The Extraction Using Deep Eutectic Solvents and Evaluation of Tea Saponin.

Tea saponins have high surface-active and biological activities and are widely used in chemicals, food, pharmaceuticals, and pesticides. Tea saponins are usually extracted using ethanol or water, but both methods have their disadvantages, including a negative impact on the environment, high energy consumption, and low purity. In this study, we explored an effective process for extracting tea saponins from tea meal using deep eutectic solvents combined with ultrasonic extraction and enzymatic techniques. The experimental results showed that a high extraction efficiency of 20.93 ± 0.48% could be achieved in 20 min using an ultrasonic power of 40% and a binary DES consisting of betaine and ethylene glycol (with a molar ratio of 1:3) at a material-liquid ratio of 1:35 and that the purity of the tea saponins after purification by a large-pore adsorption resin reached 95.94%, which was higher than that of commercially available standard tea saponin samples. In addition, the extracted tea saponins were evaluated for their antioxidant and bacteriostatic activities using chemical and biological methods; the results showed that the tea saponins extracted using these methods possessed antioxidant properties and displayed significant antibacterial activity. Therefore, the present study developed a method for using deep eutectic solvents as an environmentally friendly technological solution for obtaining high-purity tea saponins from tea meal oil. This is expected to replace the current organic solvent and water extraction process and has great potential for industrial development and a number of possible applications.

Effect of Deep Eutectic Solvents on the Activity and Stability of Cellulases and Pectinases.

Deep eutectic solvents (DESs) have physicochemical characteristics similar to those of ionic liquids but are more cost-effective, easier to produce, and less harmful to the environment, making them viable alternatives to ionic liquids. In this study, various DESs have been created to assess their potential as storage media for enzymes. The impact of the DES composition and water content on the thermal and storage stability of cellulase and pectinase was also investigated. Molecular simulation was used to examine the kinetic parameters of cellulase and pectinase in DESs with varying water levels based on choline chloride. The results demonstrated that the stability of the enzymes initially increased and then decreased with an increase in water content in DESs. The enzymes experienced secondary structural changes, leading to variations in fluorescence values. Ultimately, DESs can be utilized as a stabilizers for long-term enzyme preservation, and this study provides a theoretical basis for the coapplication of DESs and enzymes.

The Effect of Polyhydroxy Fullerene Derivative on Human Myeloid Leukemia K562 Cells.

The use of nanomedicines for cancer treatment has been widespread. Fullerenes have significant effects in the treatment of solid tumors. Here, we are going to study the effects of hydroxylated fullerene C60(OH)n(n = 18-22) treatment on chronic myeloid leukemia cell proliferation and investigate its toxicity. The results showed that hydroxylated fullerene C60(OH)n (n = 18-22) at low concentrations (less than 120 µM) not only had apparent toxic side effects, but also promoted the growth of K562 cells, while a high concentration of C60(OH)n had different degrees of inhibition on K562 cells. When the concentration is higher than 160 µM, the K562 cells showed morphological changes, the mitochondrial membrane potential decreased, the cell cycle was blocked in the stage of G2-phase, and cell apoptosis occurred, which may cause apoptosis, autophagy, and a variety of other damage leading to cell death. Meanwhile, it also indicated that its inhibition of solid tumors might be related to the tumor microenvironment; we verified the safety of fullerene without apparent cellular toxicity at a specific concentration.

Antitumor Activity and Potential Mechanism of Novel Fullerene Derivative Nanoparticles.

The development of novel nanoparticles as a new generation therapeutic drug platform is an active field of chemistry and cancer research. In recent years, fullerene nanoparticles have received extensive attention due to their unique physical and chemical properties. Properly modified fullerene nanoparticles have excellent biocompatibility and significant anti-tumor activity, which makes them have broad application prospects in the field of cancer therapy. Therefore, understanding the anti-tumor mechanism of fullerene nanoparticles is of great significance for the design and development of anti-tumor drugs with low toxicity and high targeting. This review has focused on various anti-tumor mechanisms of fullerene derivatives and discusses their toxicity and their distribution in organisms. Finally, the review points out some urgent problems that need solution before fullerene derivatives as a new generation of anti-tumor nano-drug platform enter clinical research.

Nondestructive determination of soluble solids content and pH in red bayberry (Myrica rubra) based on color space.

Color has strong relationship with food quality. In this paper, partial least square regression (PLSR) and least square-support vector machine (LS-SVM) models combined with six different color spaces (NRGB, CIELAB, CMY, HSI, I1I2I3, and YCbCr) were developed and compared to predict pH value and soluble solids content (SSC) in red bayberry. The results showed that PLSR and LS-SVM models coupled with color space could predict pH value in red bayberry (r = 0.93-0.96, RMSE = 0.09-0.12, MAE = 0.07-0.09, and MRE = 0.04-0.06). In addition, the minimum errors (RMSE = 0.09, MAE = 0.07, and MRE = 0.04) and maximum correlation coefficient value (r = 0.96) were found with the PLSR based on CMY, I1I2I3, and YCbCr color spaces. For predicting SSC, PLSR models based on CIELAB color space (r = 0.90, RMSE = 0.91, MAE = 0.69 and MRE = 0.12) and HSI color space (r = 0.89, RMSE = 0.95, MAE = 0.73 and MRE = 0.13) were recommended. The results indicated that color space combined with chemometric is suitable to non-destructively detect pH value and SSC of red bayberry.

Gadofullerene inhibits the degradation of apolipoprotein B100 and boosts triglyceride transport for reversing hepatic steatosis.

Hepatic steatosis is a widespread metabolic disease characterized by excessive accumulation of triglyceride (TG) in liver. So far, effective approved drugs for hepatic steatosis are still in development, and removing the unnecessary TG from the hepatocytes is an enormous challenge. Here, we explore a promising anti-hepatic steatosis strategy by boosting hepatocellular TG transport using ß-alanine-modified gadofullerene (GF-Ala) nanoparticles. We confirm that GF-Ala could reverse hepatic steatosis in oleic acid-induced hepatocytes, fructose-induced mice, and obesity-associated transgenic ob/ob mice. Observably, GF-Ala improves hepatomegaly and hepatic lipid accumulation, reduces lipid peroxidation, and repairs abnormal mitochondria. Of note, we demonstrate that GF-Ala markedly inhibits the posttranslational degradation of apolipoprotein B100 (ApoB100) and boosts hepatocellular TG transport based on their superior antioxidant property. Together, we conclude that GF-Ala could potently ameliorate hepatic TG transport and maintain hepatic metabolic homeostasis without apparent toxicity, being beneficial for treatments of hepatic steatosis and other fatty liver diseases.

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal.

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal-a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.

Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)-N-Boc-3-Hydroxypiperidine.

The recombinant carbonyl reductase from Rhodococcus erythropolis WZ010 (ReCR) demonstrated strict (S)-stereoselectivity and catalyzed the irreversible reduction of N-Boc-3-piperidone (NBPO) to (S)-N-Boc-3-hydroxypiperidine [(S)-NBHP], a key chiral intermediate in the synthesis of ibrutinib. The NAD(H)-specific enzyme was active within broad ranges of pH and temperature and had remarkable activity in the presence of higher concentration of organic solvents. The amino acid residue at position 54 was critical for the activity and the substitution of Tyr54 to Phe significantly enhanced the catalytic efficiency of ReCR. The kcat/Km values of ReCR Y54F for NBPO, (R/S)-2-octanol, and 2-propanol were 49.17 s-1 mM-1, 56.56 s-1 mM-1, and 20.69 s-1 mM-1, respectively. In addition, the (S)-NBHP yield was as high as 95.92% when whole cells of E. coli overexpressing ReCR variant Y54F catalyzed the asymmetric reduction of 1.5 M NBPO for 12 h in the aqueous/(R/S)-2-octanol biphasic system, demonstrating the great potential of ReCR variant Y54F for practical applications.

Identification differential behavior of Gd@C82(OH)22 upon interaction with serum albumin using spectroscopic analysis.

The interaction between Gd@C82(OH)22 and serum albumin (HSA and BSA) were investigated by spectroscopic analysis. From the characteristic feature of fluorescence quenching spectra at different temperatures, the inherent binding information including quenching mechanism, association constants, number of binding site, fraction of initial fluorescence and basic thermodynamic parameters were calculated. The binding of Gd@C82(OH)22 to serum albumin caused strong quenching of protein intrinsic fluorescence and the structural changes of serum albumin. At lower concentrations, Gd@C82(OH)22 was likely to rise fluorescence quenching of serum albumin through individual static quenching process by forming a ground-state complex, while dynamic and static coexisting quenching mechanism occurred in high concentration. Bimolecular quenching (Kq) value is twice the diffusion-controlled quenching constant (2.0 × 1010 L mol-1 s-1); binding sites of BSA were slightly more than those of HAS, and all of them reached to 1; the distance r between donor and acceptor was found to be 3.1494 nm and 3.6479 nm for HSA and BSA, respectively, both of which were fewer than 7 nm. It is confirmed that binding interaction for proteins in the presence of drugs was strong, the binding ratio was 1:1, and non-radiative energy transfer from protein to drug was extremely high probability in lower density. Binding process of Gd@C82(OH)22-HSA was driven mainly through van der Waals forces and hydrogen bonding formation, however more likely to be electrostatic interaction involved in the Gd@C82(OH)22-BSA binding process; Binding sites of Gd@C82(OH)22 to serum albumin were near tryprophan (HSA) and tyrosine residues (BSA), respectively. Moreover, a theoretical model of predicting the binding rate of drug to serum albumin was estimated, further analyzed that the binding rate was dynamically altered in various dose of protein and drug. Overall, these results provide potentially significant information for elucidating the distribution, transportation, the apparent relationship between pharmacologic activity and total plasma drug concentration as well as anti-carcinogenic activity and mechanisms in vivo.

Chemical Probe to Identify the Cellular Targets of the Reactive Lipid Metabolite 2- trans-Hexadecenal.

Lipid-derived electrophiles (LDEs) are reactive metabolites, which can covalently modify proteins and DNA and regulate diverse cellular processes. 2- trans-Hexadecenal (2-HD) is a byproduct of sphingolipid metabolism, involved in cytoskeletal reorganization, DNA damage, and apoptosis. In addition, the loss of ALDH3A2, an enzyme removing 2-HD in cells, is responsible for Sjörgen-Larsson Syndrome (SJS), suggesting that accumulation of 2-HD could lead to pathogenesis. However, the targets and the precise mechanisms of 2-HD are not well characterized. Herein, we report an alkyne-2-HD derivative as a bioorthogonal probe to explore the functions of 2-HD. We identified more than 500 potential cellular targets. Among them, the pro-apoptotic protein Bax can be covalently modified by 2-HD directly at the conserved Cys62 residue. Our work provided new chemical tools to explore the cellular functions of LDEs and revealed new mechanistic insights of the deregulation of lipid metabolism in diseases.

Asymmetric reduction of ketopantolactone using a strictly (R)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy.

OBJECTIVES: To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL]. RESULTS: The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%. CONCLUSIONS: Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

Action modes of recombinant endocellulase, EGA, and its domains on cotton fabrics.

OBJECTIVES: The action modes of an endocellulase, EGA, and its domains (CD9 and CBM3) during enzymatic treatment of cotton fabrics were investigated. RESULTS: EGA, CD9 and CBM3 had the binding capacity to cellulose substrates, of which the filter paper was the substrate with the strongest binding capacity. Analyses of scanning electronic microscopy indicated that EGA and its catalytic domain CD9 etched the surface of cotton fabrics and broke the fibers of long chains. On the other hand, the binding domain CBM3 only resulted in swelling of cotton fibers. Both EGA and its catalytic domain CD9 had minimal effect on the weight loss of cotton fabrics, whereas the effect of EGA and CD9 on the degree of polymerization and breaking strength was significant. After 12 h enzymatic action, the values of weight loss ratio for EGA and CD9 were 2.07 and 2.21 %, respectively, meanwhile the reductions in fabric strength were 27.04 % for EGA and 17.23 % for CD9. CONCLUSIONS: In contrast to the action of EGA and CD9, CBM3 showed no significant changes in terms of the weight loss ratio, degree of polymerization, and fabric strength.

Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase from Rhodococcus erythropolis WZ010.

The gene encoding a (2R,3R)-2,3-butanediol dehydrogenase from Rhodococcus erythropolis WZ010 (ReBDH) was over-expressed in Escherichia coli and the resulting recombinant ReBDH was successfully purified by Ni-affinity chromatography. The purified ReBDH in the native form was found to exist as a monomer with a calculated subunit size of 37180, belonging to the family of the zinc-containing alcohol dehydrogenases. The enzyme was NAD(H)-specific and its optimal activity for acetoin reduction was observed at pH 6.5 and 55 °C. The optimal pH and temperature for 2,3-butanediol oxidation were pH 10 and 45 °C, respectively. The enzyme activity was inhibited by ethylenediaminetetraacetic acid (EDTA) or metal ions Al3+, Zn2+, Fe2+, Cu2+ and Ag+, while the addition of 10% (v/v) dimethyl sulfoxide (DMSO) in the reaction mixture increased the activity by 161.2%. Kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2R,3R)-2,3-butanediol and NAD+. The activity of acetoin reduction was 7.7 times higher than that of (2R,3R)-2,3-butanediol oxidation when ReBDH was assayed at pH 7.0, suggesting that ReBDH-catalyzed reaction in vivo might favor (2R,3R)-2,3-butanediol formation rather than (2R,3R)-2,3-butanediol oxidation. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2R,3R)-2,3-butanediol via (R)-acetoin, demonstrating its potential application on the synthesis of (R)-chiral alcohols.

Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium.

This paper is concerned with optimization of fermentation conditions for lipstatin production with Streptomyces toxytricini zjut011 by the single factor and orthogonal tests. Five single factors of important effects on lipstatin production were explored. L-Leucine was identified to be the most suitable precursor for lipstatin biosynthesis and for the first time the divalent cations Mg(2+), Co(2+) and Zn(2+) were found to have significant effect on enhancing lipstatin fermentation titer. The effects of the additives on the lipstatin production were in the order of L-leucine ＞ Mg(2+) ＞ Co(2+) ＞ Zn(2+) ＞ octanoic acid. The optimized conditions for lipstatin production were determined as 45.72 mmol/L of L-leucine (added on the 4 th day), 31.1985 mmol/L of octanoic acid (added on the 6th day), 12 mmol/L of Mg(2+), 1 mmol/L of Co(2+) and 0.25 mmol/L of Zn(2+). Under these conditions, a maximum lipstatin of 4.208 g/ml was achieved in verification experiments in 500 ml shake flasks.

Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,ß-unsaturated alcohols from allylic aldehydes and ketones.

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,ß-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase.

Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol.

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K(m) values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.

Pinellia pedatisecta agglutinin targets drug resistant K562/ADR leukemia cells through binding with sarcolemmal membrane associated protein and enhancing macrophage phagocytosis.

Pinelliapedatisecta agglutinin (PPA) has previously been used in labeling fractions of myeloid leukemia cells in our laboratory. We report here that a bacterial expressed recombinant PPA domain b tagged with soluble coxsackie and adenovirus receptor (sCAR-PPAb) preferentially recognized drug resistant cancer cells K562/ADR and H460/5Fu, as compared to their parental cell lines. Pretreatment of K562/ADR cells with sCAR-PPAb significantly enhanced phagocytosis of K562/ADR by macrophages in vivo. Meanwhile, in a K562/ADR xenograft model, intratumoral injection of sCAR-PPAb induced macrophage infiltration and phagocytosis. Furthermore, immunoprecipitation, mass spectrometry and Western blot identified the membrane target of PPA on K562/ADR as sarcolemmal membrane associated protein (SLMAP). An antibody against SLMAP significantly promoted the phagocytosis of K562/ADR by macrophages in vitro. These findings suggest that PPA not only could be developed into a novel agent that can detect drug resistant cancer cells and predict chemotherapy outcome, but also it has potential value in immunotherapy against drug resistant cancer cells through inducing the tumoricidal activity of macrophages.

X-ray crystallographic characterization of new soluble endohedral fullerenes utilizing the popular C82 bucky cage. Isolation and structural characterization of Sm@C3v(7)-C82, Sm@C(s)(6)-C82, and Sm@C2(5)-C82.

Three isomers of Sm@C(82) that are soluble in organic solvents were obtained from the carbon soot produced by vaporization of hollow carbon rods doped with Sm(2)O(3)/graphite powder in an electric arc. These isomers were numbered as Sm@C(82)(I), Sm@C(82)(II), and Sm@C(82)(III) in order of their elution times from HPLC chromatography on a Buckyprep column with toluene as the eluent. The identities of isomers, Sm@C(82)(I) as Sm@C(s)(6)-C(82), Sm@C(82)(II) as Sm@C(3v)(7)-C(82), and Sm@C(82)(III) as Sm@C(2)(5)-C(82), were determined by single-crystal X-ray diffraction on cocrystals formed with Ni(octaethylporphyrin). For endohedral fullerenes like La@C(82), which have three electrons transferred to the cage to produce the M(3+)@(C(82))(3-) electronic distribution, generally only two soluble isomers (e.g., La@C(2v)(9)-C(82) (major) and La@C(s)(6)-C(82) (minor)) are observed. In contrast, with samarium, which generates the M(2+)@(C(82))(2-) electronic distribution, five soluble isomers of Sm@C(82) have been detected, three in this study, the other two in two related prior studies. The structures of the four Sm@C(82) isomers that are currently established are Sm@C(2)(5)-C(82), Sm@C(s)(6)-C(82), Sm@C(3v)(7)-C(82), and Sm@C(2v)(9)-C(82). All of these isomers obey the isolated pentagon rule (IPR) and are sequentially interconvertable through Stone-Wales transformations.

Towards the discovery of alcohol dehydrogenases: NAD(P)H fluorescence-based screening and characterization of the newly isolated Rhodococcus erythropolis WZ010 in the preparation of chiral aryl secondary alcohols.

A simple and reliable procedure was developed to screen biocatalysts with high alcohol dehydrogenase activity, efficient internal coenzyme regeneration, and high stereoselectivity. The strategy of activity screening in a microtitre plate format was based on the detection of fluorescence of NAD(P)H originating from the oxidation of alcohols. The primary and secondary screenings from soil samples yielded a versatile bacterial biocatalyst Rhodococcus erythropolis WZ010 demonstrating potential for the preparation of chiral aryl secondary alcohols. In terms of activity and stereoselectivity, the optimized reaction conditions in the stereoselective oxidation were 30 °C, pH 10.5, and 250 rpm, whereas bioreduction using glucose as co-substrate was the most favorable at 35 °C and pH 7.5 in the static reaction mixture. Under the optimized conditions, fresh cells of the strain stereoselectively oxidized the (S)-enantiomer of racemic 1-phenylethanol (120 mM) to acetophenone and afforded the unoxidized (R)-1-phenylethanol in 49.4 % yield and >99.9 % enantiomeric excess (e.e.). In the reduction of 10 mM acetophenone, the addition of 100 mM glucose significantly increased the conversion rate from 3.1 to 97.4 %. In the presence of 800 mM glucose, acetophenone and other aromatic ketones (80 mM) were enantioselectively reduced to corresponding (S)-alcohols with excellent e.e. values. Both stereoselective oxidation and asymmetric reduction required no external cofactor regeneration system.

Comparative proteomic analysis of colon cancer cell HCT-15 in response to all-trans retinoic acid treatment.

Colon cancer is one of the most common malignances. In vitro and in vivo study show that retinoic acids inhibit a wide variety of cancer cells but the molecular mechanism of their anti-tumor effects are not yet fully understood. Alltrans retinoic acid (ATRA), an isomer of retinoic acid, can inhibit the proliferation of HCT-15 human colon cancer cell line. A proteomic analysis was performed using HCT-15 treated with ATRA to further elucidate the retinoic acid signaling pathway and its anti-tumor effect mechanism. MTT results showed that the growth of HCT-15 cells were significantly inhibited by ATRA. The alkaline phosphatase activity assay showed that ATRA failed to induce the differentiation of HCT-15. The DNA ladder detection showed that ATRA induced apoptosis in HCT-15. Two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry identified 13 differentially expressed proteins in HCT-15 cells after all-trans retinoic acid treatment. Among the identified differentially expressed proteins, there were four scaffold proteins (YWHAE, SFN, YWHAB, and YWHAZ), two ubiquitin modification related proteins (ISG-15 and UBE2N), two translational initiation factors (EIF1AX and EIF3K), two cytoskeleton related proteins (EZRI and CNN3), two proteinmodification related proteins (TXNDC17 and PIMT), and one enzyme related to phospholipid metabolism (PSP). Both EZRI and UBE2N were rendered to western-blot validation and the results were consistent with the two-dimension electrophoresis analysis. In this study, the differentially expressed proteins in HCT-15 treated by ATRA were identified, which will assist the further elucidation of the anti-tumor mechanism of retinoic acids.