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.Keywords: acylation; antioxidant ability; Aspergillus niger lipase; deep eutectic solvent; dihydromyricetin;

Preparation of biodiesel from waste oils containing 72% of free fatty acids catalyzed by a novel Brønsted 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.The addition of [BHSO3MIM][HSO4] in the system of methanol and waster oils with high acid value favors the formation of biodiesel by esterification and trans-esterification reaction.Download high-res image (72KB)Download full-size image

•Crude glycerol was efficiently transformed into microbial oil by Lipomyces starkeyi.•L. starkeyi was tolerance to high level of methanol present in the crude glycerol.•Addition of surfactant can enhance the microbial oil production by L. starkeyi.•Kinetic model for lipid production by L. starkeyi in a 5 L fermentor was built.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.Download high-res image (164KB)Download full-size image

Deep eutectic solvents (DESs) are eutectic mixtures of salts and hydrogen bond donors with melting points low enough to be used as solvents. DESs have proved to be a good alternative to traditional organic solvents and ionic liquids (ILs) in many biocatalytic processes. Apart from the benign characteristics similar to those of ILs (e.g., low volatility, low inflammability and low melting point), DESs have their unique merits of easy preparation and low cost owing to their renewable and available raw materials. To better apply such solvents in green and sustainable chemistry, this review firstly describes some basic properties, mainly the toxicity and biodegradability of DESs. Secondly, it presents several valuable applications of DES as solvent/co-solvent in biocatalytic reactions, such as lipase-catalyzed transesterification and ester hydrolysis reactions. The roles, serving as extractive reagent for an enzymatic product and pretreatment solvent of enzymatic biomass hydrolysis, are also discussed. Further understanding how DESs affect biocatalytic reaction will facilitate the design of novel solvents and contribute to the discovery of new reactions in these solvents.

Deep eutectic solvents (DESs) are eutectic mixtures of salts and hydrogen bond donors with melting points low enough to be used as solvents. DESs have proved to be a good alternative to traditional organic solvents and ionic liquids (ILs) in many biocatalytic processes. Apart from the benign characteristics similar to those of ILs (e.g., low volatility, low inflammability and low melting point), DESs have their unique merits of easy preparation and low cost owing to their renewable and available raw materials. To better apply such solvents in green and sustainable chemistry, this review firstly describes some basic properties, mainly the toxicity and biodegradability of DESs. Secondly, it presents several valuable applications of DES as solvent/co-solvent in biocatalytic reactions, such as lipase-catalyzed transesterification and ester hydrolysis reactions. The roles, serving as extractive reagent for an enzymatic product and pretreatment solvent of enzymatic biomass hydrolysis, are also discussed. Further understanding how DESs affect biocatalytic reaction will facilitate the design of novel solvents and contribute to the discovery of new reactions in these solvents.

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 the secondary structure analysis of the enzyme. The optimun pH and tempearture of SEH@UiO-66-NH2 were significantly superior to the corresponding levels of its free counterpart. Also, SEH@UiO-66-NH2 manifested markedly enhanced enzyme–substrate affinity and catalytic efficiency compared to free SEH, as supported by a lower apparent Km value (6.5 vs 19.2 mM) and an increased Vmax/Km value (8.0 × 10–3 vs 5.8 × 10–3 min–1), respectively. Furthermore, the as-prepared SEH@UiO-66-NH2, for the first time, was successfully applied as an efficient biocatalyst for the asymmetric hydrolysis of 1,2-epoxyoctane to (R)-1,2-octanediol in a novel deep eutectic solvent (DES) with a yield of around 41.4% and a product e.e. value of 81.2%. Remarkably, the nano-/microscale UiO-66-NH2 MOFs as novel enzyme support materials are promising for enzyme immobilization, and the prepared SEH@UiO-66-NH2 exhibited great potential for efficient biosynthesis of enantipure (R)-1,2-octanediol.Keywords: Deep eutectic solvent; Dipeptide; Epoxide hydrolase; Green chemistry; Metal organic framework; Optically active vicinal diols; Sustainable chemistry;

Whole-cell-based biocatalysis in ionic liquids (ILs)-containing systems has attracted increasing interest in recent years. Compared to bioreactions catalyzed by isolated enzymes, the major advantages of using whole cell in biocatalytic processes are that cells provide a natural intracellular environment for the enzymes to function with cofactors regeneration in situ. An increasing number of renewable ILs are now accessible as a result of the ongoing progress in designing strategy of ILs and the sustainable environment requirement. The toxicity of ILs to microbial cells and the biodegradability are two of the crucial factors that allow the biocatalysis in ILs being applied in practice rather than in bench-scale. Applications of whole-cell biocatalysis in IL-containing systems have, to date, been focused on the production of valuable compounds, mainly through reduction, oxidation, and hydrolytic reactions. The mechanism research of ILs affecting the whole-cell biocatalysis offers the possibility to integrate effectively ILs with biotransformation. Thus, a comprehensive understanding of the whole-cell-based biocatalytic process with ILs will contribute to the discovery of novel solvent for enzymatic reaction and the synthesis of more valuable compounds.Keywords: Biocatalysis; Biocompatibility; Biodegradability; Ionic liquid; Whole-cell;

Magnetic nanocrystalline cellulose (MNCC), a novel biobased nanocomposite, was prepared via a simple coprecipitation-cross-linking technique and structurally characterized. Papain (PA) was successfully immobilized onto the MNCC. The resulting nanobiocatalyst PA@MNCC showed high PA loading (333 mg/g) and enzyme activity recovery (more than 80%). The stability of PA@MNCC was greatly superior to that of its free counterpart. Also, PA@MNCC manifested markedly enhanced solvent tolerance. The secondary structure study of the enzyme proved that these enhancements were attributed to the increase of structure rigidity of PA@MNCC. The observed optimum pH and temperature of PA@MNCC were significantly higher than the corresponding levels of free PA. A kinetic study demonstrated that PA@MNCC had an increase in enzyme-substrate affinity. Furthermore, the as-prepared PA@MNCC was successfully used as an efficient biocatalyst for the synthesis of N-(benzyloxycarbonyl)-alanyl-glutamine (Z-Ala-Gln) dipeptide in deep eutectic solvent (DES), choline chloride (ChCl):urea(1:2), with a high yield (about 71.5%), which, to our knowledge, was greatly higher than that reported previously. Besides, the novel PA@MNCC was easily recycled from the reaction medium by magnetic forces. Obviously, MNCC is a promising and competitive enzyme carrier and the as-prepared nanobiocatalyst PA@MNCC exhibited great potential for efficient biosynthesis of dipeptide.Keywords: Deep eutectic solvent; Dipiptide; Green chemistry; Magnetic nanoparticle; Nanocrystalline cellulose; Protease; Sustainable chemistry;

Use of deep eutectic solvents (DESs) to improve biocatalytic asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol catalyzed by whole-cell of Acetobacter sp. CCTCC M209061 was successfully performed. The cells immobilized on PVA-sodium sulfate exhibited markedly enhanced stability. Diverse DESs, as cosolvents, manifested significantly different influences on the reaction. Among them, the DES choline chloride/urea ([ChCl][U]) showed the best biocompatibility and moderately increased the cell member permeability, as demonstrated by MAR and flow cytometry assays, and consequently gave the best results. For the bioreduction conducted in the [ChCl][U]-containing system, the optimum [ChCl][U] content, substrate concentration, glucose concentration, pH and temperature were 5% (v/v), 10.0 mmol/L, 60 mmol/L, 5.5 and 30 °C, respectively. Under the optimized conditions, the obtained yield and product e.e. were 82.3% and above 99.0% at a reaction time of 6 h, respectively, and the productivity was 1.37 mmol/L/h. The efficient whole-cell biocatalytic process proved to be feasible on a 500 mL preparative scale. Moreover, the combination of water-immiscible ionic liquid C4MIM·PF6 with [ChCl][U] in a biphasic system further enhanced substrate concentration (16.0 mmol/L), product yield (93.3%) and productivity (1.87 mmol/L/h) significantly, showing to be very promising for biocatalytic synthesis of (S)-3-chloro-1-phenylpropanol with immobilized Acetobacter sp. CCTCC M209061 cells.Keywords: 3-chloropropiophenone; Biocatalysis; deep eutectic solvent; immobilized Acetobacter sp. CCTCC M209061; reduction; sustainable chemistry;

The development of novel green solvents has been one of the hottest subjects in green chemistry. Deep eutectic solvents (DESs) have logically and naturally emerged in the search for more biocompatible and biodegradable solvents. In this study, some basic physical properties, including viscosity, conductivity, and density, of 20 DESs prepared from choline chloride and various hydrogen-bond donors were investigated systematically. In addition, the biocompatibility of the tested DESs was qualitatively and quantitatively evaluated using two Gram-positive (Staphylococcus aureus and Listeria monocytogenes) and two Gram-negative (Escherichia coli and Salmonella enteritidis) bacteria. A closed bottle test was used to assess the biodegradability of these DESs. The results demonstrated that these choline chloride-based DESs were excellent solvents with extremely low toxicity and favorable biodegradability. Finally, DESs were used to extract a flavonoid (rutin) from the flower buds of Sophora japonica. An extraction efficiency of 194.17 ± 2.31 mg·g–1 was achieved using choline chloride/triethylene glycol containing 20% water. The excellent properties of DESs indicate their potential as promising green solvents for the extraction of rutin with favorable prospects for wide use in the field of green technology.Keywords: Biocompatibility; Biodegradability; Choline chloride; Deep eutectic solvent (DES); Physical properties; Rutin;

Alkaline protease was successfully immobilized onto amino-functionalized Fe3O4 nanoparticles. The enzyme loading was 388.2 mg of protein/g of support and the activity recovery was more than 54.2%. After immobilization, the affinity of alkaline protease toward substrate and its stability were significantly enhanced. The immobilized enzyme still retained 50.1% of its initial activity after 10 cycles of successive reuse, exhibiting excellent operational stability. The immobilized enzyme was capable of efficiently catalyzing hydrolysis of oat bran into oat polypeptides. Under the optimized conditions, the maximum DPPH radical scavenging rate (antioxidant activity) of oat polypeptides (8.4 mg/mL) was 82.3%, which was much higher than the reported result. Moreover, the prepared oat polypeptides by immobilized enzyme showed higher antioxidant activity than those prepared by free enzyme, owing to an increase of relatively hydrophobic components of oat polypeptides. Furthermore, the immobilized enzyme was demonstrated to be very promising for production of oat polypeptides on a preparative scale.

The asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] using immobilized Acetobacter sp. CCTCC M209061 cells was successfully conducted in a hydrophilic ionic liquid (IL)-containing system. The best one of all the tested watermiscible ILs was 1-(hydroxyethyl)-3-methylimidazolium hydrochloride (C2OHMIM·Cl). In C2OHMIM·Cl-aqueous buffer hybrid system, it was found that the optimal IL concentration, substrate and co-substrate concentration, reaction temperature, buffer pH and shaking rate were 0.5mol/L, 45 mmol/L, 80 mmol/L, 35°C, pH 5.5 and 200 rpm, respectively. Under the optimized reaction conditions, the initial reaction rate, the yield and the product e.e. reached 4.90 µmol/min, 95.3 and > 99.0%, respectively, which were much higher than the corresponding values reported previously. The efficient biocatalytic process mediated by the immobilized cells was feasible on 500 mL preparative scale, and the biocatalysts showed good operational stability and could be recycled for at least 10 batches.

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.

A new sucralose-derived solid acid catalyst (SUCRA-SO3H), containing −Cl and −SO3H functional groups, has been shown to be highly effective for hydrolyzing β-1,4-glucans, completely hydrolyzing cellobiose (1) into glucose (2) in 3 h and converting the microcrystalline cellulose pretreated by the ionic liquid into glucose (2) with a yield of around 55% and a selectivity of 98% within 24 h at a relatively moderate temperature (393K). The enhanced adsorption capacity that the catalyst has for glucan by virtue of the presence of chloride groups that act as cellulose-binding sites offers the possibility of resolving the existing bottleneck in heterogeneous catalysis to hydrolyze cellulose, namely, the low accessibility of cellulose to the reaction position in typical solid catalysts. The apparent activation energy for hydrolysis of cellobiose (1) with SUCRA-SO3H was 94 kJ/mol, which was much lower than that with sulfuric acid (133 kJ/mol) and the corresponding sucrose-derived catalyst (SUCRO-SO3H) without chlorine groups (114 kJ/mol).

The asymmetric hydrolysis of racemic styrene oxide (SO) to (R)-1-phenyl-1,2-ethanediol using cross-linked enzyme aggregates (CLEAs) of epoxide hydrolases (EHs) from mung bean (mbEHs) was successfully conducted using ionic liquids (ILs) as cosolvents in biphasic systems. Of all the tested ILs, the best results were observed in the biphasic system containing [C4MIM][PF6] with better biocompatibility to the CLEAs of mbEHs. In the [C4MIM][PF6]/buffer biphasic system, it was found that the optimal volume ratio of IL to buffer, reaction temperature, buffer pH, and substrate concentration were 1:5, 40 °C, 7.5, and 120 mM, respectively. Under the optimized reaction conditions, the initial reaction rate, yield, product ee, and E value reached 3.35 mmol/min, 49%, 95.8%, and 151, respectively, which were much higher than the corresponding values reported previously. Furthermore, the CLEAs exhibited markedly enhanced operational stability in a [C4MIM][PF6]-based biphasic system as compared with an n-hexane-based biphasic system. Additionally, the CLEAs of mbEHs-catalyzed process with the IL [C4MIM][PF6] was shown to be feasible on a 500 mL preparative scale, demonstrating their great potential for biosynthesis of chiral ortho-diols.

To give an insight into the kinetics of microbial oil fermentation with lignocellulosic hydrolysate as substrate, lipid production by Trichosporon fermentans in rice straw hydrolysate was investigated in a 5 L fermentor. For comparison, fermentation in its simulated medium was also performed to evaluate the effect of inhibitors present in the rice straw hydrolysate on the cell growth and lipid accumulation of T. fermentans. The optimum fermentation time, maximum biomass, and lipid content of T. fermentans in the rice straw hydrolysate were 10.5 days, 28.6 g/L, and 43.9%, respectively, while the corresponding values in the simulated medium were 8.5 days, 27.0 g/L, and 65.0%, respectively, indicating that the inhibitors in the rice straw hydrolysate did show some inhibition on the cell growth and lipid accumulation of T. fermentans. Kinetic models of lipid fermentation with T. fermentans showed the maximum specific growth rate and the correlation coefficient of lipid synthesis to cell growth in the rice straw hydrolysate were 0.40 and 0.51, respectively, which are much lower than those in the simulated medium (0.58 and 0.73). To better understand the influential mechanism of inhibitors in the rice straw hydrolysate on the growth and lipid accumulation of T. fermentans, the physiological and biochemical changes of cells in the fermentation were further investigated. The reduced activities of adenosine triphosphate (ATP) citrate lyase, malic enzyme, and xylose reductase, and the elongation of cells at the beginning of fermentation, partly account for the inhibitory effect of inhibitors in the rice straw hydrolysate.

Biocatalytic reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] with Acetobacter sp. CCTCC M209061 cells was successfully conducted in ionic liquid (IL)-based biphasic systems. Several water-immiscible ILs were used to construct biphasic systems. The best IL investigated was 1-butyl-3-methylimidazolium hexafluorophosphate (C4mim·PF6), which also had good biocompatibility. Several influential variables were examined. The optimum parameters were as follows: volume ratio of buffer to C4mim·PF6, 1/2 (v/v); substrate concentration, 55 mmol/L; buffer pH, 5.5; cosubstrate concentration, 80 mmol/L; reaction temperature, 35 °C; and shaking speed, 220 rpm. Under these optimal conditions, the initial reaction rate, the yield, and the product e.e. were 0.39 mmol/(L min), 90.8%, and >99%, respectively, which were much better than results reported previously. This efficient whole-cell biocatalytic process was feasible on a 450-mL preparative scale, and the immobilized cells showed excellent operational stability and could be reused for at least 10 batches.

Ionic liquids (ILs) containing a range of 1-alkyl-3-methylimidazolium cations and various anions affect papain's catalytic performance and thermostability in a manner that correlates closely with the effects of the ILs on the conformation of the enzyme as assessed by using ATR-FTIR and fluorescence techniques.

The two typical ionic liquids (ILs), one hydrophobic (BMIM·PF6) and one hydrophilic (BMIM·BF4), were tested as solvents for use in the asymmetric reduction of acetyltrimethylsilane (ATMS) to enantiopure (S)-1-trimethylsilylethanol {(S)-1-TMSE} catalyzed by immobilized Saccharomyces cerevisiae cells. The results demonstrate that BMIM·PF6 and BMIM·BF4 can markedly boost the activity and the stability of the immobilized cells. To better understand the reaction performed in these IL-containing systems, various variables that influenced the performance of the reaction were examined. The optimal buffer pH, reaction temperature and substrate concentration were 7.3, 30 °C and 84 mM, respectively, for the BMIM·PF6/buffer (1/6, v/v) biphasic system, and 7.5, 30 °C and 77 mM, respectively, for the 10% (v/v) BMIM·BF4–buffer co-solvent system. Under the optimal conditions, the initial reaction rate, maximum yield and product e.e. were 63.4 mM h−1, 99.9% and >99.9% with the former system, while those with the latter system were 74.5 mM h−1, 99.2% and >99.9%, respectively, which were much higher than those achieved with either n-hexane/buffer (2/1, v/v) biphasic system or aqueous buffer. It was also found that the optimal pH and substrate concentration changed when n-hexane was replaced by BMIM·PF6 or BMIM·BF4. Although the optimal reaction temperature remained the same in the four kinds of reaction systems, the temperature profile of the reaction varied from case to case. Additionally, BMIM·BF4 and especially BMIM·PF6 exhibited greater biocompatibility with Saccharomyces cerevisiae than n-hexane, and could be used repeatedly for economically interesting whole-cell biocatalytic processes with in situ coenzyme regeneration.

A comparative study was made of Mung bean epoxide hydrolases-catalyzed asymmetric hydrolysis of styrene oxide to (R)-1-phenyl-1,2-ethanediol in an n-hexane/buffer biphasic system containing various hydrophilic ionic liquids (ILs). Compared to the n-hexane/buffer biphasic system alone, addition of a small amount of hydrophilic ILs reduced the amount of non-enzymatic hydrolysis, and improved the reaction rate by up to 22%. The ILs with cation containing an alkanol group, namely [C2OHMIM][BF4] and [C2OHMIM][TfO], and the choline amino acid ILs [Ch][Arg] and [Ch][Pro] were found to be the most suitable co-solvents for the reaction, owing to their good biocompatibility with the enzyme, which led to high initial rates (0.99–1.25 μmol/min) and high product e.e.s (95%). When substrate concentration was around 30 mM, where optimal performance was observed with the IL-containing systems, the product e.e. was improved from 90% without ILs to ≥95% in the presence of ILs.Graphical abstractDownload full-size imageHighlights► Addition of a small amount of hydrophilic ILs improved Mung bean EHs-catalyzed hydrolysis of SO. ► ILs effectively reduced the amount of non-enzymatic hydrolysis of SO. ► Diverse ILs showed different biocompatibility to EHs and effects on the reaction. ► ILs substantially increased the thermostability of Mung bean EHs. ► ILs affected the partition coefficients of the substrate between the two phases.

•Highly active and stable CLEAs of Mung bean EHs were successfully prepared.•CLEAs were superior to free enzyme in terms of activity, stability and catalytic efficiency.•CLEAs of Mung bean EHs were more efficient in catalyzing asymmetric hydrolysis of SO to (R)-PED.•The efficient biocatalytic process with CLEAs was feasible on a 250-mL preparative scale.A highly active and stable cross-linked enzyme aggregates (CLEAs) of epoxide hydrolases (EHs) from Mung bean, which plays a crucial role in synthesis of valuable enantiopure diols, were successfully prepared and characterized. Under the optimum preparation conditions, the activity recovery of CLEAs recorded 92%. The CLEAs were more efficient than the free enzyme in catalyzing asymmetric hydrolysis of styrene oxide to (R)-1-phenyl-1,2-ethanediol in organic solvent-containing biphasic system. The biocatalytic reaction performed in n-hexane/buffer biphasic system had a clearly faster initial reaction rate, much higher product yield and product e.e. value than that in aqueous medium. Moreover, the optimal volume ratio of n-hexane to buffer, reaction temperature, buffer pH value and substrate concentration for the enzymatic hydrolysis were found to be 1:1, 40 °C, 7.5 and 30 mM, respectively, under which the initial reaction rate, product yield and product e.e. value were 13.26 mM/h, 46% and 93.5%, respectively. The CLEAs retained more than 50% of their initial activity after 8 batches of re-use in phosphate buffer and maintained 53% of their original activity after 8 reaction cycle in biphasic system. The efficient biocatalytic process with CLEAs proved to be feasible on a 250-mL preparative scale, exhibiting great potential for asymmetric synthesis of chiral diols.

•The novel carbonyl reductase (AcCR) from our isolated Acetobacter sp. CCTCC M209061 was, for the first time, successfully expressed in E. coli. with markedly enhanced enzymatic activity.•A co-expression system harboring AcCR and GDH was effectively constructed to obtain an efficient whole-cell biocatalyst with in situ coenzyme regeneration.The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9 U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0 U/mg-protein in the recombinant E. coli. As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for (S)-enantiomer, and the initial rate and product yield reached 8.04 μmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.The general process of the asymmetric reduction of carbonyl compounds to chiral alcohols with the recombinant E. coli BL21 (DE3) pLysS (pETDuet-accr-gdh).Download full-size image

The biocatalytic anti-Prelog stereoselective reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate {(R)-EHB} was successfully conducted in the aqueous system using immobilized Acetobacter sp. CCTCC M209061 cells. Among various microorganisms tested, Acetobacter sp. CCTCC M209061 gave the best performance and showed great potential for the bioreduction of EAA to (R)-EHB. Acetobacter sp. CCTCC M209061 cells were immobilized on calcium alginate and then coated with chitosan, and the immobilized cells clearly surpassed the free cells in terms of better thermal stability, storability and recyclability. The optimal co-substrate for the reaction was found to be glucose. The optimal substrate concentration, buffer pH, glucose concentration, reaction temperature, biocatalyst concentration and shaking speed were 35.0 mmol/L, 5.5, 80.0 mmol/L, 35 °C, 0.45 g/mL and 200 rpm, respectively. Under the optimized conditions, the initial reaction rate, the yield and the enantiomeric excess (e.e.) of the product were 1.28 μmol/min, 82.6% and above 99.0%, respectively, which were much higher than those reported previously. The efficient whole-cell biocatalytic process was feasible on a 500-mL preparative scale, and the immobilized cells showed excellent operational stability and could be re-used for at least 10 batches. Additionally, the product e.e. constantly remained above 99.0% regardless of re-use cycles of the biocatalyst.Highlights► Acetobacter sp. efficiently catalyzed asymmetric reduction of EAA to (R)-EHB. ► The immobilization clearly boosted the stability and recyclability of the cells. ► Higher yield and product e.e. were obtained with immobilized Acetobacter sp. cell. ► The efficient biocatalytic process was feasible on a 500-mL preparative scale.

Dihydromyricetin is the principle component of the Chinese herbal tea Teng-cha and a promising ingredient for functional food and nutraceuticals, but its low solubility limits its application potentials. This study explored enzymatic acylation of dihydromyricetin to improve its solubility in lipid systems. Acylation was achieved with several lipases with the synthesis of a major (>86%) product and a minor product. Isolation and purification of the products by preparative HPLC followed by LC–MS, 13C NMR, 1H NMR and 2 D-HSQC NMR analyses showed that the major product was a dihydromyricetin monoester with the acylation site at the 3-OH group of C ring. Quantum chemical calculations revealed that the 3-OH had the lowest antioxidant activity, and therefore acylation at this site was expected to have minimum impact on the antioxidant activity. Several factors, including solvent, acyl donor, enzyme origin, molar ratio of substrates and reaction temperature and time, exhibited significant effects on the initial rate, conversion yield and regioselectivity of the reaction. Acylation occurred only with vinyl acetate as the acyl donor, and highest conversion yields were achieved with immobilized Penicillium expansum lipase and Novozyme 435 with DMSO and acetonitrile being the best solvents. In general, the acylation results were found to be superior to previous reports on acylation of aglycone flavonoids with respects to conversion yield and regioselectivity.

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.