Electrolytic process for regenerating nadp from nadph in a polymer matrix-bound form

Regeneration of nicotinamide adenine dinucleotide phosphate (NADP) from its reduced form (NADPH) was performed in a matrix-bound form by an electrolytic method. NADP was immobilized to alginic acid. No significant loss of coenzymic function was induced by the immobilization of NADP on the matrix. Bound NADP was soluble in water. Glucose-6-phosphate dehydrogenase (G-6-PDH) was taken as a model system of coenzyme requiring enzyme. G-6-PDH immobilized on alumina particles was coupled with the soluble form of bound NADP in a fluidized bed type of reactor. The enzymatically reduced coenzyme was electrolytically oxidized in the coenzyme regenerator of NADP from NADPH, which was found to cause no harmful loss of coenzymic function.     

Immobilization-stabilization of Penicillin G acylase fromEscherichia coli

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.     

新型乳酸固定化酶荧光毛细生物传感器

对长45 mm、内径0.9 mm的医用毛细管进行γ-氨丙基三乙氧基硅烷氨基化和戊二醛醛基化后,再将乳酸脱氢酶(LDH)的氨基与戊二醛的醛基结合,使其固定在毛细管内壁,构成一种新型固定化酶乳酸荧光毛细生物传感器(IE-LFCBS),实现了对乳酸的微量、快速测定.IE-LFCBS吸入辅酶Ⅰ与乳酸的混合液,在固定化酶催化下使乳酸与辅酶Ⅰ反应,生成荧光物质还原型辅酶Ⅰ;激发波长353 nm、发射波长466 nm.适用于IE-LFCBS的优化条件为:辅酶Ⅰ浓度4 mmol/L、用于固定化的LDH浓度60 kU/L、反应时间15 min、反应温度38 ℃、测定范围为1.0～5.0 mmol/L、回收率95%～98%,IE-LFCBS的相对标准偏差为RSD<1.5%(n=11),检出限为0.45 mmol/L.IE-LFCBS的试液用量极少(18 μL),并能重复使用,可望用于发酵食品、药品、血液标本等各类样品中乳酸的快速检测.     

Immobilization of single-strand specific nuclease (S1 nuclease) fromAspergillus oryzae

S1 nuclease fromAspergillus oryzae (EC 3.1.30.1) was coupled to gelatin-alginate composite matrix using the residual free aldehyde groups on the surface of glutaraldehyde crosslinked matrix. The immobilized enzyme retained approximately 10% activity of the soluble enzyme. When partially purified enzyme was bound to the matrix, the immobilized preparation did not show any detectable enzyme activity. However, the activity could be restored when the coupling was carried out in the presence of a coprotein or substrate. The optimum pH of the immobilized S1 nuclease shifted to 3.8 from 4.3 for the soluble enzyme. Also, optimum temperature increased to 65°C after immobilization. Bound S1 nuclease showed increased pH and temperature stabilities. Immobilization brought about a twofold decrease in the Michaelis-Menton constant (K _m).     

Grapefruit Debittering by Simultaneous Naringin Hydrolysis and Limonin Adsorption Using Naringinase Immobilized in Agarose Supports

Naringin and limonin are the two main bitter compounds of citrus products such as grapefruit juice. The aim of this investigation was to evaluate the reduction in both bitter components simultaneously using a combined biochemical and physical approach. The proposed strategy was based on the use of heterofunctional supports with glyoxyl groups that allow for the covalent immobilization of naringinase, which hydrolyses naringin and alkyl groups that allow for the adsorption of limonin. The supports were butyl-glyoxyl agarose (BGA) and octyl-glyoxyl agarose (OGA), which were characterized in terms of aldehyde group quantification and FTIR analysis. The optimal pH and temperature of free and immobilized enzymes were assessed. The maximum enzyme loading capacity of supports was analyzed. Debittering of grapefruit juice was evaluated using soluble enzyme, enzyme-free supports, and immobilized catalysts. Enzyme immobilized in BGA reduced naringin and limonin concentrations by 54 and 100%, respectively, while the use of catalyst immobilized in OGA allowed a reduction of 74 and 76%, respectively, obtaining a final concentration of both bitter components under their detection threshold. The use of OGA biocatalyst presented better results than when soluble enzyme or enzyme-free support was utilized. Biocatalyst was successfully applied in juice debittering in five repeated batches.     

Determination of acetaldehyde by flow injection analysis with soluble or immobilized aldehyde dehydrogenase

Acetaldehyde (0.18–7.7 × 10^?4 M) in water is determined by using a double injection technique with the soluble enzyme or with a mini-column of aldehyde dehydrogenase immobilized on cyanogen-activated Sepharose 4B. The NADH produced is monitored spectrophotometrically. The sample throughput is ca. 40 h^?1, and the immobilized enzyme is stable for at least a month. Ethanol up to 5% (v/v) does not interfere.     

Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence

Fluorescence spectra and soluble quenching of intrinsic protein fluorescence were used as indexes of conformational changes suffered by frog epidermis tyrosinase. The activation process and the immobilization of the enzyme involving either free amino groups or its carbohydrate moiety were studied. The conformational changes resulting from denaturation of each one of the protein derivatives, as well as the effect of active center copper extraction, were followed by fluorescence studies. The results showed that: (a) both activation and immobilization were accompanied by conformational changes of the protein leading to more unfolded states; (b) neither enzyme nor immobilized enzyme were fully unfolded upon denaturation although enzymic activity was lost; (c) the enzyme immobilized through its carbohydrate moiety was more unfolded upon denaturation than the enzyme immobilized through amino groups, thus pointing to a higher conformational stabilization in the last situation; and (d), that tryptophyl residues moved to a localization near the active site upon activation.     

NMR observations of 13C-enriched coenzyme B12 bound to the ribonucleotide reductase from Lactobacillus leichmannii

The 13C NMR resonance and one-bond 1H-13C coupling constants of coenzyme B12 enriched in 13C in the cobalt-bound carbon have been observed in the complex of the coenzyme with the B12-dependent ribonucleotide reductase from Lactobacillus leichmannii. Neither the 13C NMR chemical shift nor the 1H-13C coupling constants are significantly altered by binding of the coenzyme to the enzyme. The results suggest that ground-state Co-C bond distortion is not utilized by this enzyme to activate coenzyme B12 for C-Co bond homolysis.     

Aldehyde oxidase assay by capillary electrophoresis: From off‐line,online up to immobilized enzyme reactor

Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis. A number of magnetic nano/microparticle types were tested for this purpose, showing that aldehyde oxidase was most active when immobilized on bare silica magnetic nanoparticles. Primarily, the reusability of the enzyme immobilized on bare silica nanoparticles was tested. Three consecutive incubations with substrate could be performed, but the activity considerably dropped after the first incubation. One reason could be an enzyme detachment from the nanoparticles, but no release was detected neither at 4°C nor at 37°C during 5 h. The drop in enzymatic activity observed in consecutive incubations, could also be due to inactivation of the enzyme over time at given temperature. For the immobilized enzyme stored at 4°C, the activity decreased to 83% after 5 h, in contrast with a steep decrease at 37°C to 37%.     

Immobilization of Horseradish Peroxidase on Nonwoven Polyester Fabric Coated with Chitosan

The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The physico-chemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after 4 weeks of storage at 4°C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40°C and were stable up to 40 and 50°C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications.     

Immobilization of protein on cellulose hydrogel

A technique of immobilizing an enzyme/antibody was developed using cellulose hydrogel prepared from an aqueous alkali-urea solvent. Partial oxidation by sodium periodate activated the cellulose gel for introducing aldehyde groups. Proteins were covalently introduced to cellulose gel by a Schiff base formation between the aldehyde and the amino groups of proteins, and stabilized by a reduction of imines. Coloring reactions confirmed the high activity of the immobilized enzymes. The activity of the immobilized enzymes increased with aldehyde content, but the effect leveled off at a low degree of oxidation, at approximately 8.1 of oxidized glucose/100 glucose unit. The amount of immobilized peroxidase calculated from the activity was 8.0 ng/g for an aldehyde content of 0.18 mmol/g: 14.6 ng/g for both 0.46 mmol/g and 1.04 mmol/g. The same method could be applied to the peroxidase antibody. Thus, various active proteins could be immobilized on cellulose gels by mild and facile processing. Owing to high mechanical and chemical stability of cellulose, this technique and resulting materials are potentially useful in biochemical processing and sensing technologies.     

Characterization of Immobilized β-N-Acetylhexosaminidase1

Abstract

The properties of fl-N -acetylhexosaminidase chemically bound to Sepharose 4B were determined and compared to those of the soluble enzyme. External diffusion effects on the kinetics of the immobilized enzyme were eliminated by assaying in a recirculation reactor with rapid flow rates. The immobilized 15-N-acety Hexosaminidase exhibited a broad pH optimum quite similar to that of the soluble enzyme. Compared to the soluble enzyme the immobilized enzyme demonstrated a markedly enhanced stability at each pH and temperature investigated. Immobilization caused an increase in both the apparent K and K., with rrm. A preliminaty account of this work was presented at the Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J., April, 197 3.     

Preparation,characterization, and application of a novel immobilized carboxypeptidase B

Pig pancreas carboxypeptidase B has been immobilized by covalent attachment to a polyacrylamide-type bead support possessing carboxylic functional groups activated by water-soluble carbodiimide. The optimum conditions of immobilization were determined. The activation of the support and the coupling reaction were performed in 0.1 M sodium citrate/sodium phosphate buffer (pH 4.5) using a support-carbodiimide-enzyme weight ratio 4:8:1 at 0-4 degrees C. Under such conditions, the highest activity achieved was 6700 U/g solid. The catalytic properties and stability of immobilized carboxypeptidase B were studied and compared with the corresponding properties of the soluble enzyme. The specific activity of the immobilized enzyme calculated on bound protein basis was about 70% of that of soluble enzyme. The optimum pH for the catalytic activity of the immobilized carboxypeptidase B was practically identical with that of soluble enzyme (pH 7.6-7.7). The apparent optimum temperature of the immobilized carboxypeptidase B was about 7 degrees C higher than that of the soluble enzyme. With hippuryl-L-arginine as substrate, Kmapp value of the immobilized enzyme was tenfold higher than the Km value of the soluble enzyme. The conformational stability of the enzyme was markedly enhanced by the strongly hydrophylic microenvironment in a wide temperature and pH range. The immobilized carboxypeptidase B was used for stepwise digestion of cytochrome C.     

The ammonia freeze explosion (AFEX) process

A partially purified phophotriesterase was successfully immobilized onto nylon 6 and 66 membranes, nylon 11 powder, and nylon tubing. Up to 9000 U of enzyme activity was immobilized onto 2000 cm2 of a nylon 6 membrane where 1 U is the amount of enzyme necessary to catalyze the hydrolysis of 1.0 mumol of paraoxon/min at 25 degrees C. The nylon 66 membrane-bound phosphotriesterase was characterized kinetically where the apparent Km value for the immobilized enzyme was 0.35 mM. This is 5-6 times higher than that observed for the soluble enzyme. However, nylon immobilization limited the maximum rate of paraoxon hydrolysis to less than 10% of the value measured for the soluble enzyme. The addition of the cosolvent, methanol, resulted in an increase in the apparent Km value for paraoxon hydrolysis but concentrations up to 40% had no negative effect on the catalytic effectiveness with the soluble or immobilized phosphotriesterase. Based on the kinetic analysis, methanol appears to be a competitive inhibitor for both forms of enzyme. The nylon powder immobilized enzyme was shown to be stable for at least 20 mo. The immobilization of the phosphotriesterase onto nylon provides a practical method for the detoxification of organophosphate pesticides.     

蛋白质微阵列芯片技术及其在抗体筛选中的应用

以兔IgG为模式蛋白质,对其在醛基修饰玻片表面的固定浓度、固定时间和温度等条件进行了优化,结果表明：在室温下,当固定蛋白质的浓度为1g／L、固定时间为4h时,可获得理想的蛋白质固定效果;蛋白质的定量检测范围为1μg／L～10mg／L。按优化的蛋白质微阵列芯片制作条件将规模化制备的抗体制作成抗体微阵列芯片,通过与荧光标记的人球蛋白和人白蛋白的相互作用,实现了对不同抗体株抗球蛋白和抗白蛋白活性的快速筛选与比较。     

ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.     

Identification of Antioxidant and Anti-α-amylase Components in Lotus (Nelumbo nucifera,Gaertn.) Seed Epicarp

Nano-sized Fe₃O₄ was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of “interface activation” by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.

     

酸性接枝淀粉固定化糖化酶及稳定性研究

糖化酶(EC.3.2.1.3,GA)是工业上应用规模最大的3种酶制剂之一.目前,工业生产中大多采用游离GA,这给产物的分离和纯化带来诸多不便.因此,采用固定化糖化酶(IGA)是酶制剂工业发展的必然趋势.     

New insights into ergot alkaloid biosynthesis in Claviceps purpurea: an agroclavine synthase EasG catalyses, via a non-enzymatic adduct with reduced glutathione, the conversion of chanoclavine-I aldehyde to agroclavine

Ergot alkaloids are indole derivatives with diverse structures and biological activities. They are produced by a wide range of fungi with Claviceps purpurea as the most important producer for medical use. Chanoclavine-I aldehyde is proposed as a branch point via festuclavine or pyroclavine to clavine-type alkaloids in Trichocomaceae and via agroclavine to ergoamides and ergopeptines in Clavicipitaceae. Here we report the conversion of chanoclavine-I aldehyde to agroclavine by EasG from Claviceps purpurea, a homologue of the festuclavine synthase FgaFS in Aspergillus fumigatus, in the presence of reduced glutathione and NADPH. EasG comprises 290 amino acids with a molecular mass of about 31.9 kDa. The soluble monomeric His(6)-EasG was purified after overproduction in E. coli by affinity chromatography and used for enzyme assays. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses.     

Hydrogels based on schiff base formation between an amino‐containing polyphosphazene and aldehyde functionalized‐dextrans

Hydrogels generated by the interaction of two different water‐soluble polymers offer access to a new group of soft materials. A prototype amino‐functionalized polyphosphazene with both tyramine and ferulic acid‐based side groups was coupled to aldehyde functionalized‐dextrans to form hydrogels crosslinked via Schiff base chemistry. Synthesis of the polyphosphazene was accomplished by macromolecular substitution and protection‐deprotection chemistry, with characterization by ¹H NMR, ³¹P NMR, solid state ¹³C NMR, and DSC techniques. Combination of the aqueous polyphosphazene and aldehyde functionalized‐dextran solutions at room temperature caused gelation with different gelation times and crosslink densities dependent on the aldehyde content of the dextran. The hydrogel properties were evaluated using rheology, thermal characterization, and cryo‐microscopy. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2984–2991