Shubai Li

Comparison: Adsorption of papain using immobilized dye ligands on affinity membranes

Novel membranes that involve the immobilization of Reactive Red 120 or Reactive Brown 10 as dye ligands were prepared. These were used in the purification of papain from papaya powder extracts. Papain adsorption capacities for the Red 120 and Brown 10 membranes were 143.6 mg/g and 107.3mg/g, respectively. The effectiveness of adsorption was demonstrated by Freundlich isotherm proficiency. The enzyme was eluted from the respective dye membranes using 1.0M NaCl at pH 6.0 and yields of over 80% were found for the Red 120-CS (chitosan)-nylon membrane whereas only a 50% recovery was possible using the Brown 10-CS-nylon membranes. It is concluded that Red 120-CS-nylon membranes could play an active role in the separation and purification of papain from crude extracts. This system has the potential to be developed for the commercial isolation of the protein.

Controlled release from thermo-sensitive PNVCL-co-MAA electrospun nanofibers: The effects of hydrophilicity/hydrophobicity of a drug

The thermo-sensitive copolymer poly(N-vinylcaprolactam-co-methacrylic acid) (PNVCL-co-MAA) was synthesized by free radical polymerization and the resulting nanofibers were fabricated using an electrospinning process. The molecular weight of the copolymer was adjusted by varying the content of methacrylic acid (MAA) while keeping that of N-vinylcaprolactam (NVCL) constant. Hydrophilic captopril and hydrophobic ketoprofen were used as model drugs, and PNVCL-co-MAA nanofibers were used as the drug carrier to investigate the effects of drug on its release properties from nanofibers at different temperatures. The results showed that slow release over several hours was observed at 40°C (above the lower critical solution temperature (LCST) of PNVCL-co-MAA), while the drugs exhibited a burst release of several seconds at 20°C (below the LCST). Drug release slowed with increasing content of the hydrophobic monomer NVCL. The hydrophilic captopril was released at a higher rate than the hydrophobic ketoprofen. The drug release characteristics were dependent on the temperature, the portion of hydrophilic groups and hydrophobic groups in the copolymer and hydrophilicity/hydrophobicity of drug. Study on the mechanism of release showed that Korsmeyer-Peppas model as a major drug release mechanism. Given these results, the PNVCL-co-MAA copolymers are proposed to have useful applications in intellectual drug delivery systems.

Preparation and characterization of a novel sodium alginate incorporated self-assembled Fmoc-FF composite hydrogel.

Dipeptides and their derivatives have attracted tremendous attention owning to their excellent abilities of self-assemble assembling into various structures which have great potentials for applications in biology and/or nanotechnology. In the present study, we dedicate to fabricate a rigid and structure controllable Fmoc-FF/SA composite hydrogel. We found that the modified dipeptide, fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Phe-Phe) can self-assemble into rigid hydrogels with structures of nanowires, layered thin films or honeycombs as the change of sodium alginate (SA) concentration. Meanwhile, CD-spectroscopy demonstrated that SA appeared to control the process, but it did not change the arrangement of the Fmoc-FF peptide. Our results demonstrated that the formed hydrogel showed physical and chemical stability as well as possessing good biocompatibility. Rheological measurements showed that the addition of SA could improve the stability of the hydrogel. Cell viability assay revealed that the Fmoc-FF and Fmoc-FF/SA hydrogels are both beneficial for cell proliferation in-vitro. Our results indicated that the fabricated Fmoc-FF/SA composite hydrogels could be used in tissue engineering and drug delivery in the future.

SH-Methylation of SH-Containing Heterocycles with Dimethyl Carbonate via Phase-Transfer Catalytic Reaction

Abstract A reaction of SH-containing heterocycles with dimethyl carbonate (DMC) in the presence of K2CO3 and tetrabutylammonium bromide (Bu4NBr) gave heteroaryl methyl thioethers in 44–93% yields. The reaction was carried out under mild conditions. This method provided a useful synthetic method for preparation of various heteroaryl methyl thioethers without the use of toxic methylic halides or dimethyl sulfate.

Kinetic Evaluation of Aminoethylisothiourea on Mushroom Tyrosinase Activity

This study demonstrates that aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase, is an irreversible competitive inhibitor of mushroom tyrosinase by chelation to the active site of tyrosinase when l-3,4-dihydroxyphenylalanine was assayed spectrophotometrically. The spectrophotometric recordings of the inhibition of tyrosinase by AET were characterized by the presence of a lag period prior to the attainment of an inhibited steady-state rate. The lag period corresponded to the time in which AET was reacting with the enzymatically generated o-quinone. Increasing AET concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady-state rate were dependent on AET, substrate, and tyrosinase concentrations. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET. The UV-visible spectrum of a mixture of tyrosinase and AET exhibited a characteristic shoulder peak ascribed to the chelation of AET to the active site of tyrosinase. Moreover, the presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition when CuSO4 was added to the assay medium on tyrosinase activity.

Enzyme Design by Chemical Modification of Papain Lysine

Papain (EC 3.4.22.2) has been chemically modified using two reagents including different anhydrides of maleic and poly-maleic acids. The average ratio of modified-NH2 was tested by trinitrobenzenesulfonic acid (TNBS) method. The native and modified papain were purified by dialysis and their structures were characterized by FTIR. The factor related with the activity of the modified papain, such as temperature, pH value and kinetic constant were studied and compared with the native papain. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with native papain, the thermal stability and the resistance to alkali of modified enzyme were improved considerably. Among the enzyme, poly-maleic anhydride modified enzyme was found to be the best in catalytic efficiency. Keywords-chemical modification; papain; maleic anhydride;

Papain Immobilization on Chitosan-Coated Nylon Membrane: Preparation and Its Application in Cystatin Separation

Nylon membranes are first hydrolyed with dilute HCl and then treated with chitosan before being used as the affinity carrier. Papain as a ligand has been immobilized on the activated membranes with glutaraldehyde as a crosslinking agent. The factors involving with the activity of immobilized papain, such as concentration of glutaraldehyde, pH, temperature, reaction time, and the amount of added papain have been studied. The results show that the optimum conditions for the preparation of immobilized papain nylon affinity membranes are as follows: pH 9.0, 0.5% glutaraldehyde solution, the concentration of added papain is 10 mg/ml, the reaction time is 6 h at 45degC. The nylon membrane prepared is then used to purify cystatin from potato juice, and shows that they are high efficiency affinity membranes base for cystatin separation.

Kinetic Analysis of Aminoethylisothiourea on Diphenolase of Mushroom Tyrosinase

This study demonstrates that Aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase (NOS), is an irreversible competitive inhibitor of the diphenolase activity of mushroom tyrosinase when L -DOPA was assayed spectrophotometrically in vitro. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET.

Novel Affinity Membrane Chromatography Technology: Purification of Pepsin Using Immobilized Reactive Dye Ligands

Novel membranes were prepared through covalently coupling of chitosan (CS) to activate nylon membrane after the reaction of the microporous nylon membrane with formaldehyde. Non-specific adsorption on the CS-coated nylon membrane decreased greatly. Yellow 3G-P/Reactive Red 120 were then covalently immobilized onto composite membrane as dye ligands. These Red 120/Yellow 3G-P-CS-membranes were used in the Pepsin adsorption studies. According to the results, it appears that Yellow 3G-P-CS-nylon membranes could be applied for separation and purification of Pepsin without causing any denaturation, much better than Red 120-CS-nylon membrane. Keywords-affinity chromatography; pepsin; adsorption;

4-Hydroxyphenylacetic Acid as a Monophenolase Inhibitor and a Diphenolase Activator on Mushroom Tyrosinase

4-Hydroxyphenylacetic acid (4-HPA) can inhibit monophenolase (IC 50 =0.79 mM) and activate diphenolase of mushroom tyrosinase with its increasing concentrations. The activation kinetics, analyzed by Lineweaver-Burk plots, indicated 4-HPA to be a competitive and uncompetitive mixed-type activator of diphenolase when L -DOPA was used as substrate. 4-HPA can not only inhibit the oxy form but also activate the met form of mushroom tyrosinase.