Search

Lignocellulose, the most abundant biomass resource, was a promising alternative to oil resources. The acid catalyst can hydrolyze lignocellulose to reduce sugars, the raw material for lipid production via fermentation. However, the by-product yield during hydrolysis would inhibit yeast activity when directly applying lignocellulose hydrolysate from dilute acid-catalyzed to microbial lipid production. In this paper, a solid acid catalyst highly selective for lignocellulose (rice straw) hydrolysis was prepared to minimize the effects of by-products. Also, a combination of solid acid catalysts to generate glucose with subsequent microbial fermentation was studied to obtain the higher efficiency of lipid production. A carbon-based solid acid catalyst with an enhanced hydrolysis capacity of cellulose and lignocellulose was efficiently prepared. It resulted in 71% yield of glucose with 97% of selectivity with optimal catalyst in hydrolyzing milled cellulose. The optimal preparation conditions of the catalyst were: carbonization temperature 642 K, sulfonation temperature 412 K, initial sulfonation time 12.5 h, and liquid-to-solid ratio 11.5. To expand the catalyst’ application, the rice straw hydrolysate catalyzed by the prepared catalyst was then applied to the microbial lipid production for the first time. Both dilute sulfuric acid and Amberlyst-15 resin as hydrolysis catalysts caused a decrease in yeast biomass, lipid content, and lipid yield. However, when the hydrolysate was obtained from co-heating rice straw with sucralose-derived solid acid catalyst, the yeast biomass, lipid content, and lipid yield in the presence of 5-HMF (0.39 g/L) were 12.6 g/L, 48.1%, and 7.6 g/L, respectively, and were almost similar to the control group. Our results showed that a combination of solid acid catalysts with subsequent microbial fermentation to generate lipid was confirmed to be environmentally friendly and low cost.

In this study, three kinds of Maillard reaction products (MRPs) have been, for the first time, successfully prepared by conjugating soy protein isolate (SPI) with isomaltooligosaccharide, xylooligosaccharide, or galactooligosaccharide at 80 °C for 30 or 60 min and applied for the construction of Lactobacillus casei (L. casei) microcapsules. The results showed that MRPs exhibited enhanced antioxidative activities compared with their physically mixed counterparts. The digested MRPs displayed excellent resistance to pathogenic bacteria and promoted the growth of L. casei. Moreover, MRP-encapsulated L. casei showed a higher survival rate than free L. casei under tested adverse conditions including heat treatment, storage, and mechanical forces. Under simulated digestion conditions, the viability of L. casei decreased from 8.8 log cfu/mL to 1.6 log cfu/mL, while that of MRP-encapsulated L. casei was maintained at 7.4 log cfu/mL. Thus, MRP-based SPI-oligosaccharide conjugates exhibited great potential for microencapsulation of probiotics.

A copper analogue of Prussian blue incorporated multienzyme system was successfully established via a biomimetic mineralization process, whereas CPB with peroxidase-like activity provided protecting effect for encapsulated glucose oxidase from attack in hostile conditions. The hybrid composite was developed as a colorimetric biosensor for ultrasensitive detection of glucose, with a low detection limit (0.48 μM).

A sucralose-derived magnetically separable carbon-based solid acid catalyst with cellulose-binding and catalytic sites has been successfully prepared by incompletely carbonizing the sulfonated sucralose with Fe3+ at high temperature to load ɤ-Fe2O3 nanoparticles onto the surface of the catalyst. The resultant magnetic catalyst was fully characterized and found to be suitable in the hydrolysis of cellulose. Microcrystalline cellulose pretreated by ball milling achieved the highest glucose yield of 32% using this neoteric catalyst at 363 K. Catalysts retained 90% of their activity after five runs recycle. The introduction of magnetic properties into the catalyst system will combine the high catalytic capacity for hydrolyzing cellulose and magnetic materials’ separation convenience.

Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce KgBDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming KgBDH-SC-ST-FDH-SiO2 with the capability of reducing 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, KgBDH-SC-ST-FDH-SiO2 showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of (R)-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity.

In this study, the purification and characterization of a novel polysaccharide-based bioflocculant BM2 produced by a bacterium Bacillus megaterium strain PL8 with self-flocculating property were investigated. The results showed that BM2 was an acidic polysaccharide composed of Gal, GalUA, Glc, GlcUA and Man at a molar ratio of 45.1: 33.8:9.3:9.2:2.4, respectively. The molecular weight of BM2 was 4.55 × 106 Da. BM2 had high flocculation efficiencies across a wide pH ranged from 4 to 11 and a wide temperature ranged from 20 to 100 °C towards kaolin clay. BM2 was a cation-independent bioflocculant which could achieve high flocculation activity without the addition of other cations. Adsorption bridging was the main mechanism in the flocculation process of BM2 towards kaolin clay. The BM2 also displayed a high removal efficiency in terms of Congo red (88.14%) and Pb2+ ions (82.64%). These results suggested that BM2 had a great potential as an efficient bioflocculant candidate in wastewater treatment.

The epoxide hydrolase gene (SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity (E-value) of the recombinant SpEH was 7.7 and the yield of the remaining (R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether (rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues (V195, V196, F218, N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEHV196A/N226A/M332A had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg−1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and pH for purified SpEHV196A/N226A/M332A to catalyze the hydrolysis of rac-PGE were 25 °C and 7.0 with 200 U·mg−1. The enantioselectivity and yield of the remaining (R)-PGE of E. coli_SpEHV196A/N226A/M332A increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEHV196A/N226A/M332A has a greater affinity toward (S)-PGE than (R) - PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of (R)-PGE, resulting in its improved enantioselectivity.

Human milk fat substitutes (HMFSs) are the structured lipids intended for infant formula. It provides energy and essential fatty acid for infant. HMFSs are mainly prepared by enzymatic method. In this study, we aim to explore the potential for producing HMFSs by fermentation using R. opacus. The results indicated that different compounds with chain length from 12 to 18, used as carbon source, could be incorporated into triacylglycerols directly. Polyunsaturated fatty acids in term of ARA, EPA, DHA could enter the kennedy pathway directly and involved in the biosynthesis of triacylglycerols. GC, UPLC-MS and 13C-NMR analysis demonstrated that typical structured lipids β-OPL (40.09%) was synthesized in R. opacus. Transcriptome analysis revealed that β-oxidation, fatty acid elongation and kennedy pathways existed in R. opacus. It was concluded that fatty acid supplied as carbon source could enter the kennedy pathways directly or via the de novo fatty acid biosynthesis pathway depending on the chain length, thus, affect the triacylglycerol species formed in the Rhodococcus opacus.

Microbial contamination, especially in large-scale processes, is partly a life-or-death issue for industrial fermentation. Therefore, the aim of this research was to create an anti-microbial contam...

Human milk fat substitute (HMFS) is a class of structured lipids widely used in infant formulas. Herein, HMFS was prepared by Rhodococcus opacus fermentation. The substrate oils suitable for HMFS production were coconut oil (66.1-57.5%), soybean oil (17.5-26.5%), high oleic acid sunflower oil (5.4-4.5%), Antarctic krill oil (9-9.5%), and fungal oil (2%). Six HMFSs were prepared, among which HMFS V and VI were similar to human milk fat from Chinese in terms of fatty acid composition and triacylglycerol species. The sn-2 position of HMFS was occupied by palmitic acid (49.31 and 43.48% in HMFS V and VI, respectively). The major triacylglycerols were OPL, OPO, and LPL, accounting for 15.90, 9.49, and 6.84 and 17.52, 8.44, and 8.55% in HMFS V and VI, respectively. This study is the first to prepare structured lipids intended for infant formula through fermentation, providing a novel strategy for the edible oil industry.

Papain (PA) immobilized onto magnetic nanocrystalline cellulose (PA@MNCC) was successfully fabricated and adopted as an efficient biocatalyst for the synthesis of N-(benzyloxycarbonyl)-alanyl-histidine (Z-Ala-His) dipeptide. Introducing deep eutectic solvents (DESs) as reaction media promoted the synthesis of the Z-Ala-His dipeptide. The effects of reaction conditions on the yield of papain catalytic Z-Ala-His were systematically investigated with the highest yield of 68.4%, which was higher than free papain (63.3%). Besides, this novel PA@MNCC composite can be easily recycled from the reaction system by magnetic forces. In a word, the PA@MNCC composite exhibited great potential for efficient biosynthesis of dipeptide in DESs.

Microbial contamination is a serious challenge that needs to be overcome for the successful biosynthesis of 2,3-butanediol (2,3-BD). However, traditional strategies such as antibiotic administration or sterilization are costly, have high energy demands, and may increase the risk of antibiotic resistance. Here, we intend to develop a robust strategy to achieve non-sterilized fermentation of 2,3-BD. Briefly, the robust strain can metabolize unconventional chemicals as essential growth nutrients, and therefore, outcompete contaminant microbes that cannot use unconventional chemicals. To this end, Klebsiella pneumoniae OU7, a robust strain, was confirmed to rapidly exploit urea and phosphite (unconventional chemicals) as the primary sources of nitrogen (N) and phosphorus (P), and withstand deliberate contamination in the possibly contaminated systems. Secondly, metabolic engineering, pathogenicity elimination and adaptive laboratory evolution were successively performed, endowing the best strain with an excellent fermentation performance for safe 2,3-BD production. Finally, 84.53 g L−1 of 2,3-BD was synthesized with a productivity of 1.17 g L−1 h−1 and a yield of 0.38 g g−1 under the non-sterilized system. In summary, our technique reduces labor and energy costs and simplifies the fermentation process because sterilization does not need to be performed. Thus, our work will be beneficial for the sustainable synthesis of 2,3-BD.

The asymmetric addition of ammonia to unsaturated acids using engineered methylaspartate ammonia lyase (MAL) is a particularly attractive and atom-economic method for the synthesis of unnatural ami...

Two polysaccharides, DGS1 and DGS2, were obtained by solid-state fermentation (SSF) of Fusarium solani DO7, an endophytic fungus isolated from the orchid Dendrobium officinale. Structural characterizations revealed that DGS1 consisted of arabinose, glucose, mannose and galactose with a molar ratio of 2.9:13.4:3.0:1, respectively, and contained (1 → 5)-Araf, (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 3)-Manp, (1 → 2,6)-Manp and (1 → 6)-Galp glycosidic linkages, while DGS2 was composed of arabinose, glucose, mannose and galactose in a molar ratio of 3.5:8.1:2.1:1, respectively, and contained (1 → 5)-Araf, (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 3)-Manp and (1 → 6)-Galp glycosidic linkages. Neither polysaccharide was toxic to human embryonic kidney cells or mouse RAW 264.7 macrophage cells. An immunomodulatory activity assay indicated that both polysaccharides could significantly enhance the levels of TNF-α, IL-6 and NO by activating TNF-α, IL-6 and iNOs gene expression, respectively, especially DGS2. Interestingly, DGS2 also possessed relatively high antioxidant activity. These results illustrate that, due to its cost-effectiveness and environmentally friendly features, SSF has significant potential as a commercially competitive source of natural products, including fungal polysaccharides, with immunomodulatory activity.

Glucaric acid (GA) is a major value-added chemicals feedstock and additive, especially in the food, cosmetics, and pharmaceutical industries. The increasing demand for GA is driving the search for a more efficient and less costly production pathway. In this study, a new in vitro multi-enzyme cascade system was developed, which converts sucrose efficiently to GA in a single vessel. The in vitro system, which does not require adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD+ ) supplementation, contains seven enzymes. All enzymes were chosen from the BRENDA and NCBI databases and were expressed efficiently in Escherichia coli BL21(DE3). All seven enzymes were combined in an in vitro cascade system, and the reaction conditions were optimized. Under the optimized conditions, the in vitro seven-enzyme cascade system converted 50 mm sucrose to 34.8 mm GA with high efficiency (75 % of the theoretical yield). This system represents an alternative pathway for more efficient and less costly production of GA, which could be adapted for the synthesis of other value-added chemicals.

Biocatalysis has found enormous applications in sorts of fields as an alternative to chemical catalysis. In the pursue of green and sustainable chemistry, ionic liquids (ILs) have been considered as promising reaction media for biocatalysis, owing to their unique characteristics, such as nonvolatility, inflammability and tunable properties as regards polarity and water miscibility behavior, compared to organic solvents. In recent years, great developments have been achieved in respects to biocatalysis in ILs, especially for preparing various chemicals. This review tends to give illustrative examples with a focus on representative chemicals production by biocatalyst in ILs and elucidate the possible mechanism in such systems. It also discusses how to regulate the catalytic efficiency from several aspects and finally provides an outlook on the opportunities to broaden biocatalysis in ILs.

Pullulanase (EC 3.2.1.41) is a starch-debranching enzyme in the α-amylase family and specifically cleaves α-1,6-glycosidic linkages in starch-type polysaccharides, such as pullulan, β-limited dextrin, glycogen, and amylopectin. It plays a key role in debranching and hydrolyzing starch completely, thus bring improved product quality, increased productivity, and reduced production cost in producing resistant starch, sugar syrup, and beer. Plenty of researches have been made with respects to the discovery of either thermophilic or mesophilic pullulanases, however, few examples meet the demand of industrial application. This review presents the progress made in the recent years from the first aspect of characteristics of pullulanases. The heterologous expression of pullulanases in different microbial hosts and the methods used to improve the expression effectiveness and the regulation of enzyme production are also described. Then, the function evolution of pullulanases from a protein engineering view is discussed. In addition, the immobilization strategy using novel materials is introduced to improve the recyclability of pullulanases. At the same time, we indicate the trends in the future research to facilitate the industrial application of pullulanases.

A newly immobilized Aspergillus niger lipase (ANL@ZnGlu-MNPs) was employed for the preparation of 1,2-epoxycyclooctane by oxidation of cyclooctene. The chosen variables, including substrate concentration, reaction temperature, immobilized enzyme dose, and H2O2 dose, were optimized in the reaction system of ethyl acetate. The yield and the enantiomeric excess of the product were achieved at 56.8% and 84.1%, respectively, under the following optimum reaction conditions: the concentration of substrate (cyclooctene) was 150 mM, the dosages of immobilized enzyme (ANL@ZnGlu-MNPs) and hydrogen peroxide were respectively 100 mg and 4.4 mmol, and the reaction was carried out in the system of 4 mL ethyl acetate at 40 &deg, C. Further study on the operational stability of ANL@ZnGlu-MNPs showed that more than 51.6% of product yield was obtained after reusing for ten batches. A novel immobilized lipase was prepared and applied to synthesize 1,2-epoxycyclooctane from cyclooctene. Although ANL@ZnGlu-MNPs performs well in operational stability and the reaction can achieve high enantiomeric purity of the product, the yield of the catalytic reaction needs to be further improved.

A novel biocompatible magnetic cellulose nanocrystal (MCNC) composite was in situ prepared via a simple co-precipitation-electrostatic-self-assembly technique and was structurally characterized. The results showed that the anionic cellulose nanocrystals (CNCs) were successfully composited with cationic chitosan-coated Fe3O4 by self-assembly technology. The electrostatic interaction between CNCs and chitosan, and that between chitosan and Fe3O4, were the key driving forces for the formation of the composite. Papain, a widely used protease, could be successfully immobilized on the activated MCNCs with formaldehyde. The immobilized papain exhibited higher thermal stability than the free enzyme, with the relative activity being higher than 80% after incubation at 40 °C for 7 h while that of free papain was less than 30%. Also, the pH stability of immobilized papain was superior to that of free papain. Moreover, the immobilized papain showed significantly better tolerance to the three solvents tested compared with its free counterpart. The optimum range of pH for immobilized papain (pH 5–10) was remarkably wider than that of free enzyme (pH 5–7). The relative activities of immobilized papain at 50–70 °C were more than 90%, which significantly surpassed those of free papain. The immobilized papain also manifested excellent storage stability, with relative activity being as high as 93.6% after 16 days of storage at 4 °C. Furthermore, the obtained kinetic constant values showed that papain immobilized on the MCNCs had relatively high catalytic efficiency. Additionally, the immobilized papain could be easily separated and recycled from the reaction system through magnetic forces. Obviously, the prepared MCNCs as novel supports are promising and competitive for enzyme immobilization.

Food contamination caused by microorganisms has become a threat to consumers' health. Exploring antagonistic endophytes from plants of food raw-material and applying bioactive metabolites to inhibit the contamination has been an alternative and safer solution. In this study, we isolated and screened potential antagonistic endophytes from fresh Camellia assamica leaves, which were widely used in tea beverage production. We focused on a strain that showed visible inhibitory activity to Gram-positive bacteria, Gram-negative bacteria, and fungi. It was identified as a member of Bacillus velezensis and named FZ06. The results of genome analysis showed the strain FZ06 had 167 single-copy specific genes, much higher than those of most related strains. Also, 11 potential gene clusters of antimicrobial metabolites were found. Three groups of lipopeptides (surfactin, iturin, and fengycin) were identified by UPLC-MS/MS in purified antimicrobial methanol fraction of strain FZ06. The results of minimum inhibitory concentration (MIC) test proved the lipopeptide extract showed significant inhibitory effect on food spoilage bacteria (MIC 512−2048 μg/mL) and toxigenic fungi (MIC 128−256 μg/mL). In conclusion, this study suggests that the endophytic B. velezensis FZ06 and its lipopeptide extract hold great potential applications in the inhibition of food spoilage bacteria and toxic fungi in food industry.

(R)-1-phenyl-1,2-ethanediol is an important synthon for the preparation of β-adrenergic blocking agents. This study identified a (2R,3R)-butanediol dehydrogenase (KgBDH) from Kurthia gibsonii SC0312, which showed high enantioselectivity for production of (R)-1-phenyl-1,2-ethanediol by reduction of 2-hydroxyacetophenone. KgBDH was expressed in a recombinant engineered strain, purified, and characterized. It showed good catalytic activity at pH 6–8 and better stability in alkaline (pH 7.5–8) than an acidic environment (pH 6.0–7.0), providing approximately 73 and 88% of residual activity after 96 h at pH 7.5 and 8.0, respectively. The maximum catalytic activity was obtained at 45°C; nevertheless, poor thermal stability was observed at >30°C. Additionally, the examined metal ions did not activate the catalytic activity of KgBDH. A recombinant Escherichia coli strain coexpressing KgBDH and glucose dehydrogenase (GHD) was constructed and immobilized via entrapment with a mixture of activated carbon and calcium alginate via entrapment. The immobilized cells had 1.8-fold higher catalytic activity than that of cells immobilized by calcium alginate alone. The maximum catalytic activity of the immobilized cells was achieved at pH 7.5, and favorable pH stability was observed at pH 6.0–9.0. Moreover, the immobilized cells showed favorable thermal stability at 25–30°C and better operational stability than free cells, retaining approximately 55% of the initial catalytic activity after four cycles. Finally, 81% yields (195 mM product) and >99% enantiomeric excess (ee) of (R)-1-phenyl-1,2-ethanediol were produced within 12 h through a fed-batch strategy with the immobilized cells (25 mg/ml wet cells) at 35°C and 180 rpm, with a productivity of approximately 54 g/L per day.

An efficient double-chitinase hydrolysis of crab shell particles (CSP) pretreated by ionic liquids was investigated in this work. First, two family 18 chitinase genes (chip1 and chip2) from Paeniba...

In the study, the extraction, purification, identification and microencapsulation of anthocyanins from saskatoon berries were investigated. 4.48 ± 0.03 mg/ml of anthocyanins were obtained under the optimum extraction conditions that were 69% (v/v) acidified ethanol, 1:15 solid-liquid ratio and 3 times continuous extraction at 68 °C for 90 min. The AB-8 resin was selected to further purify and enrich anthocyanins based on its superior adsorption and desorption capacity. The purity of the obtained fraction by 80% acidized ethanol reached 92.1%, from which cyanidin-3-O-galactoside was identified by FTIR, LC-MS and NMR. In addition, microencapsulated anthocyanins, obtained using a deep eutectic solvent (DES), showed significantly improved stability in response to changes in light, oxygen, moisture content and pH. Further, as reaction media, DES could be recycled. The results showed that the present anthocyanins’ modification process could be used for the preparation of anthocyanins with a high purity and stability from berry fruits as well as other plants.

Magnetic nanocrystalline cellulose (MNCC) was prepared and used as an enzyme support for the immobilization of penicillin acylase (PA). A novel coupling agent, tri(hydroxymethyl)phosphine(THP) instead of the conventional glutaraldehyde(GA), was used as a crosslinker in this study. The obtained results showed that the immobilized PA with THP (PA-THP-MNCC) had high enzyme loading (172.3 mg/g) and activity recovery (77.6%) in the optimal preparation conditions, which were remarkably superior to those of the counterpart using GA (PA-GA-MNCC, 148.4 mg/g and 48.7%, respectively). Compared with free PA and PA-GA-MNCC, PA-THP-MNCC displayed a higher optimum pH and temperature, and manifested relatively higher enzyme-substrate affinity and catalytic efficiency. In addition, PA-THP-MNCC exhibited significantly enhanced stability. Furthermore, PA-THP-MNCC was successfully employed for synthesis of cefaclor, an important second-generation cephalosporin antibiotic, affording a significantly higher yield of 84% than that reported previously.

The extraction conditions and antioxidant activities of rutin from Sophora japonica bud by deep eutectic solvents were investigated. Box–Behnken design was used to optimize the extraction conditions and the scavenging activities of DPPH, O2− and ·OH of purified rutin were evaluated. The highest yield of 279.8 mg/g was achieved in the extraction medium of choline chloride/triethlene glycol (1/4) under the optimum conditions: water content of the DES 18.1%, extraction time 28.3 min, extraction temperature 70 °C and liquid–solid ratio 10 mg/1 g. The highest extraction amount was slightly different from the predicted value of the established second-order polynomial equation. In addition, The EC50 of DPPH scavenging, O2− scavenging and ·OH scavenging of rutin were 5.68 µg/mL, 0.19 and 0.28 mg/mL, respectively. The above results indicate rutin extracted by the choline chloride/triethylene glycol has excellent antioxidant activity and was an admirable free radical scavenger.

In order to obtain high-efficiency and sustainable adsorbents for both heavy metal ions and dyes, Millettia speciosa Champ cellulose and chitosan were used to prepare magnetic hydrogels with in-situ synthesis of Fe3O4 in this study. The results of characterization by vibrating sample magnetometer (VSM), Fourier transform infrared reflection (FTIR), X-ray diffraction (XRD), thermogravimetry analysis (TG and DTG), scanning electron microscope (SEM), mercury intrusion porosimetryand (MIP) and texture analysis indicated the magnetic hydrogels with high strength, high porosity and macroporous structure were successfully prepared, and they could be recovered by magnetic field. Besides, the prepared hydrogels had excellent adsorption effect on Cu2+ and Congo red (CR). The adsorption process for Cu2+ conformed to pseudo-first-order kinetics model better, whereas that for CR conformed to pseudo-second-order kinetics model better. Adsorption isotherm analysis showed that Freundlich model could better describe the adsorption process for both CR and Cu2+. The thermodynamic study results proved that the adsorption process for CR or Cu2+ was spontaneous and endothermic. Besides, the increase of pH had a negative effect on CR adsorption and a positive effect on Cu2+ adsorption. Adsorption mechanisms analysis revealed that electrostatic attraction and hydrogen bonding were involved in the CR adsorption, and amino groups acted dominantly in the process of Cu2+ adsorption. Furthermore, the prepared hydrogels exhibited excellent reusability, remaining their adsorption efficiency above 75% in five consecutive cycles. Therefore, the prepared hydrogels were expected to be promising materials for both heavy metal ions and dyes removal from waste water.

Treatment of textile water containing organic molecules as contaminants still remains a challenge and has become a central issue for environment remediation. Here, a nucleotide incorporated zeolitic imidazolate frameworks (NZIF) featuring hierarchically porous structure served as a potential adsorbent for removal of organic dye molecules. Adsorption isotherms of organic dyes were accurately described by Langmuir adsorption model with correlation coefficients of 0.98 and kinetic data followed the pseudo-second-order model. The maximum adsorption capacity of NZIF for Congo red (CR) and methylene blue (MB) reached 769 and 10 mg/g, respectively, which were 6 and 5 times higher than that of ZIF-8. The adsorption behavior of sunset yellow and crystal violet was examined for mechanism investigation. Analysis of pore size, molecular size, zeta potential and FTIR measurement together revealed that mesopores in NZIF provided more interaction sites and led to enhanced adsorption capacity. Hydrogen bonding and π-π stacking which resulted from the interaction between introduced nucleotide monophosphate and dyes dominated the driving forces for adsorption, where electrostatic interaction was also involved. Moreover, the introduced nucleoside monophosphate enabled NZIF to function under acidic condition whereas ZIF-8 collapsed. This study opens a new avenue for design of porous materials for environment remediation.

Preparation of biodiesel from waste oils containing 72% of free fatty acids catalyzed by a novel Bronsted acidic ionic liquid 1-sulfobutyl-3-methylimidazolium hydrosulfate ([BHSO3MIM][HSO4]) was systematically investigated. The optimum molar ratio of methanol to waste oils, catalyst amount, reaction temperature and reaction time were 8/1, 10% (based on the mass of waste oils), 140 °C and 6 h, respectively, under which the obtained yield of biodiesel reached 94.9%. Also, [BHSO3MIM][HSO4] as a catalyst still retained around 97% of its original catalytic activity after successive re-use of 5 batches (6 h per batch), showing the excellent operational stability. Moreover, the acidic IL [BHSO3MIM][HSO4] was able to efficiently catalyze conversions of waste oils with different amounts of FFAs (free fatty acids) into biodiesel, and showed tremendous application potential. Therefore, an efficient and environmentally friendly catalyst is provided for the synthesis of biodiesel from waste oils with high acid value.

A composite zein nanofibrous mat containing fish oil and ferulic acid was successfully fabricated by electrospinning. The process became more fluent and continuous by adding 30 g/L glycerol into the polymer solutions and using modified coaxial electrospinning. The average diameter of nanofibers was 440 nm. The loading capacity and encapsulation efficiency of fish oil were 20% and 94%, respectively. FTIR data demonstrated that fish oil and ferulic acid were successfully embedded into the nanofibers and there were interactions among the molecules of zein, fish oil and ferulic acid. Addition of ferulic acid into the nanofibers significantly improved the oxidative stability of encapsulated fish oil; moreover, it did not change the release behavior of fish oil. The release of encapsulated fish oil was controlled by a combination of diffusion and macromolecular chain relaxation. This composite nanofibrous mat with favorable oxidation stability and release property is potential in application as nutrition additive.

The capability of using crude glycerol as the sole carbon source for microbial oil production by Lipomyces starkeyi AS 2.1560 was investigated. The optimal crude glycerol concentration, nitrogen source, C/N ratio, inoculum concentration, culture temperature, and pH for lipid production were 70 g/L, yeast extract + peptone, 60, 10.0%, 30 °C, and 6.0, respectively. Under the optimal condition, the maximum biomass, lipid content, lipid yield, and lipid coefficient were 21.1 g/L, 35.7%, 7.5 g/L, and 17.3%, respectively. Methanol present in the crude glycerol had minor inhibition on the lipid production. Addition of 0.05 g/L PEG-200, 0.1 g/L potassium oleate or sodium stearate into the medium enhanced the lipid production by 6.4%, 7.7% and 10.4%, respectively. Lipid fermentation was further performed in a 5 L fermentor and the biomass, lipid content, and lipid yield after 10 days’ fermentation were 29.2 g/L, 42.9%, and 12.5 g/L, respectively. The corresponding kinetic models for the cell growth, lipid synthesis, and glycerol consumption were built. The maximum specific growth rate and the correlation coefficient of lipid synthesis to cell growth were 0.40 and 0.56, respectively. This study shows that L. starkeyi AS 2.1560 is a promising strain for lipid production on crude glycerol.

An extracellular chitinase produced by Paenibacillus pasadenensis CS0611 was purified by ammonium sulfate precipitation, HiTrap DEAE FF and HiLoad 26/600 Superdex 200pg column chromatography. The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 69 kDa. The optimum pH and optimum temperature of the chitinase were 5.0 and 50 °C, respectively. The enzyme showed high stability at alkaline pH values and temperatures below 40 °C. Additionally, the metal ions Mn2+, Mg2+, and Co2+ inhibited activity of the chitinase. The chitinase was active on colloidal chitin with an apparent Km of 4.41 mg/mL and Vmax of 1.08 mg/min. Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidic bond between GlcNAc-GlcNAc. The enzymatic hydrolysate was analyzed by high-performance liquid chromatography and thin layer chromatography, and clearly showed that a subunit of (GlcNAc)2 was the main hydrolysis product.

A novel deep eutectic solvent (DES)-DMSO cosolvent system has been, for the first time, successfully used as the reaction medium for the enzymatic acylation of dihydromyricetin (DMY) catalyzed by the immobilized lipase from Aspergillus niger (ANL). The cosolvent mixture, ChCl:Glycerol-DMSO (1:3, v/v) proved to be the optimal medium. With the newly developed cosolvent, the initial reaction rate of enzymatic acylation of DMY achieved 11.1 mM/h and the conversion of DMY was 91.6%. ANL@PD-MNPs is stable and recyclable in this cosolvent, offering 90% conversion rate after repeated use of 5 times. The lipid-solubility of DMY-16-acetate was 10 times higher than that of its raw materials DMY. The results showed that the DMY-16-acetate product exhibits good antioxidative activity. The present research illustrated that the use of DES-DMSO cosolvent may become a feasible alternative for the synthesis of DMY ester.

Cold-adapted pullulanase with high catalytic activity and stability is of special interest for its wide application in cold starch hydrolysis, but few pullulanases displaying excellent characteristics at ambient temperature and acidic pH have hitherto been reported. Here, a novel pullulanase from Bacillus methanolicus PB1 was successfully expressed in Escherichia coli BL21 (DE3) and determined to be a cold-adapted type I pullulanase (PulPB1) with maximum activity at 50 °C and pH 5.5. The recombinant PulPB1 showed great stability, its half-life at 50 °C was 137 h. PulPB1 can efficiently hydrolyze pullulan and amylopectin, with activities of 292 and 184 U/mg at 50 °C and pH 5.5, respectively. Moreover, the N-terminal domain of PulPB1 was found to significantly affect the enzymatic performance. Following truncation of the N-terminal domain, the activity towards pullulan decreased markedly from 292 to 141 U/mg and the half-life at 50 °C decreased from 137 to 10 h. Compared to the hydrolysis system with amyloglucosidase alone, the catalytic efficiency showed a 2.4-fold increase on combining PulPB1 with amyloglucosidase for amylopectin hydrolysis at 40 °C. This demonstrates that PulPB1 is promising for development as a superior candidate for cold amylopectin hydrolysis.

Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T1/215min compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides.

The asymmetric resolution of racemic 1-phenyl-1,2-ethanediol (PED) to (S)-PED by Kurthia gibsonii SC0312 (K. gibsonii SC0312) was conducted in a biphasic system comprised of an organic solvent and aqueous phosphate buffer. The impacts of organic solvents on the whole cell catalytic activity, metabolic activity, membrane integrity, and material distribution were first evaluated. The results showed that all organic solvents, except for dibutyl phthalate, showed a detrimental effect on the metabolic activity of the cells, especially for those with low log P values. All organic solvents were capable of changing the membrane permeability and membrane integrity of the cells. Moreover, some organic solvents showed a good extraction of the oxidation product. Finally, a high yield of 47.7 % of (S)-PED was obtained by the asymmetric resolution of racemic PED using K. gibsonii SC0312 in a biphasic system under the optimal conditions: racemic PED 120 mM, temperature 35 °C, reaction time 6 h, 180 rpm, and a volume ratio of dibutyl phthalate to aqueous phosphate buffer of 1:1. The optical purity of (S)-PED increased from 51.3 % to >99 %. This work described an efficient approach to improve reaction efficiency, and constructed a highly effective biphasic reaction system for the fabrication of (S)-PED via K. gibsonii SC0312.

A novel polysaccharide fraction (MSCP2) was extracted and isolated from the roots of Millettia Speciosa Champ. Structural characterization revealed that MSCP2 had an average molecular weight of 2.85 × 104 Da and was composed of fucose, arabinose, galactose, glucose and xylose with a ratio of 2.20: 2.52: 4.04: 87.29: 3.96. Methylation analysis and nuclear magnetic resonance (NMR) analysis showed that the main glycosidic linkage types of MSCP2 were proved to be α-D-Glcp-(1→, →4)-α-D-Glcp-(1→, →4)-α-D-Xylp-(1→, →6)-β-D-Galp-(1→, α-L-Araf-(1→, →3,4)-β-L-Fucp-(1→ and →4)-α-D-GalpA-(1→. The immunomodulatory assay suggested that MSCP2 could significantly improve the pinocytic capacity and increase the secretion of nitric oxide (NO) and cytokines by regulating the corresponding mRNA expression in RAW 264.7 cells. The data from the membrane receptor assay demonstrated that the potential mechanisms of MSCP2-induced macrophage activation were mainly through toll-like receptor 4 (TLR4), scavenger receptor type A (SRA) and glucan receptor (GR)-mediated signaling pathways. These results suggested that MSCP2 can be developed as a promising immunomodulatory agent in functional foods.

Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

Odd-chain fatty acids (OCFAs) have been reported to possess pharmacological activity and have been used in the manufacture of agricultural and industrial chemicals. We here provided a new method to increase the OCFAs content in oil produced by Rhodococcus opacus PD630 through addition of 1-propanol to the fermentation media. The OCFAs in oil of R. opacus PD630 are primarily pentadecanoic acid (C15:0), heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1). After adding 0.5–1.5% (v/v) 1-propanol, the production of oil increased from 1.27 g/L to 1.31–1.61 g/L, and the OCFAs content in oil increased by 46.7–55.1%. Metabolic intermediates determination and transcriptome analysis revealed that R. opacus assimilated 1-propanol through methylmalonyl-CoA pathway. When the nitrogen source was limited, propionyl-CoA was converted to propionyl-acyl carrier protein (ACP) which could be used as primer during the elongation of fatty acid synthesis. Then OCFAs were produced when odd number of propionyl-ACP was incorporated in the cycles of fatty acid synthesis.

Deep eutectic solvent (DES), has been considered as a new type of green solvent applied in enzymatic systems. Here, we reported DES-buffer co-solvent as a novel reaction medium for high efficient synthesis of cefaclor by penicilin acylase immobilized on magnetic nanocrystalline cellulose. Effect of DES composition, DES-buffer ratio, temperature, pH, substrate ratio and substrate concentration was systematically investigated. In co-solvent consisting of choline chloride (ChCl):glycol-buffer (7:3, v/v), conversion of 7-ACCA was 94%, synthesis to hydrolysis ratio was 1.8, and yield of cefaclor reached 91%, higher than that in aqueous buffer with optimized yield of 84%, showing the great potential of DES as organic solvent alternative. To the best of our knowledge, this is the first example of biosynthesis of cefaclor in the DES-buffer co-solvent.

Sequential immobilization of multiple enzymes can not merely improve the enzymatic activity and enzyme stability but also facilitate the recovery for reuse, well imitating the natural enzyme complexes. Here, we developed a novel approach of preparing sequential multienzyme nanomachineries based on chemical crosslinking and protein assembly, and then characterized the nanodevice. In the preparation process, KgBDH-SC (SpyCatcher-fused (2R,3R)-2,3-butanediol hydrogenase) was firstly immobilized onto the functionalized silicon dioxide nanoparticles with glutaraldehyde, and then FDH-ST (SpyTag-fused formate hydrogenase) was immobilized by the spontaneous protein reaction between SpyTag and SpyCatcher to achieve the sequential co-immobilized enzymes FDH-ST-SC-KgBDH@SiO2, which can asymmetrically reduce 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol by cofactor regeneration. In comparison to the mixture of free enzymes, the sequential co-immobilized enzymes created under the design conditions performed around 50% activity recovery. Furthermore, FDH-ST-SC-KgBDH@SiO2 presented wider pH tolerance and greater organic solvents tolerance than the mixture of free enzymes as well as retained 51.7% of overall activity after being recycled eight times. Finally, the sequential co-immobilized enzyme enabled the biosynthesis of (R)-1-phenyl-1,2-ethanediol from 80 mM 2-hydroxyacetophenone in the phosphate buffer reaction system, with 87.4% yield and over 99% optical purity.

Oxidized high-amylose starch macrogels (OHASMs) were successfully prepared and, for the first time, used as novel delivery vehicles for nutraceuticals. Oxidized high-amylose starch (OHAS) fabricated by 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) oxidation could crosslink Ca2+, Al3+, Fe2+, Fe3+, Cu2+, Zn2+ and Ba2+. For OHASM, the oxidation degree (DO) of OHAS had great effects on its morphology and controlled release properties except for the variety and concentration of metal ions. OHASM, formed by Zn2+ crosslinking OHAS (DO~90%), was suitable for encapsulating various nutraceuticals, such as hydrophobic and hydrophilic bioactive substances as well as probiotics, evidenced by 62.4%, 77.9% and 88.8% encapsulation efficiency for β-carotene, tea polyphenols and Lactobacillus paracasei, respectively. Furthermore, the outstanding pH-responsiveness and resistance of OHASM against gastric acid, bile salt, and digestive enzymes were demonstrated by swelling and simulative digestive experiments in vitro. The development of OHASM in this study suggests an attractive strategy for designing potential vehicles to encapsulate a variety of nutrients in food industries.

An efficient one-pot approach for the production of levulinate ester from renewable carbohydrates is demonstrated over heteropolyanion-based ionic liquid (IL-POM) catalysts with alcohols as the promoters and solvents. The relationships between the structure, acidic strength, and solubility of the IL-POM in methanol and the catalytic performance were studied intensively. A cellulose conversion of 100 % could be achieved with a 71.4 % yield of methyl levulinate over the catalyst [PyPS]3PW12O40 [PyPS=1-(3-sulfopropyl)pyridinium] at 150 °C for 5 h. This high efficiency is ascribed to the reasonably high activity of the ionic liquid (IL) catalyst and reaction coupling with rapid in situ esterification of the generated levulinic acid with the alcohol promoter, which allows the insolubility of cellulose encountered in biomass conversion to be overcome. Furthermore, the present process exhibits high feedstock adaptability for typical carbohydrates and handy catalyst recovery by a simple self-separation procedure through temperature control.

Valorization of biomass-based platform chemicals into high value-added products has attracted increasing attention recently. In this work, upgrading of 5-hydroxymethylfurfual (HMF) and levulinic acid, two important biomass-based platform chemicals, to HMF levulinate via a green and efficient enzymatic approach was reported. Novozym 435 was found to be the best biocatalyst for the enzymatic esterification. The enzymatic esterification progressed smoothly in t-butanol, 2-methyl-2-butanol, and cyclopentyl methyl ether as well as in the ecofriendly biomass-derived 2-methyltetrahydrofuran (2-MeTHF), while no enzymatic reaction occurred in deep eutectic solvents. When HMF concentration was up to 500 mM, a good conversion of 72% was achieved in 2-MeTHF. The reaction temperature exerted a significant effect on the enzymatic esterification. When the reaction temperature is below 40 °C, high HMF conversions (>85%) were obtained. Besides, significant inactivation of the enzyme was observed at more than 50 °C, result...

The nano-/microscale UiO-66-NH2 metal–organic framework (MOF) materials were successfully prepared with a uniform size of about 350–400 nm and structurally characterized. Soybean epoxide hydrolase (SEH), a useful hydrolase for synthesis of valuable vicinal diols, was for the first time efficiently immobilized onto the prepared UiO-66-NH2 MOF. The resulting novel nano-/microbiocatalyst SEH@UiO-66-NH2 manifested high SEH loading (87.3 mg/g) and enzyme activity recovery (88.0%). The novel SEH@UiO-66-NH2 greatly surpassed the free SEH with resepct to pH stability, thermostability, and tolerance to organic solvents. SEH@UiO-66-NH2 retained more than 17.6 U activity after 2 h of incubation at 45 °C, whereas free SEH maintained around 10.1 U activity under the same conditions. After storage at 4 °C for 4 weeks, the prepared SEH@UiO-66-NH2 still retained around 97.5% of its initial activity. The significant enhancements resulted from the increase of structural rigidity of SEH@UiO-66-NH2, which was demonstrated by...

A support made of mussel-inspired polydopamine-coated magnetic iron oxide nanoparticles (PD-MNPs) was prepared and characterized. The widely used Aspergillus niger lipase (ANL) was immobilized on the PD-MNPs (ANL@PD-MNPs) with a protein loading of 138 mg/g and an activity recovery of 83.6% under optimized conditions. For the immobilization, the pH and immobilization time were investigated. The pH and thermal and storage stability of the ANL@PD-MNPs significantly surpassed those of free ANL. The ANL@PD-MNPs had better solvent tolerance than free ANL. The secondary structure of free ANL and ANL@PD-MNPs was analyzed by infrared spectroscopy. A kinetic study demonstrated that the ANL@PD-MNPs had enhanced enzyme-substrate affinity and high catalytic efficiency. The ANL@PD-MNPs was applied as a biocatalyst for the regioselective acylation of dihydromyricetin (DMY) in DMSO and gave a conversion of 79.3%, which was higher than that of previous reports. The ANL@PD-MNPs retained over 55% of its initial activity after 10 cycles of reuse. The ANL@PD-MNPs were readily separated from the reaction system by a magnet. The PD-MNPs is an excellent support for ANL and the resulting ANL@PD-MNPs displayed good potential for the efficient synthesis of dihydromyricetin-3-acetate by enzymatic regioselective acylation.

Artificial enzyme mimics have recently emerged as alternative biocatalysts for overcoming the intrinsic fragility of natural enzyme in practical applications. However, current researches regarding mimetic enzymes are still confined to very few reaction types, with artificial oxidoreductases as dominance. Herein, inspired by nature, we designed and fabricated a range of Zn-Triazole coordination polymers (ZnTazs) that presented similar coordination structures with the active site of natural carbonic anhydrase Ⅱ (CA Ⅱ). These synthesized compounds exhibited inherently mimetic function with natural CA for catalyzing the hydrolysis of p-nitrophenyl acetate (p-NPA). Especially, the initial hydrolysis rate (V0) of p-NPA catalyzed by ZnTaz-1 (Zn5(bta)6(NO3)4 · H2O) and ZnTaz-2 (Zn3(OH)2(btca)2 · DMF · 4 H2O) reached 42.1 and 73.8 nM · s−1, respectively. Meanwhile, ZnTaz-1 and ZnTaz-2 showed favorable recyclability, and excellent stability towards various pH values and organic solvents, which are of great significance for practical employment. Moreover, they could also promote the efficient hydration and sequestration of greenhouse gas CO2 in aqueous medium. Based on this work, we aim to provide more theoretical and practical basis for rational design of CA mimics from the inspiration of natural enzyme, as well as propose a potential strategy for tackling the CO2 crisis.

(S)-1-Phenyl-1,2-ethanediol (PED) is a vital chiral block in specialty chemical industries. The biotransformation of 2-hydroxyacetophenone (2-HAP) to (S)-PED was conducted successfully catalyzed with BL21(DE3)(pETDuet-gst-mut-accr-gdh) which harboring a carbonyl reductase mutant (mut-AcCR). The catalytic activity of the biocatalyst was 15.9 times higher than the recombinant cells harboring original AcCR. 250 mM 2-HAP was reduced, under optimal conditions, and the yield and space-time yield were 95.2 % and 1.89 M/d in the buffer system. Then C4MIMI·PF6 was chosen to be the second phase to improve the catalytic efficiency, and the substrate concentration was increased to 450 mM in the C4MIMI·PF6/buffer system. After a reaction duration of 3 h, the product yield was over 92 %, and the space-time yield increased to 2.88 M/d which was 1.52 folds higher than that in the buffer system. Also, the product e.e. was over 99 % consistently. Overall, the preparative scale reduction of 2-HAP (450 mM) in C4MIMI·PF6/buffer system was conducted with promising result. The developed effective C4MIMI·PF6/buffer system of asymmetric reduction catalyzed by BL21(DE3)(pETDuet-gst-mut-accr-gdh) will promote the preparation of the enantiomeric pureity chiral alcohol at industrial scale.

A robust Escherichia coli (E. coli) strain, with a dual protection system to fight against microbial contamination and T7 phage infection, is used as a chassis to overexpress various proteins in auxotrophic MOPS medium. Among them, a robust EH (epoxide hydrolase)-producing E. coli was used as a biocatalyst to enantioselectively resolve racemic glycidyl o-methylphenyl ether (rac-o-GMPE) for preparing (R)-o-GMPE with 6416.7 U/g wet cells. The optimal temperature, pH and biocatalyst dosage were 25 °C, 7.0 and 0.2 mg/mL in aqueous buffer, respectively. The maximal yield (23.0 %) and ee (99.2 %) were achieved within 12 min in 20 mM rac-o-GMPE. Additionally, 98.0 % ee and 38.7 % yield were observed by cell-supplemental strategy with substrate concentration of 100 mM. Furthermore, to alleviate substrate inhibition and product toxicity toward the cells, a biphasic system (aqueous buffer: butyl acetate = 0.5:1) was developed to catalyze 3 M rac-o-GMPE with the initial reaction rate of 549.7 mmol/L/h, 91.7 % ee, 17.1 % yield and 3.66 g/L/h space-time yield.

Background Deep eutectic solvents have attracted considerable attention in numerous fields. There is little information on mung bean epoxide hydrolase-catalyzed epoxides in deep eutectic solvent-containing system. Results Adding deep eutectic solvents with hydrogen bond donor of acids to phosphate buffer resulted in an obvious decrease in the optical purity of product; nevertheless, the relative slight change was observed by the addition of the deep eutectic solvents with hydrogen bond donor of alcohols and urea. Of the tested deep eutectic solvents, 10% additional amount of choline chloride/trithylene glycol can cause a significant improvement in the enantiopurity of product, from 83.2 ± 1.3% to 87.9 ± 0.3%. Moreover, with the increase in the addition amount of choline chloride/triethylene glycol from 10 to 30%, the enantiomeric excess of product enhanced from 87.9 to 94%, but a decline of product yield was observed. Finally, the evaluation of enzyme stability showed that the additional amount from 10% to 30% was not beneficial to the activity recovery. Conclusion In short, adding choline chloride/triethylene glycol contributes to the improvement in the optical purity of (R)-1-phenyl-1, 2-ethanediol in the catalysis of styrene oxide by mung bean epoxide hydrolases; meanwhile, the enzyme immobilization could be essential.

Different kinds of low transition temperature mixtures (LTTMs) were synthesized with DL-lactic acid and five different quaternary ammonium salts respectively, and their viscosity and solubility parameters change with different molar ratios of quaternary ammonium salt to DL-lactic acid were investigated and compared with anthocyanins. Then the LTTMs synthesized were used to extract anthocyanins from saskatoon berry (Amelanchier alnifolia Nutt.). It turned out that the choline chloride/DL-lactic acid (ChCl/LA) showed the best solubility of anthocyanins compared with the other LTTMs, which was in line with solubility parameter theory. Under the optimized process condition with 1 : 15 of molar ratio, 1 : 20 of solid to liquid ratio, 50 °C of extraction temperature and 45 min of extraction time, the extraction amount of anthocyanins reached 525.27 ± 0.82 mg per 100 g and the extracted rate of anthocyanins was 87.4%. Then the macroporous resin AB-8 adsorbent was used to separate and purify the anthocyanin compounds from saskatoon berry, with a recovery yield of 65.7%. Further, the confirmation of the purified anthocyanins was carried out using nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and high performance liquid chromatography-mass spectrometry (HPLC-MS). Finally, the purified anthocyanins were proved to be good antioxidants through the comparison with vitamin C and butylated hydroxytoluene.