IMMOBILIZATION OF GLUCOSE OXIDASE AND CELLULASE BY CHITOSAN— POLYACRYLIC ACID COMPLEX

This study is concerned with chitosan-polyacrylic acid complex as a carrier to immobilize glucose oxidase (GOD)and cellulase. The optimum emperature of the immobilized GOD (IG) was determined to be 60℃which is higher than that of the native GOD about 40℃. The optimum temperature of the immobilized cellulase (IC) was determined to be about 30℃higher than that of native cellulase. Both of the optimum pH of IG and IC shifted one pH unit to acid. Immobilized enzyme may be used in more wide pH range. Their storage life are much longer compared with their native states. Both of them can be reused at least 12 times.     

[Cu(2,2'-bpy)Cl2]和[Cu(1,10-phen)C12]在多金属氧酸盐上的多相化:选择性氧化四氢化萘的新催化剂

Mononuclear Cu(II)bipyridine(1)and phenantroline complexes(2)were synthesized and immobilized by different procedures on H3PW12O40 polyoxometalate(POM).Characterization by XRD and SEM-EDX were performed to assess the preservation of the Keggin structure and stoichiometry of the complex.The immobilized complexes were tested as heterogeneous catalysts for the partial oxidation of tetralin(1,2,3,4-tetrahydronaphthalene)using hydrogen peroxide as oxidant in acetonitrile/water as solvent.[Cu(2,2’-bpy)Cl][H2PW12O40] and[Cu(1,10-phen)Cl][H2PW12O40]oxidized tetralin at room temperature,with 16%conversion with(2),to 1-tetralone and 2-tetralone with 83%selectivity.However,the selectivity for 1-tetralone was only 56%.Different preparation methods for the heterogenization of these complexes on the POM Keggin unit were compared and used to enhance the selectivity to 1-tetralone to 75%.     

STRUCTURE AND CATALYTIC ACTIVITY OF RHODIUM COMPLEX COORDINATED ON 2-VINYLPYRIDINE-METHYL ACRYLATE COPOLYMER FOR THE CARBONYLATION OF METHANOL

A series of rhodium complexes coordinated on the hybrid ligand. 2-vinylpyridine-methyl acrylate copolymers, as catalyst for the carbonylation of methanol were synthesized by the reaction of the copolymers with tetracarbonyldichlorodirhodium. These complexes were in general very reactive in carbonylation of methanol to form acetic acid and methyl acetate without other by-product. The XPS studies of the complexes showed that the binding energies of Nls(399.1-399.4 eV) and that of Ols(532.5-532.9 eV) were higher than that of the copolymer ligand (Nls 398.1-398.3 eV and Ols 532.2-532.3 eV), indicating the fromation of N→Rh and O→Rh coordination bonds. The IR spectra of complexes showed that the appearance of two absorption peaks of terminal carbonyl at 1990-2100 cm~(-1), characteristic of the cis-dicarbonyl rhodium moiety. These XPS and IR studies confirm the formation of cis-dicarbonylrhodium complex with the active centers(A) and (B) as shown in the text. The increase of activity of the complex in the c     

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg2 /Al3 layered double hydroxide (LDH) material was prepared by means of amodified coprecipitation method involving a rapid mixing step followed by a separate aging process. LDH calcined at 500℃ , denoted as CLDH, was characterized by XRD, IR and BET surface area measurements.CLDH has a poor crystalline MgO-like structure with a high surface area and porosity. CLDH was used as asupport for the immobilization of penicillin G acylase(PGA). The effect of varying the immobilization conditions, such as pH, contact time and the ratio of enzyme to support, on the activity of the immobilized enzymein the hydrolysis of penicillin G has been studied. It was found that the activity of the immobilized enzyme decreased slightly with decreasing pH and reached a maximum after a contact time of 24 h. The activity of theimmobilized enzyme increased with increasing the ratio of enzyme to support. It was found that the adsorption of PGA inhibited the expected reaction of CLDH with an aqueous medium to regenerate a LDH phase. Itsoriginal activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.     

Peroxo Complexes of Molybdenum(VI) Containing Mannich Base Ligands

The molybdenum(VI)-peroxo complexes containing Mannich base ligands having a formula as [MoO(O2)2(L-L)] [where L-L＝morpholinobenzyl benzamide (MBB), piperidinobenzyl benzamide (PBB), morpholinobenzyl urea (MBU), piperidinobenzyl urea (PBU), morpholinobenzyl thiourea (MBTU), piperdinobenzyl thiourea (PBTU)] have been synthesized and characterized by physico-chemical, electrochemical techniques and TGA/DTA studies. The complexes have been prepared by stirring ammonium molybdate and excess of 30% aqueous-H2O2 and then treatment with ethanolic solution of the ligand. Studies revealed that these complexes were non-electrolytes and diamagnetic in nature. The ligands are bound to metal in a bidentate mode through carbonyl oxygen/thiocarbonyl sulphur and the ring nitrogen. The cyclic voltammograms of the complexes show two quasi-reversible steps involving complexes. The complexes have also been tested for antibacterial activity against Salmonella and Kleibsella. The antibacterial study of the ligands and complexes indicate that the complexes exhibit higher activity than the free ligands.     

SYNTHESIS OF DI（ETHYLMERCAPTOETHYL） AMINO GROUP—CONTAINING POLYSESQUISILOXANE PLATINUM COMPLEX AND ITS CATALYSIS IN HYDROSIL YLATION OF OLEFINS WITH TRIETHOXYSILANE

Three new polysesquisiloxane-bound platinum complexes were synthesized via hydrolysis of N,N-di(β-ethylmercaptoethyl)γ-(triethoxysilyl)propylamine or cohydrolysis of the monomer with dodecyltriethoxysilane or with phenylpropylthiethoxysilane and immobilixation on fumed silica,followed by reacting with potassium platinite in acetone under argon atmosphere.The platinum complexes exhibited high catalytic activity for the hydrosilylation of olefins by triethoxysilane. The effects of temperature and the amount of complex on the catalytic activity,as well as the recovery and reusability of the catalysts were investigated.     

Improved Performance of W／HZSM-5 Catalysts for Dehydroaromatization of Methane

The dehydroaramatization of methane over W-supported ZSM-5 with varying degrees of Li^ ion-exchanged catalysts was studied with and without oxygen at 1073 K and atmospheric pressure.Catalyst activity and stability were found to be influenced by the catalyst acidity related to BrSnsted acid sites and by the presence of oxygen in the feed. The NH3-TPD and FTIR-pyridine results demonstrated that partially exchanged of H^ ions by Li^ into the W/HZSM-5 catalysts could be used to control the amount of strong acid sites on the catalyst surface. Without oxygen, the 3WHLi-Z (5:1) catalyst that has strong acid sites equal to nearly 74% of the original strong acid sites in the parent HZSM-5 exhibited the highest methane conversion and selectivity towards aromatics. However, the catalyst deactivated in a five hour period. In the presence of oxygen, the catalyst activity and stability could be improved further.The results of this study revealed that a suitable amount of strong Bronsted acid sites as well as oxygen addition in the feed increased the catalyst activity and stability. The 3WHLi-Z(5:1) catalyst exhibited improved performance in the dehydroaromatization of methane.     

POLYIONIC COMPLEXES DERIVED FROM POLYVINYL ALCOHOL Ⅰ. PREPARATION AND THEIR SOLVATION BEHAVIOR*

Polycations and polyanions of polyvinyl alcohol (PVA) with various substitution degrees were pre-pared by acetalization of PVA with aldehyde derivatives such as p-formylbenzoic acid, glyoxalic acid, p-formyl-benzenesulfonic acid, p-formylpyridine, and p-dimethylaminobenzaldehyde and then neutral-ized or quaternarized with methyl iodide. The corresponding PVA type polyionic complexes wereobtained by mixing the aqueous solutions of these polycations and polyanions. The solvation behaviourof these polyionic complexes were investigated. It was found that the H_2O-DMF-NaSCN solventsystem is superior to the H_2O-DMF-NaBr system for these PVA type polyionic complexes.     

New Transition and Actinide Metal Complexes of 2‐Carboxy‐phenylhydrazo‐Benzoylacetone Ligand: Synthesis,Characterization and Biological Study

A new series of binary mononuclear complexes were prepared from the reaction of the hydrazone ligand, 2-carboxyphenylhydrazo-benzoylacetone (H2L), with the metal ions, Cd(II), Cu(II), Ni(II), Co(II), Th(IV) and UO2(VI). The binary Cu(II) complex of H2L was reacted with the ligands 1,10-phenanthroline or 2-aminopyridine to form mixed-ligand complexes. The binary complexes of Cu(II) and Ni(II) are suggested to have octahedral configurations. The Cd(II) and Co(II) complexes are suggested to have tetrahedral and/or square-planar geometries, respectively. The Th(IV) and UO2(VI) complexes are suggested to have octahedral and dodecahedral geometries, respectively. The mixed-ligand complexes have octahedral configurations. The structures of all complexes and the corresponding thermal products were elucidated by elemental analyses, conductance, IR and electronic absorption spectra, magnetic moments, 1H NMR and TG-DSC measurements as well as by mass spectroscopy. The ligand and some of the metal complexes were found to activate the enzyme pectinlyase.     

Lewis acid/base modulation in β-diiminate zinc-catalyzed switchable ring-opening polymerization of rac-lactide

Two β-diiminate zinc complexes were prepared and found to show high activity and well-controlled catalytic behavior in the ring-opening polymerization of rac-lactide. Coordination of the Lewis acid to the zinc complexes completely terminated the polymerization. The coordinated Lewis acid was easily cleaved by using a Lewis base, so that the polymerization could resume.Thus, switchable polymerization could be realized by the subsequent addition of a Lewis acid and a Lewis base. This work provides a new strategy to control the ring-opening polymerization of rac-lactide.     

Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane

Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM.     

氨基化树枝状介孔二氧化硅固定葡萄糖氧化酶用于检测葡萄糖

以三乙胺为碱源合成了树枝状介孔二氧化硅纳米粒子（DMSNs），并用3-氨基丙基三乙氧基硅烷（APTES）进行氨基修饰合成了氨基化树枝状介孔二氧化硅纳米粒子（DMSNs-NH₂），将其用于葡萄糖氧化酶（GOD）的固定化研究.采用扫描电子显微镜、透射电子显微镜、红外光谱仪、X射线衍射仪、氮气吸附仪及热重分析仪对固定化GOD（DMSNs-NH₂-GOD）进行了表征，测定了其活性及蛋白载量.结果表明，固定化GOD的直径约为200 nm，形状均一，呈分散的球形微粒；在最佳固定条件下，蛋白载量达225 mg/g，酶活性达215 U/mg；固定化GOD检测葡萄糖的最低检测限为0.0014 mg/mL.利用固定化GOD检测了血清和饮料中的葡萄糖，重复使用36次以上其相对酶活性仍剩余80%.该方法操作方便、准确度高，提高了酶的pH稳定性、热稳定性及重复使用性，降低了检测成本.     

Reversible denaturation behavior of immobilized glucose oxidase

Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25±1°C. The soluble enzyme required a GdmCl concentration of 5M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.     

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor

Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.     

固定化葡萄糖氧化酶活性的X射线微区分析

利用X射线微区分析方法,对固定化活性葡萄糖氧化酶进行了定位分析;葡萄糖作为底物,FeSO4和KI作为捕捉剂,底物经固定化葡萄糖氧化酶催化产生H2O2,后者和捕捉剂反应生成沉淀,可以确定固定化葡萄糖氧化酶的催化活性部位。结果表明：颗粒越小,酶活越高,活性葡萄糖氧化酶在凝胶内分布均匀,且绝大多数葡萄糖氧化酶固定在凝胶的内部。作者还研究了固定化活性葡萄糖氧化酶定位的最佳条件。     

Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution

Adsorption condition and enzymatic activity of glucose oxidase (GOD) on polystyrene (PS) film surfaces modified with ozone aeration and UV irradiation (O3/UV) treatment were investigated. The total amount of GOD immobilized on the PS film modified with the O3/UV treatment in distilled water (PS-W film) was approximately twice as large as that on the film treated in an aqueous ammonia solution (PS-A film), whereas the specific activity of GOD on the PS-A film was four times higher than that on the PS-W film. In contrast, no enzymatic activity of GOD on the non-treated PS film was observed because of irreversible denaturation of the adsorbed GOD. We therefore conclude that the PS films modified by the O3/UV treatment in the aqueous media are effective in immobilizing GOD.     

Direct electrochemistry of glucose oxidase immobilized on a hexagonal mesoporous silica-MCM-41 matrix

The direct electrochemistry of glucose oxidase (GOD) immobilized on a hexagonal mesoporous silica modified glassy carbon electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -417 mV in 0.1 M pH 6.1 phosphate buffer solution (PBS). The response showed a diffusion-controlled electrode process with a two-electron transfer coupled with a two-proton transfer reaction process. GOD immobilized on a hexagonal mesoporous silica retained its bioactivity and stability. In addition, the immobilized GOD could electrocatalyze the oxidation of glucose to gluconlactone by taking ferrocene monocarboxylic acid (FMCA) as a mediator in N(2) saturated solutions, indicating that the electrode may have the potential application in biosensors to analyze glucose. The sensor could exclude the interference of commonly coexisted uric acid, p-acetaminophenol and ascorbic acid and diagnose diabetes very fast and sensitively. This work demonstrated that the mesoporous silica provided a novel matrix for protein immobilization and the construction of biosensors.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Alginate beads containing pH-sensitive liposomes and glucose oxidase: glucose-sensitive release

Egg PC (EPC) liposomes bearing a copolymer of N-isopropylacrylamide, methacrylic acid, and octadecylacrylate (P(NIPAM-co-MAA-co-ODA)) were prepared as pH-sensitive liposomes. They were embedded in glucose oxidase (GOD)-immobilized alginate beads. The ratio of EPC/GOD/alginate in the beads was 7.8:1.0:140.4, and the beads were added to glucose solutions so that the concentration of GOD was 0.0068 mg/ml. The enzymatic activity of the immobilized GOD was one fifth to half of that of native enzyme. As the glucose concentration increased from 0 to 400 mg/dl, the degree of calcein release increased from 17% to 75%. The acidification induced by the enzymatic reaction would be responsible for the glucose-triggered release.