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1.
alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.  相似文献   

2.
Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.  相似文献   

3.
The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N′-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of α-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20°C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.Linus Olofsson and Pernilla Söderberg authors have contributed equally to the work.  相似文献   

4.
Direct enantioseparation of mandelic acid by high-performance liquid chromatography (HPLC) with a reversed phase column and a mobile phase containing a small amount of hydroxylpropyl-β-cyclodextrin (HP-β-CD) was studied as an efficient method for saving consumption of the CD additive. As a result, it was proposed that racemic mandelic acid can be analyzed with a phenyl column by using a mobile phase composed of 10 mM ammonium acetate buffer (pH 4.2) and 0.02% (w/v) HP-β-CD at a flow rate of 1.0 mL/min at 40°C after the passage of 10 mM ammonium acetate buffer (pH 4.2) containing 0.1% (w/v) HP-β-CD as a precoating mobile phase for 60 min. It is suggested that HP-β-CD is bound with a phenyl group on the surface of the stationary phase to allow a phenyl column to act as a transient chiral column, and injected mandelic acid can form the ternary complex with the adsorbed HP-β-CD. The longer retention time of D-mandelic acid than the L-isomer for HPLC can be explained from the higher stability of the HP-β-CD complex with D-mandelic acid, which was confirmed by CE experiment with HP-β-CD as a selector. The efficiency of a phenyl column compared with other stationary phases was also discussed.  相似文献   

5.
The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF6] at 80 °C.  相似文献   

6.
Almond β-d-glucosidase was used to catalyze alkyl-β-d-glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β-d-glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.  相似文献   

7.
The effect of various organic solvents on the hydrolytic and esterification activities of lipase A from Candida antarctica (CALA) was investigated. Compared to the control group, the esterification and hydrolytic activities of CALA both increased with treatment of acetonitrile or acetone. However, the catalytic activity decreased after treatment with other organic solvents, especially with ethanol or ethyl acetate. Moreover, with treatment by the same organic solvents, the residual esterification activity of CALA was much higher than the hydrolytic activity for the olive oil. Results of Fourier transform-infrared spectroscopy analysis implied that the catalytic activity variance was mainly attributed to the secondary structure changes of CALA. The acetonitrile treatment could also increase the thermal and pH stability of CALA.  相似文献   

8.
Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.  相似文献   

9.
The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.  相似文献   

10.

Objective

To establish a method for the recovery and reutilization of hydroxypropyl-β-cyclodextrin (HP-β-CD) to lower the cost of its industrial application in cortisone acetate bioconversion.

Results

HP-β-CD is not degraded by Arthrobacter simplex CPCC140451 (ASP) resting cells and 96.4 % HP-β-CD could be recovered by isobutyl acetate extraction. Moreover, the inclusion ability of recovered HP-β-CD barely decreased. The saccharide metabolic and catalytic activities of ASP were greater in the aqueous phase after extracting with isobutyl acetate than other organic solvents. Cyclic utilization tests showed that cortisone acetate conversion ratio was 91.0 % after eight cycles and reached 95.7 % with 0.2–0.6 mM HP-β-CD. Furthermore, >90 % conversion ratio was reached per cycle through a co-cyclic-utilization method with HP-β-CD and immobilized ASP.

Conclusion

Cortisone acetate conversion ratio in the HP-β-CD cyclic-utilization method is promising for industrial applications. The method can also be expanded to other CDs and other hydrophobic compounds bioconversion.
  相似文献   

11.
Lung cancer is notorious for high morbidity and mortality around the world. Interleukin (IL)-8, a proinflammatory chemokine with tumorigenic and proangiogenic effects, promotes lung cancer cells growth and migration and contributes to cell aggressive phenotypes. Integrin αvβ6 is a receptor of transmembrane heterodimeric cell surface adhesion, and its overexpression correlates with poor survival from non–small cell lung cancer. However, the cross talk between αvβ6 and IL-8 in lung cancer has not been characterized so far. Herein, human lung cancer samples were analyzed, and it revealed that the immunohistochemical and mRNA expression of integrin αvβ6 was significantly correlated with the expression of IL-8. Furthermore, in vitro, integrin αvβ6 increased cell proliferation, migration, and invasion by impairing the expressions of MMP-2 and MMP-9 and inhibited cell apoptosis in human lung cancer cells A549 and H460. In addition, integrin αvβ6 upregulated IL-8 expression through activating MAPK/ERK signaling. The in vivo experiment showed that integrin αvβ6 promoted tumor growth in xenograft model mice by accelerating tumor volume and reducing apoptosis. Meanwhile, lung metastasis model experiment suggested that integrin αvβ6 stimulated tumor metastasis with the increase of lung/total weight and tumor nodules. Simultaneously, integrin αvβ6 upregulated IL-8 expression detected by both Western blots and immunohistochemistry, along with the activation of MAPK/ERK signaling. Overall, these data suggested that, in vitro and in vivo, integrin αvβ6 promoted lung cancer proliferation and metastasis, at least in part, through upregulation of IL-8–mediated MAPK/ERK signaling. Thus, the inhibition of integrin αvβ6 and IL-8 may be the key for the treatment of lung cancer.  相似文献   

12.
A high-performance liquid chromatographic (HPLC) method has been developed for separation and determination of components in hydroxypropyl-β-cyclodextrin (HP-β-CD). The method involves separation on an amino-bonded HPLC column using water–acetonitrile as a mobile phase with a polarimetric HPLC detector for quantification. It provides good selectivity and sensitivity and can also be used to compare different sources of HP-β-CD and to measure batch to batch variation. The similarity of the values of molar optical rotation for β-cyclodextrin (β-CD) and HP-β-CD suggests that a polarimetric HPLC detector may be used with a straightforward area normalization method, to quantify the proportion of β-CD in any HP-β-CD sample. Trace amounts of β-CD in HP-β-CD have been measured to a precision of 0.01%. © 1993 Wiley-Liss, Inc.  相似文献   

13.
Two secondary alcohol glucosides, cyclohexyl-α-d-glucoside and cyclohexyl-β-d-glucoside, were synthesized via the condensation reaction of cyclohexanol with d-glucose in a biphase system catalyzed by α-glucosidase and β-glucosidase, respectively. The effects of pH, water content, glucose concentration and metal ions on the yield of glucosides were studied. The optimum catalytic conditions established for α-glucosidase was 25% (v/v) water content, 2.5 mol/L glucose concentration and pH 2.0, and for β-glucosidase was 30% (v/v) water content, 2.0 mol/L glucose and pH 5.0. The maximum yield of glucoside was 13.3 mg/mL for cyclohexyl-α-d-glucoside and 8.9 mg/mL for cyclohexyl-β-d-glucoside. Synthesis progress was monitored by TLC and quantitatively analyzed by pre-derived capillary gas chromatography (GC). The retention time was 12.34 min for the α isomer and 12.96 min for the β isomer, respectively. With an anomeric purity of more than 99.5%, the two glucosides display excellent site-specific catalysis by α- and β-glucosidase. Herein, we present a general method to produce anomerically pure glucosides via a one-step bio-reaction in a biphase system. This method could potentially be applied in glucosylation of primary and secondary alcohols or other reactions requiring glucosylation.  相似文献   

14.
Novel glycosides of piceid (3,4′-5-trihydroxy stilbene 3-O-β-d-glucoside) were produced by the transglycosylation reactions of cyclodextrin glucanotransferase (CGTase) from Bacillus macerans, with piceid (PicG1) and maltodextrin as the acceptor and donor substrates, respectively. The reactions were performed at 40 °C with 2.56 mM piceid (0.1% w/v) and maltodextrin (5% w/v) in 0.02 M citrate phosphate buffer, pH 6.0 containing 5% (v/v) methanol for 6 h. Glucose, maltose, sucrose, maltotriose and α-cyclodextrin (α-CD) were also used to analyze their ability to function as donor substrates, for the glycosylation of piceid. Among the different donor substrates used, the maximum transfer efficiency (TE) of glycosylation of piceid was observed for α-cyclodextrin (78.9%) followed by maltodextrin (72.1%). The partially purified piceid glycoside products (PicG2 and PicG3) were identified by mass spectrometry.  相似文献   

15.
《Process Biochemistry》2007,42(5):909-912
The effects of aqueous solutions of ethanol, acetonitrile and 1,4-dioxane in the concentration range 10–90% (v/v) on the activity of porcine pepsin were studied. The enzyme retained its activity in aqueous ethanol and aqueous acetonitrile with increasing organic solvent concentration up to 60%, and in aqueous 1,4-dioxane up to 30%, but thereafter a considerable decrease in activity was observed. The changes caused in the catalytic activity by the water-miscible organic solvents may be related to structural changes, which were followed by means of intrinsic fluorescence and circular dichroism spectroscopy measurements.  相似文献   

16.
The αvβ6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-β1 and transforming growth factor-β3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein 1 (VP1) coat protein of the O1 British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for αvβ6, and we recently reported that a peptide derived from VP1 exhibited αvβ6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to αvβ6. A 17-mer peptide of VP1 was inserted into the complementarity-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to αvβ6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with αvβ6 but not with α5β1, αvβ3, αvβ5, αvβ8 or CEA. B6-2 was internalized into αvβ6-expressing cells and inhibited αvβ6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of αvβ6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block αvβ6-mediated cancer cell invasion or to deliver and internalize toxins specifically to αvβ6-expressing tumors.  相似文献   

17.
The peptide synthesis from N-acetyl-L-tyrosine ethyl ester and amino acid amides was realized using α-chymotrypsin as a catalyst in ethanol or acetonitrile containing small amounts of water. In these reaction systems, the precipitates of phosphate salt, which was used as a component of buffer solution, are considered to act as carriers of chymotrypsin. It was found that peptide formation is competitive with hydrolysis of the substrate ester, but the secondary synthesis of the peptide from the hydrolysate was also considered to proceed. The yield of the peptide after 24 h reaction was strongly dependent on the water concentration; maximum yields of the peptide were obtained at water concentrations below 10% (v/v). The addition of tertiary amines, such as triethyl amine, markedly increased the peptide yield, probably due to the increase in the concentration of the nucleophilic amine components by neutralization of hydrohalides of amino acid amides. The effect of reaction temperature and the reactions with CT immobilized on PVA, chitosan, or TEAE-cellulose are also described.  相似文献   

18.
Pepsin, a member of the aspartate protease family, exists in a partially unfolded state at alkaline pH where the N-terminal domain of pepsin has a flexible structure while the C-terminal domain has a highly folded structure. In this work, the conformational stability of porcine pepsin in an alkaline denatured (A(D)) state against acetonitrile and ethanol solvents was studied using a combination of electronic circular dichroism (ECD) and fluorescence techniques. The ECD results demonstrate that both ethanol and acetonitrile induce secondary structural changes in pepsin at A(D) state. However, the minimum concentration required to induce significant secondary structural changes in pepsin varies for ethanol (>30%, v/v) and acetonitrile (>60%, v/v) solvents. At maximum concentration used (90%, v/v), both solvents induce predominantly β-sheet conformation. Unlike acetonitrile, ethanol induces significant amount of non-native α-helical conformations at the intermediate concentrations (50-80%). The tryptophan fluorescence results demonstrate that both acetonitrile and ethanol induce substantial changes in the tertiary structure of pepsin in the A(D) state above certain concentrations. The current results have important implications in understanding the effect of co-solvents on the conformation of proteins in the "denatured state".  相似文献   

19.
Synthesis of N-CBZ-(N-Carbobenzoxy)-1-amino-acid methyl esters from N-CBZ-amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent K(m) value of CBZ-glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S(2) subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.  相似文献   

20.
Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.  相似文献   

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