Immobilization Screening and Characterization of an Alcohol Dehydrogenase and its Application to the Multi-Enzymatic Selective Oxidation of 1,-Omega-Diols

Alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus (BsADH) is a NADH-dependent enzyme catalyzing the oxidation of alcohols, however its thermal and operational stabilities are too low for its long-term use under non-physiological conditions. Enzyme immobilizations emerges as an attractive tool to enhance the stability of this enzyme. In this work, we have screened a battery of porous carriers and immobilization chemistries to enhance the robustness of a His-tagged variant of BsADH. The selected carriers recovered close to 50% of the immobilized activity and increased enzyme stability from 3 to 9 times compared to the free enzyme. We found a trade-off between the half-life time and the specific activity as a function of the relative anisotropy values of the immobilized enzymes, suggesting that both properties are oppositely related to the enzyme mobility (rotational tumbling). The most thermally stable heterogeneous biocatalysts were coupled with a NADH oxidase/catalase pair co-immobilized on porous agarose beads to perform the batch oxidation of five different 1,ω-diols with in situ recycling of NAD+. Only when His-tagged BsADH was immobilized on porous glass functionalized with Fe3+, the heterogeneous biocatalyst oxidized 1, 5-pentanediol with a conversion higher than 50% after five batch cycles. This immobilized multi-enzyme system presented promising enzymatic productivities towards the oxidation of three different diols. Hence, this strategical study accompanied by a functional and structural characterization of the resulting immobilized enzymes, allowed us selecting an optimal heterogeneous biocatalyst and their integration into a fully heterogeneous multi-enzyme system.

Cross-Linked Chitosan/Multi-Walled Carbon Nanotubes Composite as Ecofriendly Biocatalyst for Synthesis of Some Novel Benzil Bis-Thiazoles

Aminohydrazide cross-linked chitosan (CLCS) and its MWCNTs (CLCS/MWCNTs) were formulated and utilized as a potent ecofriendly basic heterogeneous biocatalyst under ultrasonic irradiation for synthesis of two novel series of benzil bis-aryldiazenylthiazoles and benzil bis-arylhydrazonothiazolones from the reaction of benzil bis-thiosemicarbazone with 2-oxo-N′-arylpropanehydrazonoyl chlorides and ethyl 2-chloro-2-(2-phenylhydrazono) acetates, respectively. The chemical structures of the newly synthesized derivatives were elucidated by spectral data and alternative methods, where available. Additionally, their yield % was estimated using a traditional catalyst as TEA and green recyclable catalysts as CLCS and CLCS/MWCNTs composite in a comparative study. We observed that, under the same reaction conditions, the yield % of the desired products increased by changing TEA to CLCS then to CLCS/MWCNT from 72–78% to 79–83% to 84–87%, respectively. The thermal stability of the investigated samples could be arranged as CLCS/MWCNTs composite > CLCS > chitosan, where the weight losses of chitosan, CLCS and CLCS/MWCNTs composite at 500 °C were 65.46%, 57.95% and 53.29%, respectively.

PTFE-Carbon Nanotubes and Lipase B from Candida antarctica—Long-Lasting Marriage for Ultra-Fast and Fully Selective Synthesis of Levulinate Esters

An effective method for levulinic acid esters synthesis by the enzymatic Fischer esterification of levulinic acid using a lipase B from Candida antarctica (CALB) immobilized on the advanced material consisting of multi-wall carbon nanotubes (MWCNTs) and a hydrophobic polymer—polytetrafluoroethylene (Teflon, PTFE)—as a heterogeneous biocatalyst, was developed. An active phase of the biocatalyst was obtained by immobilization via interfacial activation on the surface of the hybrid material MWCNTs/PTFE (immobilization yield: 6%, activity of CALB: 5000 U∙L∙kg−1, enzyme loading: 22.5 wt.%). The catalytic activity of the obtained biocatalyst and the effects of the selected reaction parameters, including the agitation speed, the amount of PTFE in the CALB/MWCNT-PTFE biocatalyst, the amount of CALB/MWCNT-PTFE, the type of organic solvent, n-butanol excess, were tested in the esterification of levulinic acid by n-butanol. The results showed that the use of a two-fold excess of levulinic acid to n-butanol, 22.5 wt.% of CALB on MWCNT-PTFE (0.10 wt.%) and cyclohexane as a solvent at 20 °C allowed one to obtain n-butyl levulinate with a high yield (99%) and selectivity (>99%) after 45 min. The catalyst retained its activity and stability after three cycles, and then started to lose activity until dropping to a 69% yield of ester in the sixth reaction run. The presented method has opened the new possibilities for environmentally friendly synthesis of levulinate esters.

Exploring the in Vitro Operating Window of Glycosyltransferase PtUGT1 from Polygonum Tinctorium for a Biocatalytic Route to Indigo Dye

The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive by-products is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of > 98% for enzyme loadings up to 7.6 w-%. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.

Exploring the in Vitro Operating Window of Glycosyltransferase PtUGT1 from Polygonum Tinctorium for a Biocatalytic Route to Indigo Dye

The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive by-products is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of > 98% for enzyme loadings up to 7.6 w-%. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.

Toward a New Way for the Valorization of Miscanthus Biomass Produced on Metal-Contaminated Soils Part 2: Miscanthus-Based Biosourced Catalyst: Design, Preparation, and Catalytic Efficiency in the Synthesis of Moclobemide

The conception of two biosourced catalysts (biocatalysts) using stems of miscanthus from the first part of this study are described herein. The temperature and the process used to extract metals from plant as mixture of Lewis acids were investigated in detail and proved to be essential in the design of the biosourced catalysts and their catalytic efficiency. One part of the crude mixture of Lewis acids extracted from the aerial parts of miscanthus plants was used without further treatment as a homogeneous biocatalyst (M1), and the other part was supported on montmorillonite K10 to provide a heterogeneous biocatalyst (MM1). M1 and MM1 were next tested in the synthesis of moclobemide (main ingredient of a drug used to treat depression) and led to excellent yield. Additional comparative experiments with different commercial metallic salts (NaCl, KCl, CaCl2, MgCl2, CuCl2, ZnCl2, FeCl2, FeCl3, MnCl2, and AlCl3) and their mixtures were carried out and underlined the importance of the multimetallic synergy on catalytic activity. Finally, a comparison of this new synthetic method assisted by the biosourced catalyst with the previously described procedures to access moclobemide was realized by calculating their green chemistry metrics. This study revealed that the use of the biosourced catalyst led to one of the greenest synthetic methods described today to produce moclobemide.