Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids DOI Creative Commons
Javier Santiago‐Arcos, Susana Velasco‐Lozano,

Eleftheria Diamanti

et al.

Research Square (Research Square), Journal Year: 2023, Volume and Issue: unknown

Published: Nov. 23, 2023

Abstract Cell-free biocatalysis is gaining momentum in the production of value-added chemicals, particularly stepwise reaction cascades. However, stability enzyme cascades industrial settings often compromised when using free enzymes. In this study, we have developed a stable multifunctional heterogeneous biocatalyst co-immobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved operational efficiency and by fine-tuning loading spatial organization. Stability issues are overcome through post-immoblization polymer coating. The general applicability demonstrated its scale-up both batch packed bed reactors, allowing product yield > 80%. continuous process fed with H2O2 as oxygen source, reaching Space-Time Yield (STY) 0.76 g·L− 1·h− 1, maintained for first 12 hours. Finally, flow system telescoped second plug-flow reactor different biocatalyst. As result, 6-enzyme 2-reactor sequentially transforms 1,ω-aminoacids while in-situ recycling NAD+, depleting generating O2.

Language: Английский

Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis DOI Creative Commons
Alba Ledesma‐Fernandez, Susana Velasco‐Lozano, Javier Santiago‐Arcos

et al.

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: May 4, 2023

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger substrate channeling effect, allowing for efficient cofactor reuse industrial potential. However, precise nanometric organization of challenges the design scaffolds. In this study, we create nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding biocatalysis. We genetically fuse TRAP domains and program them to selectively orthogonally recognize peptide-tags fused enzymes, which upon binding form spatially metabolomes. addition, encodes sites reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, catalytic efficiency. This concept is demonstrated biosynthesis amino acids amines using up three enzymes. Scaffolded multi-enzyme systems present 5-fold higher specific productivity than non-scaffolded ones. In-depth analysis suggests that NADH between assembled enhances overall cascade throughput product yield. Moreover, immobilize biomolecular on solid supports, creating reusable heterogeneous multi-functional biocatalysts consecutive operational batch cycles. Our results demonstrate potential TRAP-scaffolding spatial-organizing tools increase efficiency cell-free biosynthetic pathways.

Language: Английский

Citations

34
Enzyme-assisted patulin detoxification: Recent applications and perspectives DOI
Shipeng Gao, Yang Zhang, Qiang Sun

et al.

Trends in Food Science & Technology, Journal Year: 2024, Volume and Issue: 146, P. 104383 - 104383

Published: Feb. 18, 2024

Language: Английский

Citations

14
Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids DOI
Javier Santiago‐Arcos, Susana Velasco‐Lozano,

Eleftheria Diamanti

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2024, Volume and Issue: 12(25), P. 9474 - 9489

Published: June 10, 2024

Cell-free biocatalysis is gaining momentum in producing value-added chemicals, particularly stepwise reaction cascades. However, the stability of enzyme cascades industrial settings often compromised when free enzymes are involved. In this study, we have developed a stable multifunctional heterogeneous biocatalyst coimmobilizing five on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved operational efficiency and by fine-tuning loading spatial organization. Stability issues overcome through postimmobilization polymer coating. The general applicability demonstrated its scale-up both batch packed bed reactors, allowing product yield >80%. continuous process fed with H

Language: Английский

Citations

5
Interactive biocatalysis achieved by driving enzyme cascades inside a porous conducting material DOI Creative Commons
Bhavin Siritanaratkul, Clare F. Megarity, Ryan A. Herold

et al.

Communications Chemistry, Journal Year: 2024, Volume and Issue: 7(1)

Published: June 10, 2024

Language: Английский

Citations

4
Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades DOI
Idania L. López, Mercedes Sánchez‐Costa, Alejandro H. Orrego

et al.

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(35)

Published: July 22, 2024

Abstract Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding consensus immobilization protocol for several enzymes forming multi‐enzyme system extremely difficult relies on combinatorial trial‐and‐error approach. Herein, we describe which 17 different carriers functionalized with reactive groups are tested 96‐well microtiter plate to screen up 21 protocols 18 enzymes. This screening includes an activity stability assay select optimal chemistry achieve most active stable The information retrieved from can be rationalized using Python‐based application CapiPy. Finally, through scoring results, find assemble immobilized four‐enzyme transform vinyl acetate into ( S )‐3‐hydroxybutyric acid. methodology opens path speed prototyping pathways chemical manufacturing.

Language: Английский

Citations

4
Self-Sufficient Heterogeneous Biocatalysis through Boronic Acid-Diol Complexation of Adenylated Cofactors DOI

Eleftheria Diamanti,

Susana Velasco‐Lozano, Daniel A. Grajales‐Hernández

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2023, Volume and Issue: 11(39), P. 14409 - 14421

Published: Sept. 21, 2023

Self-sufficient heterogeneous biocatalysts (ssHB) are promising candidates for implementing cofactor-dependent enzymes in chemical biomanufacturing. Most strategies coimmobilizing cofactors with dehydrogenases on porous agarose microbeads involve the use of cationic polymers (i.e., polyethylenimine, PEI) that interact electrostatically phosphate groups their corresponding phosphorylated cofactors. Although latter is a powerful and versatile approach, ionic bonds disrupted biotransformations operating at high strength, where screening bonded ions takes place. Harnessing ribose present adenylated cofactors, we immobilize selection these (NAD(P)H, NAD(P)+, FAD, ATP) functionalized boronic acid to establish reversible covalent between cis-diol cofactor backbone acid. To do so, functionalize cobalt-activated beads (AG-B/Co2+) coimmobilization dependent His-tagged dehydrogenases. First, demonstrate cofactor-support interactions but show resistance against salt concentrations, overcoming main limitation current self-sufficient biocatalysts. Then, coimmobilized several investigated functionality stability ssHBs reductive aminations performed under strength both batch flow reactors. As result, manage reuse immobilized 3.5 × 105 167 times, respectively. This work expands usefulness ssBHs hitherto bioprocess regardless media.

Language: Английский

Citations

7
Genetically Encoded Multienzyme Particles for the Biosynthesis of Putrescine from l-Arginine DOI Creative Commons

Xu Xiong,

Bradley S. Heater,

Wing Yin Ho

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2024, Volume and Issue: 12(2), P. 773 - 784

Published: Jan. 4, 2024

Immobilized enzyme biocatalysts have wide industrial applications, but the multiple steps involved in their preparation significantly increase time, effort, and cost to produce them. Our group has previously developed a platform for direct immobilization of enzymes based on crystal-forming Cry3Aa protein. The application this been demonstrated individual enzymes, with molecular size limitation approximately 50 kDa. However, biosynthesis many compounds involve cascade reactions utilizing various sizes including those larger than limit. Herein, we report use Cry homologue, Cry1Ab, vivo coimmobilization two putrescine─human arginase I (hArg, 35 kDa) ornithine decarboxylase (ODC, 79 kDa). We show that Cry1Ab-mediated enhanced thermostability these allowing efficient recyclable conversion arginine putrescine, yielding 30 mM putrescine per cycle at nearly 100% min first seven cycles. Furthermore, produced could be directly converted thermostable polyamide nylon-4,6.

Language: Английский

Citations

2
Directionally co-immobilizing glucose oxidase and horseradish peroxidase on three-pronged DNA scaffold and the regulation of cascade activity DOI
Zhenzhen Zhang, Yu Han, Jingjing Cao

et al.

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: 282, P. 137072 - 137072

Published: Oct. 30, 2024

Language: Английский

Citations

1
Heterogeneous biocatalytic reduction of 5-(hydroxy)methyl furfural using two co-immobilised alcohol dehydrogenases DOI Creative Commons
Jakub F. Kornecki,

André Pick,

Pablo Domı́nguez de Marı́a

et al.

RSC Sustainability, Journal Year: 2023, Volume and Issue: 1(7), P. 1883 - 1895

Published: Jan. 1, 2023

Cofactor self-sustained reduction of HMF to BHMF catalyzed by a co-immobilized bi-enzymatic system, capable in situ recycling NADH along the process.

Language: Английский

Citations

3
Removal of Emerging Contaminants by Degradation during Filtration: A Review of Experimental Procedures and Modeling DOI Open Access
Tomás Undabeytia López,

José Manuel Jiménez-Barrera,

Shlomo Nir

et al.

Water, Journal Year: 2023, Volume and Issue: 16(1), P. 110 - 110

Published: Dec. 27, 2023

Here, we review the efficient removal of organic micropollutants from water by degradation during filtration using specialized bacteria and enzymes. In both approaches, filter provides essential binding sites where can occur. A model is presented that enables simulation prediction kinetics for a given pollutant concentration, flow rate, dimensions facilitate design experiments capacity estimates; it predicts establishment steady state, which emerging concentrations pollutants remain constant. One method to remove cyanotoxins produced Microcystis cyanobacteria, pose threat at above 1.0 µg L−1, use an activated granular carbon with biofilm; this resulted in complete filtered toxins (5 L−1) long experiment (225 d). This system was analyzed predicted toxin when applied 10-fold-higher concentration. Enzymes are also used processes trace contaminants, mostly through membrane bioreactors, enzyme continuously introduced or maintained bioreactor, immobilized on membrane.

Language: Английский

Citations

2