A new application of humic substances: activation of supports for invertase immobilization

Invertase was immobilized on aminopropyl silica (APTS-SiO₂) activated with humic substances (APTS-SiO₂-HS) and on aminopropyl silica activated with glutaraldehyde (APTS-SiO₂-GA). The resulting activity of both systems was compared. Humic substances (HS) used for the activation of the silica were extracted from soil of Cananéia, São Paulo State, Brazil, according to the procedure recommended by the International Humic Substances Society. Activity was determined by measuring the rate of formation of reduced sugars using the reaction with dinitrosalicylic acid (DNS). The amount of HS bound on the APTS-SiO₂ was equal to 50 mg. The maximum amount of invertase immobilized on APTS-SiO₂-HS was 15200 U/g while in the system APTS-SiO₂-GA it was 13400 U/g. The experimental enzymatic activity was 3700 and 3300 U/g, for the systems APTS-SiO₂-HS and APTS-SiO₂-GA, respectively. Considering the increased amount and activity of immobilized enzyme compared with the glutaraldehyde method, it was concluded that this technique opens a new perspective in the preparation of supports for enzyme immobilization employing humic substances.     

固定化乙酰胆碱酯酶的制备及其特性研究

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(AChE),为天然产物复杂体系中AchE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-Si)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 h后,在给酶量5 U,28℃固定16 h条件下,得到固定化酶的活性最大.固定化酶在常温(20～40℃),以及较宽pH范围内(pH 6～10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.     

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.     

Stabilization and reutilization ofBacillus megaterium glucose dehydrogenase by immobilization

Glucose dehydrogenase (GDH) fromBacillus megaterium was immobilized using aminopropyl controlled-pore silica (CPS, average pore sizes of 170 and 500 Å) as a support and glutaraldehyde as a bifunctional crosslinking agent. The CPS-immobilized enzyme could be reused 12 times and the best results were obtained using aminopropyl CPS-500 and bovine serum albumin as a feeder for stabilizing the protein layer on the support. DEAE-Sephadex (A-25 and A-50) was also used as a support for immobilizing GDH, with yields of around 42% for A-25 and 25–30% for A-50. The effect of pH on the immobilization procedure showed pH 6.5 to be better than pH 7.5 with respect to the recovery of enzyme activity. Both preparations of DEAE-Sephadex immobilized GDH could be reused several times and were thermostable at 40°C for 7 h. The kinetic parameters as Michaelis constant and maximum rate were determined for the immobilized enzyme and compared with those for the freeform.

     

Immobilization of a lactase onto a magnetic support by covalent attachment to polyethyleneimine-glutaraldehyde-activated magnetite

A magnetic immobilized lactase has been prepared using magnetite as the magnetic material. Magnetite was functionalized by treatment with polyethyleneimine and crosslinked with glutaraldehyde. Lactase was then covalently coupled to the activated magnetic matrix via the aldehyde groups. The conditions for optimal immobilization of enzyme are described. Eighty percent of the lactase activity was lost on immobilization and is thought to be owing to the orientation of enzyme binding to the matrix. The amount of protein coupled was 80% of that applied. The maximum lactase activity retained on the matrix following immobilization was 360 U/g matrix. The immobilized lactase showed optimal activity at pH 4.5 and 65 degrees C. The immobilized lactase was more heat stable than the free enzyme, and retained 83% of its original activity after 14 d at 55 degrees C. Galactose competitively inhibited the immobilized lactase preparation (Ki 20 m/M). The presence of high initial concentrations of galactose (10% w/v) did not prevent total hydrolysis of lactose. Glucose and calcium ions were activators of the immobilized enzyme. The immobilized enzyme hydrolyzed high concentrations of lactose (up to 25% w/v) to completion within 4-6 h in a stirred batch reactor at 55 degrees C. There was no evidence of substrate inhibition at high substrate concentrations. The efficiency of hydrolysis of lactose by the immobilized lactase was better than that of the free enzyme. The magnetic immobilized lactase was demonstrated to be suitable for use in the enzymatic hydrolysis of both pure, and cheese whey permeate, lactose.     

Prediction of Properties of Soil Humic Substances from FTIR Spectra using Partial Least Squares Regression

Abstract

Humic substances (HS) play a key role in aquatic and terrestrial ecosystems. The understanding of the ecological functionalities of HS is based on the analysis of their properties, which is normally a very time-consuming procedure. Therefore we tested the possibility to apply the partial least squares regression (PLSR) method in connection with the mid infrared Fourier transform (FTIR) spectra of a series of soil humic substances for the prediction of different HS properties. The results with humic acid (HA) and fulvic acid (FA) fractions of soil HS from different environments show the possibility to predict several properties of an unknown soil HA with satisfying reliability above all the elemental composition.     

树枝状大分子聚酰胺-胺用于氨基酰化酶的固定化研究

以树枝状大分子修饰的硅胶为载体,戊二醛为交联剂,对氨基酰化酶进行固定化研究。考察了树枝状大分子的代数、戊二醛浓度、反应温度与时间对氨基酰化酶固定化效果的影响,并且考察了该固定化酶的最佳酶解条件。结果表明,随着树枝状大分子代数的增加,固定化的酶量随之增大,同时,固定后的酶仍然保持较高的活性。     

Preparation and Properties of Urease Immobilized onto Glutaraldehyde Cross-linked Chitosan Beads

Urease was immobilized onto the glutaraldehyde cross-linked chitosan beads that were prepared under microwave irradiation. The activity and the yield of activity of immobilized urease was 10.83 U/g B and 47.7%, respectively. The conditions of urease immobilization were optimized. The properties of the immobilized urease were investigated and compared with that of the free enzyme.     

壳聚糖固定化血管紧张素转化酶及其性质

以壳聚糖微球为载体, 戊二醛为交联剂固定化血管紧张素转化酶, 研究了酶固定化的最优条件和固定化酶的性质. 结果表明, 在戊二醛质量分数为2.5%、给酶量为8 mg/mL时, 固定化酶的比活性最大, 为0.085 U/g. 固定化酶在40~50 ℃, pH在7~9之间有最大活性, 其米氏常数Km为2.39 mmol/L. 同时, 固定化酶具有良好的稳定性, 可重复利用.     

Bioluminescent monitoring of detoxification processes: activity of humic substances in quinone solutions

This study deals with application of bioluminescent assay systems to evaluate the detoxifying effect of humic substances (HS) on the solutions of organic oxidizers - quinones. A series of homologous quinones with different redox characteristics: 1,4-benzoquinone, tetrafluoro-1,4-benzoquinone, methyl-1,4-benzoquinone, tetramethyl-1,4-benzoquinone, and 1,4-naphtoquinone, was used. Bioluminescent bacteria Photobacterium phosphoreum, and NADH:FMN-oxidoreductase-luciferase enzyme system isolated from these bacteria were used as assay systems. The toxicity was compared in the presence and in the absence of HS. Variation of complexity of bioassays (in vivo or in vitro) combined with spectrometric and microscopic methods, provides insight into the process of detoxification in quinone solutions. Two ways of HS effect were studied: the reduction activity of HS and intensification of self-protection of bacterial cells on HS addition.     

l-DOPA production by immobilized tyrosinase

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).     

固定化木瓜蛋白酶的制备和性质研究

多孔硅球固定化木瓜蛋白酶具有热增活性 .本文在前文研究的基础上 ,用载体交联法制备了甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶 .考察了固定化pH值、戊二醛浓度和给酶量对固定化木瓜蛋白酶活力的影响 .研究了固定化木瓜蛋白酶的性质 ,特别是热稳定性和耐热性 ,并与溶液酶和多孔硅球固定化木瓜蛋白酶进行了比较 .所制得的甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶的最适反应温度均达到了 80℃ ;90℃温育 1h后固定化酶的活力保持在 95 %以上 ;70℃温育处理 5h和 6h后固定化酶的活力也仍能保持在 90 %以上 .固定化木瓜蛋白酶的热稳定性和耐热性得到了显著提高     

Changes in morphology and activity of transglutaminase following cross-linking and immobilization on a polypropylene microporous membrane

Transglutaminase (TGase) was cross-linked with glutaraldehyde, and cross-linked crystalline transglutaminase was immobilized on a polypropylene microporous membrane by UV-induced grafting. Immobilized enzyme activity were calculated to be 0.128 U/cm2 polypropylene microporous membrane. The microstructure and enzyme characteristics of free, cross-linked and immobilized transglutaminase were compared. The optimum temperature of free transglutaminase was determined to be approximately 40 °C, while cross-linking and immobilization resulted in an increase to approximately 45 °C and 50 °C. At 60 °C, immobilized, cross-linked and free transglutaminase retained 91.7 ± 1.20%, 63.2 ± 1.05% and 37.9 ± 0.98% maximum activity, respectively. The optimum pH was unaffected by the state of transglutaminase. However, the thermal and pH stabilities of cross-linked and immobilized transglutaminase were shown to increase.     

Properties of Immobilized Laccase on Mesostructured Cellular Foam Silica and its Use in Dye Decolorization

Laccase was immobilized on mesostructured cellular foam (MCF), a kind of mesoporous silica with large pore size by adsorption–cross linking method. The effects of immobilization time, temperature, pH, amount of enzyme and content of glutaraldehyde on the immobilization were optimized. The activities and stabilities towards pH and temperature of the immobilized enzyme were studied, and significantly improved enzymatic properties and good operational stability were obtained for the immobilized laccase. Dye decolorization tests showed that the immobilized enzyme could decolorize Alizarin Red and Indigo Blue solution fast and efficiently in the presence of ABTS.     

单分散磁性纳米粒子固定化猪胰脂肪酶的研究

借助溶热法制备了一种亲水及生物相容良好的Fe3O4磁性纳米粒子,用γ-氨丙基三乙氧基硅烷直接对所得磁性粒子表面改性,然后用戊二醛偶联法制得了固定化猪胰脂肪酶.表征研究显示,所得磁性粒子粒径约200 nm,具有良好的单分散性和磁响应性.考察了戊二醛浓度、给酶量和反应时间对脂肪酶固定化过程的影响,并通过游离酶与固定化酶的比...     

Use of insoluble yeast beta-glucan as a support for immobilization of Candida rugosa lipase

In the present study, insoluble yeast beta-glucan (IYG) has been explored as a support matrix for enzyme immobilization. IYG contains mainly beta-(1-3) linkages along with some intra- or inter-molecular branches of beta-(1-6) linkages with large number of free hydroxyl groups. Epichlorohydrin was used to convert these free hydroxyl groups into activated epoxy groups that are capable of forming covalent linkages with various groups of enzyme molecule. The epoxy-activated IYG was evaluated for immobilization of Candida rugosa lipase (CRL). Post-immobilization treatment of 5% glutaraldehyde was given in order to achieve stable and irreversible binding of enzyme on the support. The resultant biocatalytic IYG support expressed lipase activity of 8136.7 U/g and 59.6% activity yield. There was 51.05% retention of synthetic activity after six repeated esterification cycles, indicating its stability and reusability in non-aqueous medium. Moreover, the immobilized lipase gave the storage half-life of about 285 days (at 4 degrees C).     

Technical and economic analysis of lignin conversion to methyl aryl ethers

Glucose oxidase (GOD) and Horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass by means of glutaraldehyde. About 700-800 U/g of immobilized GOD and 300-400 U/g of immobilized HRP were obtained. Some factors of affecting enzyme immobilization were discussed. The immobilized enzymes were packed into a plastic tube and used in flow-injection analysis (FIA) for glucose in serum. A good linearity range was observed for this immobilized enzyme system at 20 mg/mL to 1000 mg/dL D-glucose, the recovery was 95.4-103.5%, the within-batch imprecision was 0.8-2.2%, and the between-batch imprecision was 2.2-4.2%. More than 100 samples were measured within an hour. One enzyme column with five units of immobilized GOD and HRP, applied for 50 assays/d, has been used for more than 2 mo.     

Iodine emission in the presence of humic substances at the water's surface

Humic substances that preferentially adsorb at the air/water interfaces of water or aerosols consist of both fulvic and humic acid. To investigate the chemical reactivity for the heterogeneous reaction of gaseous ozone, O(3)(g), with aqueous iodide, I(-)(aq), in the presence of standard fulvic acid, humic acid, or alcohol, cavity ring-down spectroscopy was used to detect gaseous products, iodine, I(2)(g) and an iodine monoxide radical, IO(g). Fulvic acid enhanced the I(2)(g) production yield, but not the IO(g) yield. Humic acid, n-hexanol, n-heptanol, and n-octanol did not affect the yields of I(2)(g) or IO(g). We can infer that the carboxylic group contained in fulvic acid promotes the I(2)(g) emission by supplying the requisite interfacial protons more efficiently than water on its surface.