Type 1 Builtin yes yes yes 70 0 PDCOFY+CMEX10 Type 1 Builtin yes yes yes 81 0 IOLAWT+PazoMath-Italic Type 1 Builtin yes yes yes 86 0 GIGFZE+CMR10 Type 1 Builtin yes yes yes 91 0 MUUDAP+MSAM10 Type 1 Builtin yes yes yes 99 0 JKNGMM+TimesNewRoman CID TrueType Identity-H yes yes yes 111 0 JKNHCM+Arial TrueType WinAnsi yes yes no 117 0 JKNEEC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 120 0 JKNEED+PalatinoLinotype CID TrueType Identity-H yes yes yes 123 0 JKNEEE+PalatinoLinotype TrueType WinAnsi yes yes no 129 0 JKNEFG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 132 0 JKNFAD+PalatinoLinotype TrueType MacRoman yes yes no 135 0 JKNHCL+Arial CID TrueType Identity-H yes yes yes 159 0 JKNEEC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 165 0 JKNEED+PalatinoLinotype CID TrueType Identity-H yes yes yes 168 0 JKNEEE+PalatinoLinotype TrueType WinAnsi yes yes no 174 0 JKNEFG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 177 0 JKNFAD+PalatinoLinotype TrueType MacRoman yes yes no 180 0 JKODOB+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 217 0 JKNEEC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 220 0 JKNEED+PalatinoLinotype CID TrueType Identity-H yes yes yes 223 0 JKNEEE+PalatinoLinotype TrueType WinAnsi yes yes no 229 0 JKNEFG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 232 0 JKNFAD+PalatinoLinotype TrueType MacRoman yes yes no 235 0 JKNGMM+TimesNewRoman CID TrueType Identity-H yes yes yes 250 0 JKODOB+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 256 0 JKOFEJ+Marlett CID TrueType Identity-H yes yes yes 259 0 JKNEEC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 265 0 JKNEED+PalatinoLinotype CID TrueType Identity-H yes yes yes 268 0 JKNEEE+PalatinoLinotype TrueType WinAnsi yes yes no 274 0 JKNEFF+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 277 0 JKNEFG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 283 0 JKNFAD+PalatinoLinotype TrueType MacRoman yes yes no 286 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-11-14 02:53:05 CET RepresentationInformation: processes-12-02014.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-10-08 14:37:11 CEST Size: 2668872 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 457 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Materials Destination: subsection.2.1 Item: Title: Enzyme Production and Purification Destination: subsection.2.2 Item: Title: GlyDH Enzyme Activity Assay Destination: subsection.2.3 Item: Title: NOX Enzyme Activity Assay Destination: subsection.2.4 Item: Title: Protein Content Determination Destination: subsection.2.5 Item: Title: Synthesis of Ag-Ni Supports Destination: subsection.2.6 Item: Title: Immobilization of Enzymes Destination: subsection.2.7 Item: Title: Stability of Biocatalyst and Determination of Catalytic Potential Destination: subsection.2.8 Item: Title: Production of DHA from Glycerol in a Basket Reactor Destination: subsection.2.9 Item: Title: Results and Discussions Destination: section.3 Children: Item: Title: Characterization of Soluble Enzymes Destination: subsection.3.1 Item: Title: Immobilization of Enzymes Destination: subsection.3.2 Item: Title: Post-Immobilization Modifications for GlyDH and NOX Catalysts Destination: subsection.3.3 Item: Title: Determination of Operational Compromise Conditions Destination: subsection.3.4 Item: Title: Production of Dihydroxyacetone from Glycerol Using the Development Multi-Enzymatic System Destination: subsection.3.5 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: appendix.A. Info: Title: Catalytic Potential-Guided Design of Multi-Enzymatic System for DHA Production from Glycerol Author: Carolina Fernández-Pizarro, Lorena Wilson and Oscar Romero Subject: The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni+2 agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations. 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