Simultaneously Enhanced Catalytic Activity and Thermostability of a Baeyer-Villiger Monooxygenase from Oceanicola granulosus by Reshaping the Binding Pocket.

Enzymatic oxygenation of various cyclic ketones into lactones via Baeyer-Villiger monooxygenases (BVMOs) could provide a promising route for synthesizing fragrances and pharmaceutical ingredients. However, unsatisfactory catalytic activity and thermostability restricted their applications in the pharmaceutical and food industries. In this study, we successfully improved the catalytic activity and thermostability of a Baeyer-Villiger monooxygenase (OgBVMO) from Oceanicola granulosus by reshaping the binding pocket. As a result, mutant OgBVMO-Re displayed a 1.0- to 6.4-fold increase in the activity toward branched cyclic ketones tested, accompanied by a 3 °C higher melting point, and a 2-fold longer half-life time (t1/2 (45 °C)). Molecular dynamics simulations revealed that reshaping the binding pocket achieved strengthened motion correlation between amino acid residues, appropriate size of the substrate-binding pocket, beneficial surface characteristics, lower energy barriers, and shorter nucleophilic distance. This study well demonstrated the trade-off between the enzyme activity and thermostability by reshaping the substrate-binding pocket, paving the way for further engineering other enzymes.

Continuous Fatty Acid Decarboxylation using an Immobilized Photodecarboxylase in a Membrane Reactor.

The realm of photobiocatalytic alkane biofuel synthesis has burgeoned recently; however, the current dearth of well-established and scalable production methodologies in this domain remains conspicuous. In this investigation, we engineered a modified form of membrane-associated fatty acid photodecarboxylase sourced from Micractinium conductrix (McFAP). This endeavour resulted in creating an innovative assembled photoenzyme-membrane (protein load 5 mg cm-2 ), subsequently integrated into an illuminated flow apparatus to achieve uninterrupted generation of alkane biofuels. Through batch experiments, the photoenzyme-membrane exhibited its prowess in converting fatty acids spanning varying chain lengths (C6-C18). Following this, the membrane-flow mesoscale reactor attained a maximum space-time yield of 1.2 mmol L-1 h-1 (C8) and demonstrated commendable catalytic proficiency across eight consecutive cycles, culminating in a cumulative runtime of eight hours. These findings collectively underscored the photoenzyme-membrane's capability to facilitate the biotransformation of diverse fatty acids, furnishing valuable benchmarks for the conversion of biomass via photobiocatalysis.

Structure-Based Redesign of a Methanol Oxidase into an "Aryl Alcohol Oxidase" for Enzymatic Synthesis of Aromatic Flavor Compounds.

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H2O2 as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and kcat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.

A Highly Efficient Three-Liquid-Phase-Based Enzymatic One-Pot Multistep Reaction System with Recoverable Enzymes for the Synthesis of Biodiesel.

A three-liquid-phase system (TLPS) was developed and used as a novel enzymatic one-pot multistep reaction (EOMR) system. In this system, lipase and phospholipase were enriched in a single liquid phase with a high recovery (ca. 98%) and then used for the simultaneous catalysis of mutually inhibiting and interfering reactions (hydrolysis of phospholipids and glyceride in crude oil). A novel emulsion containing the two dispersed droplets (W2/O/W2 and W1/W2 emulsion structures) could be the key reason for this phenomenon because the emulsion system not only provided a new catalytic interface but also relieved the product inhibition. As a result, the content of free fatty acid (main hydrolysate of the glyceride) and the removal of phospholipid from the crude oil could be increased to 96 and 95%, respectively, within 1 h. The product obtained from the EOMR was directly used in the production of biodiesel via enzymatic esterification, and the content of fatty acid methanol ester could be increased to 93% within 2 h. Furthermore, the enzymes in the middle phase could also be reused, at least for eight rounds without significant loss in catalytic efficiency. Therefore, the TLPS could be considered as an ideal catalytic platform for the EOMR.

Optimized Extraction of Total Triterpenoids from Jujube (Ziziphus jujuba Mill.) and Comprehensive Analysis of Triterpenic Acids in Different Cultivars.

Triterpenoid compounds are one of the main functional components in jujube fruit. In this study, the optimal process for ultrasound-assisted extraction (UAE) of total triterpenoids from jujube fruit was determined using response surface methodology (RSM). The optimal conditions were as follows: temperature of 55.14 °C, ethanol concentration of 86.57%, time of 34.41 min, and liquid-to-solid ratio of 39.33 mL/g. The triterpenoid yield was 19.21 ± 0.25 mg/g under optimal conditions. The triterpenoid profiles and antioxidant activity were further analyzed. Betulinic acid, alphitolic acid, maslinic acid, oleanolic acid, and ursolic acid were the dominant triterpenoid acids in jujube fruits. Correlation analysis revealed a significant positive correlation between the major triterpenic acids and antioxidant activities. The variations of triterpenoid profiles and antioxidant activity within the jujube fruits and the degree of variation were evaluated by hierarchical cluster analysis (HCA) and principal component analysis (PCA), respectively. The results provide important guidance for the quality evaluation and industrial application of jujube fruit.

Cascade Synthesis from Cyclohexane to ϵ-Caprolactone by Visible-Light-Driven Photocatalysis Combined with Whole-Cell Biological Oxidation.

Cyclohexane was directly oxy-functionalised to ϵ-caprolactone through a cascade reaction sequence combining visible-light-driven photocatalysis with cyclohexanone monooxygenase (CHMO) whole-cell biocatalysis. Two available photocatalysts, Au-doped TiO2 (Au-TiO2 ) and graphitic carbonitride (g-C3 N4 ), were evaluated in the experiment and some optimisations to the cascade reaction were applied. In stepwise mode, the highest degree of conversion from cyclohexanol to ϵ-caprolactone can be up to 41 %, with use of g-C3 N4 . The cascade reaction from cyclohexane to ϵ-caprolactone is achievable under a light intensity of 149 µW cm-2 .

Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline Oxidase and a Peroxygenase in the Presence of Natural Deep Eutectic Solvents.

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

Natural Deep Eutectic Solvents as Multifunctional Media for the Valorization of Agricultural Wastes.

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2 O2 to promote the epoxidation reaction. The proof-of-concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.

Recovery of lead from lead paste in spent lead acid battery by hydrometallurgical desulfurization and vacuum thermal reduction.

Lead sulfate, lead oxides and lead metal are the main component of lead paste in spent lead acid battery. When lead sulfate was desulfurized and transformed into lead carbonate by sodium carbonate, lead metal and lead oxides remained unchanged. Lead carbonate is easily decomposed to lead oxide and carbon dioxide under high temperature. Namely, vacuum thermal process is the reduction reaction of lead oxides. A compatible environmental process consisted of hydrometallurgical desulfurization and vacuum thermal reduction to recycle lead was investigated in this research. Lead paste was firstly desulfurized with sodium carbonate, by which, the content of sulfur declined from 7.87% to 0.26%. Then, the desulfurized lead paste was reduced by charcoal under vacuum. Under the optimized reaction conditions, i.e., vacuum thermal reduction at temperature 850°C under 20 Pa for 45 min, a 22.11×10(-2) g cm(-2) min(-1) reduction rate, and a 98.13% direct recovery ratio of fine lead (99.77%) had been achieved, respectively.

Separation and analysis of trace volatile formaldehyde in aquatic products by a MoO3/polypyrrole intercalative sampling adsorbent with thermal desorption gas chromatography and mass spectrometry.

An in situ embedded synthesis strategy was developed for the preparation of a MoO3 /polypyrrole intercalative sampling adsorbent for the separation and analysis of trace volatile formaldehyde in aquatic products. Structural and morphological characteristics of the MoO3 /polypyrrole intercalative adsorbent were investigated by a series of characterization methods. The MoO3 /polypyrrole sampling adsorbent possessed a higher sampling capacity and selectivity for polar formaldehyde than commonly used commercial adsorbent Tenax TA. Finally, the MoO3 /polypyrrole adsorbent was packed in the thermal desorption tube that was directly coupled to gas chromatography with mass spectrometry for the analysis of trace volatile formaldehyde in aquatic products. Trace volatile formaldehyde from real aquatic products could be selectively sampled and quantified to be 0.43-6.6 mg/kg. The detection limit was achieved as 0.004 µg/L by this method. Good recoveries for spiked aquatic products were achieved in range of 75.0-108% with relative standard deviations of 1.2-9.0%.

Rapid analysis of trace volatile formaldehyde in aquatic products by derivatization reaction-based surface enhanced Raman spectroscopy.

Toxic formaldehyde is sometimes used illegally as a food preservative, however, on-site rapid analysis of trace formaldehyde in aquatic products remains a challenge. In this work, a simple on-site rapid quantification method for trace volatile formaldehyde in aquatic products was developed by a derivative reaction-based surface enhanced Raman spectroscopy (SERS) technique coupled with a homemade portable purge-sampling device. Trace formaldehyde separated from complicated aquatic matrices via a purge-sampling procedure was reacted with a derivative reagent to produce a Raman-active analyte for consequent SERS analysis. Au/SiO2 nanoparticles (NPs) were employed as the enhancement substrate to achieve significant enhancement of Raman signal intensity. Conditions of derivative reaction and SERS detection were optimized in detail, and the selectivity of this analytical method was also evaluated based on related analogs. Under optimal conditions, an extremely low detection limit of 0.17 µg L(-1) was achieved. Trace volatile formaldehyde can be found in fresh squid and shrimp samples without obvious matrix interference, and this was quantified to be 0.13-0.21 mg kg(-1) using the described method. The recoveries of spiked aquatic product samples were found to be 70.0-89.1% with RSDs of 2.3-7.2% (n = 3). The results suggest that the proposed method is reliable and suitable for on-site rapid analysis of trace formaldehyde in aquatic products.

Preparation of novel alumina nanowire solid-phase microextraction fiber coating for ultra-selective determination of volatile esters and alcohols from complicated food samples.

A novel alumina nanowire (ANW) solid-phase microextraction (SPME) fiber coating was prepared by a simple and rapid anodization-chemical etching method for ultra-selective determination of volatile esters and alcohols from complicated food samples. Preparation conditions for ANW SPME fiber coating including corrosion solution concentration and corrosion time were optimized in detail for better surface morphology and higher surface area based on scanning electron microscope (SEM). Under the optimum conditions, homogeneous alumina nanowire structure of ANW SPME fiber coating was achieved with the average thickness of 20 µm around. Compared with most of commercial SPME fiber coatings, ANW SPME fiber coatings achieved the higher extraction capacity and special selectivity for volatile esters and alcohols. Finally, an efficient gas sampling technique based on ANW SPME fiber coating as the core was established and successfully applied for the ultra-selective determination of trace volatile esters and alcohols from complicated banana and fermented glutinous rice samples coupled with gas chromatography/mass spectrometry (GC/MS) detection. It was interesting that 25 esters and 2 alcohols among 30 banana volatile organic compounds (VOCs) identified and 4 esters and 7 alcohols among 13 identified VOCs of fermented glutinous rice were selectively sampled by ANW SPME fiber coatings. Furthermore, new analytical methods for the determination of some typical volatile esters and alcohols from banana and fermented glutinous rice samples at specific storage or brewing phases were developed and validated. Good recoveries for banana and fermented glutinous rice samples were achieved in range of 108-115% with relative standard deviations (RSDs) of 2.6-6.7% and 80.0-91.8% with RSDs of 0.3-1.3% (n=3), respectively. This work proposed a novel and efficient gas sampling technique of ANW SPME which was quite suitable for ultra-selectively sampling trace volatile esters and alcohols from complicated food samples.

Preparation of polypyrrole composite solid-phase microextraction fiber coatings by sol-gel technique for the trace analysis of polar biological volatile organic compounds.

Two novel polypyrrole (PPy) composite solid-phase microextraction (SPME) fiber coatings involving polypyrrole ß-naphthalenesulfonic acid (PPy/ß-NSA) and polypyrrole graphene (PPy/GR) composite SPME fiber coatings were prepared by a simple sol-gel technique for selectively sampling relatively polar biological volatile organic compounds (VOCs). Crucial preparation conditions of the PPy composite SPME fiber coatings were optimized and are discussed in detail. Physical tests suggested that the PPy composite SPME fiber coatings possessed a porous surface morphology, stable chemical and thermal properties. Due to the inducing polar functional groups in the PPy molecule, the PPy composite SPME fiber coatings achieved a higher extraction capacity and special selectivity for the polar biological VOCs with conjugate structures, compared with commercial SPME fiber coatings. Enrichment factors of most of the VOCs by the PPy/ß-NSA and PPy/GR SPME fibers were much higher than those achieved by common commercially available SPME fiber coatings. Finally, the PPy/ß-NSA and PPy/GR SPME fiber coatings were applied for the trace analysis of typical polar VOCs from ant and coriander samples coupled with gas chromatography/mass spectrometry (GC/MS) detection, respectively. It was satisfactory that the average contents of 4-heptanone, 4-heptanol, 4-nonanone and methyl 5-methylsalicylate from ant samples were actually found to be 28.0, 58.7, 3.0 and 0.6 µg g(-1), and the average contents of nonane, decanal, undecanal and dodecanal from coriander samples were actually found to be 0.79, 0.13, 0.06 and 0.21 µg g(-1). The results suggested that PPy composite SPME coatings will be a potentially excellent sampling technique for the trace analysis of polar biological VOCs.