查看更多>>摘要：With the development of green chemistry, the demand for environmentally friendly and biocompatible solvents for non-aqueous enzymatic catalysis is increasingly urgent. Deep eutectic solvents (DESs) are viewed as the most promising alternatives to traditional organic solvents in non-aqueous biocatalysis. To expand the types of DESs and provide guidance for DESs design for non-aqueous enzymatic catalysis, the enzyme performance in aqueous buffer after incubation in nearly anhydrous DESs was associated with the properties and component structures of DESs. Almond 13- Glucosidase (13-GC) and Candida antarctica lipase B (CALB) were selected as model enzymes. Physico-chemical properties of DESs (as inferred by their solvatochromic parameters) were applied to explore the influences of DESs properties on enzyme activity. For DESs with the same HBD, the biocompability of DESs and thermal stability of enzymes in DESs were negatively associated with the polarity and hydrogen bond acidity of DESs, and were positively associated with hydrogen bond basicity of DESs. Whereas an opposite trend was observed in DESs with the same HBA. Analyzing from the DESs components, the biocompatibility of hydrophobic DESs for enzyme was much lower than that of hydrophilic DESs. Generally, the amount of hydroxyl group and the length of carbon chain represent advantageous ingredients for maintaining natural structure of enzyme molecule. The presence of carboxyl group in hydrophilic DESs and carbon-carbon double bond may impair enzyme structure and activity. This work is hoped to be helpful in expanding the applications of DESs in nonaqueous biocatalysis.
Kang, MinjuKim, Ha HyungKim, Dong-IlLim, Jin-Hyuk...
10页
查看更多>>摘要：Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and over expressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and alpha 2,6-sialyltransferase (alpha 2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and alpha 2,6-ST), and co-overexpression (GnT-IV and alpha 2,6-ST, and GnT-V and alpha 2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnTIV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (-10.9% and-18.6%, respectively), while bi-and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV.ST (cells co-overexpressing GnT-IV and alpha 2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV.ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and alpha 2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.
查看更多>>摘要：The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae.
查看更多>>摘要：Ethyl 3-hydroxy-3-phenylpropionate (EHPP), (R)-EHPP or (S)-EHPP, is an important chiral intermediate for pharmaceuticals. Its synthesis from ethyl benzoyl acetate (EBA) by alcohol dehydrogenase is regarded as a green method. However, scarcely any alcohol dehydrogenase has been reported competent in asymmetric synthesis of chiral EHPP at high EBA loading. Present study developed two robust and efficient bio-catalysts Mu-S2 and Mu -R4 for preparation of (S)-EHPP and (R)-EHPP respectively by rational design of alcohol dehydrogenase PcSDR from Pedobacter chitinilyticus based on molecular dynamics (MD) simulation analysis. BtGDH, a glucose dehy-drogenase from Bacillus toyonensis catalyzing the oxidation of glucose for cofactor regeneration, was co-expressed with the screened mutants to form enzyme systems Mu-S2-BtGDH and Mu-R4-BtGDH. After reaction condition optimization, Mu-S2-BtGDH and Mu-R4-BtGDH were efficient in the synthesis of (S)-EHPP (94% conv. and 99% e.e.) and (R)-EHPP (99% conv. and 98% e.e.) respectively in 100 mL scale under 500 mM of EBA loading in 10 h following a substrate continuous feeding mode. After purifying, the isolated yield for each EHPP enantiomer is > 93%. This work not only provides potential biocatalysts for the industrial production of (R)-EHPP and (S)-EHPP, but also enriches the constructure-function relationship of alcohol dehydrogenases.
查看更多>>摘要：Benzene, toluene, and xylene (BTX) are volatile aromatic compounds used in industries, however, they are hazardous when released into the environment. BTX degradation by Aspergillus niger cells combined with semiconducting zinc sulfide (ZnS) nanoparticles was explored in batch systems. Experiments were conducted individually for benzene, toluene, and xylene as well as in binary and trinary mixtures using A. niger cells-ZnS nanobiohybrids. The mechanism governing the removal of BTX by both A. niger cells and A. niger cells-ZnS nanobiohybrids were elucidated. Complete BTX degradation was achieved in 75 min and 60 min, respectively, by nanobiohybrids composed of chemical and biological ZnS nanoparticles in the presence of UV-A light at 1.83 * 1018 photons/second and 1.68 * 1018 photons/second, respectively. The removal efficiency was in the order of the molecular weight for A. niger cells, whereas for the light-driven A. niger-ZnS nanobiohybrids, the removal efficiency was according to the methyl group number. Further, the respiratory coefficient and volumetric mass transfer coefficient (Ka) values are higher for A. niger cells compared to the light-driven A. niger-ZnS nanobiohybrids.
查看更多>>摘要：Hitherto, the contribution of C-terminal amino acids in structure, stability and function of GH26 endomannanases has not been demonstrated. Semi-logarithmic plot of endo-mannanase activity showed a progressive decline with increase in the number of truncated amino acids [ManB-CA5 (129 U/mL), ManB-CA10 (47 U/ mL), ManB-CA15 (0.05 U/mL) and ManB-CA20 (0.02 U/mL)]. ManB-CA5 and ManB-CA10 exhibited similar temperature and pH optima and product profile but biochemical properties (kinetic constants, mannan hydrolysis, response to metal ions and enzyme inhibitors) and stability (in presence of commercial detergents, anionic surfactants and organic solvents and half-life) were markedly affected. Interaction of truncated proteins with anionic surfactants was probed using intrinsic, Nile red, acrylamide quenching, resonance light scattering and synchronous fluorescence spectroscopy studies. Truncation of ten amino acids increased vulnerability to anionic surfactants as conformational changes, exposure of the hydrophobic core and susceptibility to unfolding process were observed. The microenvironment around Trp residues was affected more with surfactants as compared to Tyr residues in truncated proteins. Zn2+ coordination might not play a role in providing stability against SDS. MD simulation studies corroborated that C-terminal amino acids (327-336) helps in structure stabilization, regulating flexibility of loops around the active site and preventing denaturation in the presence of SDS.
Hong, Seong-JinPark, Bo-RamLee, Ha-NulJang, Da Eun...
7页
查看更多>>摘要：Thermoanaerobacter thermocopriae-derived thermostable cycloisomaltooligosaccharide (CI)-forming enzymes catalyze the production of CIs from dextran. The primary structure of the enzyme is comprised of CI glucanotransferase (TtCITase) at the N-terminal region and long isomaltooligosaccharide-forming enzyme (TtTGase) at the C-terminal region connected by carbohydrate-binding module family 35 (CBM, TtCBM). Three truncated mutants of CI-forming enzymes were successfully produced in Corynebacterium glutamicum, a food-grade host system, and their biochemical properties were characterized. The enzymes had optimum at pH 6.0 and pH-stability (5.0-12.0). Three enzymes had optimum temperature over 55 degrees C and they maintained 80% activity at 55 degrees C for 2 h, 12 h, and 18 h, respectively. Enzymes without CBM showed weaker allosteric behavior than those of other enzymes, which suggests the important role of CBM in allosteric behavior. However, CBM bearing enzymes showed high production of CIs with various degree of polymerization. These enzymes have potential application as the encapsulating material for insoluble pharmaceutical biomaterials.
查看更多>>摘要：Enzyme immobilization technology has a key role in improving the stability of enzyme reaction systems and biocatalyst utilization rates. In this study, polyvinyl alcohol/sodium alginate@Fe3O4 (PVA/SA@Fe3O4) magnetic immobilized-enzyme hydrogel beads were prepared. Their structure and morphology were characterized by scanning electron microscopy, surface area and porosity analyses, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and a vibrating sample magnetometer. The capability of PVA/SA@Fe3O4 to adsorb neutral protease was investigated with variations in composition, temperature, pH, stirring speed, enzyme concentration, and crosslinking concentration. The optimal parameters of the immobilization process were determined by response surface methodology (3% neutral protease, 4% crosslinking at 200 rpm, 45 degrees C and pH 7.2), under which an immobilization rate of 41.98 mg/g was obtained. The thermal stability, acid-base stability, and reusability of the immobilized enzyme were improved significantly. After seven cycles, the immobilized enzyme activity remained at 30.8% that of the initial enzyme activity. The results indicate that the immobilization of NP onto magnetic PVA/SA@Fe3O4 hydrogel beads improves enzyme efficiency, giving this process potential industrial applications.
查看更多>>摘要：This study aimed the enzymatic decyl esters production by hydroesterification, a two-step process consisting of hydrolysis of refined soybean (RSBO) or used soybean cooking (USCO) oils to produce free fatty acids (FFA) and further esterification of purified FFA. Using free lipase from Candida rugosa (CRL), about 98% hydrolyses for both oils have been observed after 180 min of reaction using a CRL loading of 50 U g-1 of reaction mixture, 40 degrees C, and a mechanical stirring of 1500 rpm. FFA esterification with decanol in solvent-free systems was performed using lipase from Thermomyces lanuginosus (TLL) immobilized by physical adsorption on silica particles extracted from rice husk, an agricultural waste. For such purpose, non-functionalized (SiO2) or functionalized rice husk silica bearing octyl (Octyl-SiO2) or phenyl (Phe-SiO2) groups have been used as immobilization supports. Protein amounts between 22 and 28 mg g-1 of support were observed. When used in the esterification, they enabled a FFA conversion of 81.3-87.6% after 90-300 min of reaction. Lipozyme TL IM, a commercial immobilized TLL, exhibited similar performance compared to TLL-Octyl-SiO2 (FFA conversion approximate to 90% after 90-120 min of reaction). However, high operational stability after fifteen successive esterification batches was observed only for TLL immobilized on Octyl-SiO2 (activity retention of approximate to 90% using both FFA sources). The produced decyl esters presented good characteristics as potential biolubricants according to standard methods (ASTM) and thermal analysis.
NSTL
Elsevier