Radiation sterilization of enzyme hybrids with biodegradable polymers

Ionizing radiations, which have already been utilized for the sterilization of medical supplies as well as gas fumigation, should be the final candidate to decontaminate “hybrid” biomaterials containing bio-active materials including enzymes because irradiation induces neither heat nor substances affecting the quality of the materials and our health. In order to check the feasibility of ⁶⁰Co-gamma rays on these materials, we selected commercial proteases including papain and bromelain hybridized with commercial activated chitosan beads and demonstrated that these enzyme-hybrids suspended in water showed the significant radiation durability of more than twice as much as free enzyme solution at 25-kGy irradiation. Enhanced thermal and storage stability of the enzyme hybrids were not affected by the same dose level of irradiation, either, indicating that commercial irradiation sterilization method is applicable to enzyme hybrids without modification.     

Influence of gamma radiation onto polymeric matrix with papain

Papain is a proteolytic enzyme that has been widely used as debridement agent for scars and wound healing treatment. However, papain presents low stability, which limits its use to extemporaneous or short shelf-life formulations. The purpose of this study was to entrap papain into a polymeric matrix in order to obtain a drug delivery system that could be used as medical device. Since these systems must be sterile, gamma radiation is an interesting option and presents advantages in relation to conventional agents: no radioactive residues are formed; the product can be sterilized inside the final packaging and has an excellent reliability. The normative reference for the establishment of the sterilizing dose determines 25 kGy as the inactivation dose for viable microorganisms. A silicone dispersion was selected to prepare membranes containing 2% (w/w) papain. Irradiated and non-irradiated membranes were simultaneously assessed in order to verify whether gamma radiation interferes with the drug-releasing profile. Results showed that irradiation does not affect significantly papain release and its activity. Therefore papain shows radioresistance in the irradiation conditions applied. In conclusion, gamma radiation can be easily used as sterilizing agent without affecting the papain release profile and its activity onto the biocompatible device is studied.     

THERMAL ACTIVATION OF IMMOBILIZED PAPAIN

Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beadsby cross linking with glutaraldehyde. The thermal activation of this immobilized papainin aqueous system was found at a temperature range from 50 to 90℃. The higher thetemperature, the more active the immobilized papain will possess. At the same time,the durability of the immobilized papain on heating was greatly improved. The effect ofadditives and salts on the activity of the immobilized papain were also studied. The resultsshowed that the additives and some of the salts studied could markedly enhance the activityof the immobilized papain at elevated temperature.     

A new hybrid nanocomposite prepared by graft copolymerization of butyl acrylate onto chitosan in the presence of organophilic montmorillonite

Organophilic montmorillonite (OMMT) was synthesized by cationic exchange between Na-MMT and tricetadecylmethyl ammonium bromide in an aqueous solution. A new nanocomposite consisting of poly(butyl acrylate)-modified chitosan and OMMT was prepared by γ-ray irradiation polymerization in acetic acid aqueous solution. The degree of dispersion and the intercalation spacing of these nanocomposites were investigated using X-ray diffraction. The enhanced thermal stabilities of nanocomposites were characterized by the thermal gravimetric analysis. The improved mechanical properties of nanocomposites were characterized by static tensile studies and dynamic mechanical analysis. The nanocomposites showed improved resistance to water absorption.     

吸附-纤维素覆膜联合固定化酶

以食品工业中常用的木瓜蛋白酶为模式酶, 建立了吸附-纤维素覆膜联合固定化酶方法. 通过对吸附载体类别、 纤维素种类及溶剂、 保护剂种类及其浓度、 干燥方式及时间等的优化, 得到最佳的吸附-纤维素覆膜联合固定化酶工艺. 以硅藻土或HPD-417(大孔树脂)作为吸附载体, 甲基纤维素(分子量40000~50000)丙酮溶液作为覆膜溶液, 加入6%(质量分数)的聚乙二醇或麦芽糖作为覆膜保护剂, 于4 ℃干燥9 h, 制得固定化木瓜蛋白酶, 硅藻土吸附-纤维素覆膜固定化酶酶活回收率达到96.50%, HPD-417吸附-纤维素覆膜固定化酶酶活回收率达到93.92%. 对吸附-纤维素覆膜固定化酶的性质进行了研究, 发现纤维素覆膜后固定化酶具有良好的热稳定性, 于80 ℃下保存12 h后, 固定化酶活残余率仍然能保持90%左右; 在pH=4.5~9.5的范围内, 固定化酶的稳定性较好; 连续使用9次后, 固定化酶活残余率仍能保持95%左右.     

木瓜蛋白酶的化学修饰及对其酶活力的影响

用不同酸酐对木瓜蛋白酶进行化学修饰, 以三硝基苯磺酸法(TNBS)测定修饰酶的平均氨基修饰度, 对修饰前后的木瓜蛋白酶分别纯化并通过UV-vis和IR对其结构进行了表征. 考察了温度、pH值和表面活性剂SDS对化学修饰的木瓜蛋白酶活力的影响, 并与天然木瓜蛋白酶进行了比较, 对天然酶和修饰酶进行了动力学研究. 结果表明, 化学修饰木瓜蛋白酶的最适反应温度为80 ℃; 最适pH值为9.0; 在SDS浓度为5 mg•mL－1时修饰酶酶活仍能保持在50%左右; 在所有酶中, 均苯四甲酸酐修饰木瓜蛋白酶的催化效率最高, 为2.442×102. 与天然木瓜蛋白酶相比, 化学修饰木瓜蛋白酶的热稳定性、耐碱性和耐洗涤性得到了显著提高.     

Dynamic Modulation of Enzyme Activity by Near-Infrared Light

Engineering near-infrared (NIR) light-sensitive enzymes remains a huge challenge. A photothermal effect-associated method is developed for tailoring the enzymatic activity of enzymes by exposure to NIR light. An ultrasmall platinum nanoparticle was anchored in an enzyme to generate local heating upon NIR irradiation, which enhanced the enzyme activity without increasing bulk temperature. Following NIR irradiation, the enzyme activity was tailored rapidly and reversibly, and was modulated by varying laser power density and irradiation time. Four enzymes were engineered, including glucoamylase, glucose oxidase, catalase, and proteinase K with NIR-light sensitivity, and demonstrated their utility in practical applications such as photolithography and NIR light-responsive antibacterial or anticancer actions. Our investigation suggests that this approach could be broadly used to engineer enzymes with NIR-light sensitivity for many biological applications.     

磁性纳米粒子负载木瓜蛋白酶的制备、表征及用于果汁澄清(英文)

The preparation,characterization,and application of silica-coated magnetic nanoparticles for papain immobilization is reported.Papain was covalently attached onto the(3-chloropropyl) trimethoxysilane-modified silica-coated magnetic nanoparticles. The enzyme-immobilized nanoparticles were characterized by Fourier transform infrared spectroscopy,X-ray powder diffraction,scanning electron microscopy,and vibrating sample magnetometry techniques.Response surface methodology combined with statistical analyses using Minitab were employed to evaluate optimum operating conditions to immobilize papain on the magnetic nanoparticles.The optimum conditions were: temperature = 27.3℃,pH of the enzyme solution = 7.1,concentration of papain = 3.3 mg/mL,and immobilization time = 10 h.Compared with the free papain,the immobilized papain displayed enhanced enzyme activity,better tolerance to variations in the medium pH and temperature,improved storage stability,and good reusability.Both the free and immobilized enzymes were effective for the clarification of pomegranate juice.     

ZnO·WO3悬浮水溶液中SO2的光催化氧化

本文考察了ZnO·WO₃悬浮水溶液中污染物SO₂的可见光催化氧化并建议通空气处理较高浓度的SO₃^2-扩水溶液。含0.15mol/LSO₃^2-水溶液可见光照2h可将SO₃^2-基本上光催化氧化成SO₄^2-。对这光助SO₃^2-自发氧化反应中可能平行存在的自发、多相催化、光化学和光催化等反应进行实验对比;筛选出克分子比0.5ZnO·WO为最佳掺杂配比;考察了光催化剂热处理、空气流量、pH对光催化活性的影响,得出pH7.5为最佳值;太阳光催化SO₃^2-氧化速率比室内实验更快,展现了利用太阳能于环保的应用前景,对所有实验结果都给以理论解释,并提出一个较详细的反应机理。     

Radiolysis of cyanocobalamin (vitamin B12)

Research on the radiolysis of vitamins is of considerable interest since these compounds are important nutritional constituents in foods and in dietetic supplements. In spite of these considerations there are few data and very often difficult to compare for the radiolytic behavior of vitamins. In this work we focused our attention on to the study of the radiolysis of cyanocobalamin (vitamin B₁₂) in solid state and in aqueous solutions. The procedure was followed by HPLC and UV-spectroscopy. The results obtained in aqueous solutions showed a dependence of the decomposition as a linear function of the dose. The G of decomposition for a 1×10⁻⁵ M solution was 3.3. In the solid state the vitamin was very stable towards the irradiation in the conditions used in this study with a G=2.1×10⁻³. A study made with Serratia marcescens as a microbiological contaminant showed that at the sterilization dose there is a destruction of the vitamin in aqueous solution. In the solid state the degree of decomposition was 7%.     

A new method of immobilizing enzymes by radiopolymerization under low temperature

A new method of immobilizing enzymes by ionizing radiation is described. The mixed aqueous solution of enzyme and polymerizing reagents were quickly frozen at about -70°C then were irradiated with 200 to 500 Krad by⁶⁰Co γ ray. Irradiation was conducted aerobically under the low temperature. The enzyme was entraped in the resulting polymer. As the polymerizing reagent some water soluble polymers having vinyl bonds were also applicable. By this method an immobilized enzyme was prepared in bead, membrane, bag, or tube form having high enzymic activity. When the bead form was to be prepared, the mixture of enzyme and reagents were injected into precooled solvent such as n-hexane, toluene, or petroleum ether. the size of the bead was controlled freely from 10 μm to 1 cm in diameter. The surface of the bead had numerous small holes and the cross section of the bead showed a spongy structure. some acrylates were suitable for the immobilization of enzymes which required the corresponding metal ion as the essential substance. Microorganisms and multienzymes will be immobilized by this technique. This method is inexpensive, quick, simple, and reliable. Immobilized microbial cells can be sterilized by γ irradiation. Invertase was immobilized and the application test was conducted in an enzyme column.     

Dynamic Modulation of Enzyme Activity by Near‐Infrared Light

Engineering near‐infrared (NIR) light‐sensitive enzymes remains a huge challenge. A photothermal effect‐associated method is developed for tailoring the enzymatic activity of enzymes by exposure to NIR light. An ultrasmall platinum nanoparticle was anchored in an enzyme to generate local heating upon NIR irradiation, which enhanced the enzyme activity without increasing bulk temperature. Following NIR irradiation, the enzyme activity was tailored rapidly and reversibly, and was modulated by varying laser power density and irradiation time. Four enzymes were engineered, including glucoamylase, glucose oxidase, catalase, and proteinase K with NIR‐light sensitivity, and demonstrated their utility in practical applications such as photolithography and NIR light‐responsive antibacterial or anticancer actions. Our investigation suggests that this approach could be broadly used to engineer enzymes with NIR‐light sensitivity for many biological applications.     

Native and modified glucose oxidase in reversed micelles

The enzymatic activity of the native and modified glucose oxidase (GOx) from Aspergillus niger in the system of reversed micelles of Aerosol OT in octane was investigated. Two forms of the modified enzyme were studied: a hydrophobized form obtained by the attachment of palmitic chains to lysine amino groups by the reaction with palmitic acid ester of N-hydroxysuccinimide and a glycosylated (hydrophilized) form obtained by the attachment of the cellobiose moieties. The native glucose oxidase and its derivatives, while incorporated into micelles in a surfactant concentration range from 0.05 to 0.3 M, display an enzymatic activity, which is comparable with the activity in aqueous solution. The dependence of the enzymatic activity on hydration degree of surfactant (the molar ratio of water to surfactant, W₀) does not indicate the formation of qualitatively new associated forms of the enzyme subunits inside the micelles. The apparent size of Aerosol OT micelles obtained by dynamic light scattering gradually increases from 10±3 nm at low W₀ up to 25±5 nm at high W₀. Incorporation of the native and hydrophobized glucose oxidase into micelles does not affect their mean size. Kinetic analysis shows that the enzyme specificity is about an order of magnitude greater in the system of reversed micelles as compared with aqueous solution.     

PHOTOCHEMISTRY OF URACIL. INTERSYSTEM CROSSING AND DIMERIZATION IN AQUEOUS SOLUTION

Abstract— Ultraviolet irradiation of ¹⁴C-uracil in aqueous solution results in the formation of hydrate and dimer photoproducts. The rate of dimerization increases with increasing uracil concentration, and decreases with increasing concentration of oxygen in solution. The kinetics are in agreement with a model previously proposed to account for the reactions, in which dimerization occurs by a reaction involving the triplet state of uracil, but hydration occurs from an excited singlet state. Oxygen reduces dimer formation by quenching the triplet. The quantum yield for intersystem crossing (ISC) to the triplet depends on the irradiation wavelength, increasing from 0.0014 at 280 nm to 0.016 at 230 nm. The ratio of rate constants for reaction of the triplet with oxygen and for dimerization is 1.1; the ratio of rate constants for triplet decay and for dimerization is 5.9 × 10^-5 M. The increase in ISC with photon energy suggests that ISC is favoured from excited vibrational levels. The quantum yield for hydration is about 0.002 at pH 4.5 for all wavelengths, but increases as the pH is decreased.     

Sythesis and crystal structure of a new nickel(II)-terpyridine complex: [Ni(terpy)(NO2)(ONO)(H2O)]

The compounds Ni(terpy)Cl₂ · 3H₂O and NaNO₂ react readily in aqueous solution to give the complex [Ni(terpy)(NO₂)(ONO)(H₂O)], which has been structurally characterized by an X-ray diffraction study. The hexacoordinate nickel atom bonds to three nitrogen atoms of the terpyridine ligand, to a “nitro” group, to a “nitrite” group and to a water molecule. Distortion from an octahedron to a trigonal prism, for the complex, has been evaluated. An infrared study of the complex has been carried out and shows bands of nitro and nitrite coordination. Magnetic study reveals a Curie-Weiss behavior, giving an effective magnetic moment at room temperature 3.12 B.M.     

A study on preparation of glucoamylase by irradiation

A study on preparation of industrial glucoamylase irradiated by ⁶⁰Co gamma-ray showed that an abvious change took place in the extraviolet absorption spectrum and the pH - value of the enzyme solution and the activity of the enzyme preparation was much more resistant to irradiation than that of microbes contained in the preparation. The result indicates that irradiated glucoamylase preparation is not only favourable to activity preservation but also is improved in the hygienic quality of products. A comparison of the various properties of irradiated enzyme preparation with those of unirradiated one has proved that irradiation does not change the normal application of glucoamylase preparation.     

Study on sterilization and storage of enzymatic preparations by irradiation

Effects of irradiation with cobalt-60 γ-rays on sterilization, storage and enzymes activity of four kinds of biological enzymatic preparations, namely trypsin, pepsin, amylase and liquid carbohydrase, were investigated. The results showed that these enzymes have different sensitivity to irradiation, amylase being the most sensitive. The enzyme activity and enzyme effect of amylase were lowered with irradiation dose higher than 7 KGY. With irradiation doses between 7–10 KGY, enzyme activity of trypsin and pepsin were stable. Liquid carbohydrase can be stored more than half a year under a natural temperature with a stable enzyme activity without molding.     

Synthesis and study of Bi2Ti2O7/TiO2 composites as efficient visible-light-active photocatalysts in the reduction of aqueous Cr(VI)

In this work, Bi_2Ti_2O_7/TiO_2 composites were synthesized and studied as potential visible-light-activated photocatalysts in the reduction of aqueous Cr(VI). Bi_2Ti_2O_7/TiO_2 composites with tunable compositions were synthesized via a solvothermal-calcination two-step method, simply by changing the molar ratios of Bi(NO_3)_3·5H_2O to tetrabutyl titanate in the reactants. The compositions, structures and optical properties of the as-synthesized Bi_2Ti_2O_7/TiO_2 composites were characterized by X-ray diffraction, field emission scanning electron microscopy and UV–vis diffuse reflectance spectra. The photocatalytic activity of the as-synthesized Bi_2Ti_2O_7/TiO_2 composites was tested in the reduction of aqueous Cr(VI)under visible-light(λ420 nm) irradiation, and compared with that of TiO_2 nanoparticles. It was observed that the as-synthesized Bi_2Ti_2O_7/TiO_2 composites exhibited much higher photocatalytic activity than TiO_2 nanoparticles, and the most efficient composite(300 mg) can achieve the complete reduction of Cr(VI) in 300 mL of 50 mg/L K_2Cr_2O_7 aqueous solution under visible-light(λ420 nm)irradiation in 90 min.     

原位聚合碳掺杂改性石墨态氮化碳光催化活性研究

本文通过在双氰胺前驱体中添加聚乙二醇,在缩聚过程实现碳掺杂形成含氮空位的g-C₃N₄光催化剂。通过X射线衍射(XRD)、红外光谱(FTIR)、光电子能谱(XPS)、紫外-可见吸收光谱(UV-Vis)和荧光谱(FL)等表征手段,考察了原位聚合碳掺杂形成氮空位对g-C₃N₄物相结构、组分与化学态、光吸收性能及光催化活性的影响。研究结果表明,采用该方法可实现原位聚合碳掺杂,有效拓展g-C₃N₄的可见光吸收至850 nm,在紫外-可见光与可见光照射下光降解RhB及光催化产氢性能均显著提高,尤其可见光条件下的性能提升更为显著。