Preparation of phenylalanine imprinted polymer by the sol–gel transition method

A phenylalanine (Phe) imprinted polymer was prepared by the wet-phase inversion and sol–gel transition method to endow a copolymer matrix with a large uptake capacity of template molecules and prominent adsorption selectivity at the high concentration of the racemate solution. A copolymer bead prepared by wet-phase inversion was shrunken in a hydrochloric acid solution containing a large amount of template molecules after swelling in a sodium hydroxide solution. Template molecules were effectively implanted in the polymer matrix during shrinking after swelling. The adsorption selectivities of Phe-imprinted copolymer bead were 2.1 and 1.33 at 1 g and 10 g Phe/l racemate solution, respectively, and the Phe uptake capacity reached about 1 g Phe/g dry weight of the copolymer. The adsorption selectivity of the copolymer was retained after five batches of adsorption/desorption in 1 g Phe/l solution composed of 5% D-Phe and 95% L-Phe.     

Characterization of sol–gel encapsulated lipase using tetraethoxysilane as precursor

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of the precursor tetraethoxysilane (TEOS) in the presence of polyethylene glycol (PEG) as additive. The properties of silica and their derivatives with regard to mean pore diameter, specific surface area, mean pore size, weight loss upon heating (thermogravimetric analysis, TGA) and ²⁹Si and ¹³C NMR are reported. The pH optimum shifted from 7.8 to 6.7 and optimum temperature jumped from 36 to 60 °C upon enzyme encapsulation. Encapsulated lipase in presence of PEG (EN-PEG) exhibited higher stability in the range of 37–45 °C, but from 50 to 65 °C the EN-PEG was inactivated after seven cycles. Hydrolytic activity during long-term storage at room temperature decreased to 50% after 94 days. High diffusional resistance was observed for large oil concentration reducing hydrolytic effectiveness by 60% in the case of the encapsulated lipase. NMR, pore size and specific surface area data suggested an active participation of the lipase enzyme during gelling of the silica matrix. This lead to reduction of available Si–OH groups, larger pores and smaller surface area. Larger pores increase substrate diffusion that correlates well with higher hydrolytic activity of the TEOS–PEG sol–gel matrix encapsulated enzyme in comparison with other sol–gel supports.     

Influence of ionic liquids as additives on sol–gel immobilized lipase

The immobilization of lipase from Candida rugosa, using ionic liquids as additives to protect the inactivation of lipase by released alcohol and shrinking of gel during sol–gel process, was investigated. The influence of various factors, such as structure of ionic liquids, content of ionic liquids and types of precursor in the sol–gel process on the activity and stability of immobilized lipase was also studied. The highest hydrolytic activity of immobilized lipase was obtained when the hydrophilic ionic liquid, [C₂mim][BF₄], was used as an additive, while the highest stability of immobilized lipase was obtained by using hydrophobic ionic liquid, [C₁₆mim][Tf₂N]. Therefore, the binary mixtures of these ionic liquids as additives were used to obtain the optimal immobilized lipase, which shows both high activity and stability. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1:1 at molar ratio of [C₂mim][BF₄] and [C₁₆mim][Tf₂N] were 10-fold and 14-fold greater than in silica gel without ionic liquids (ILs), respectively. After 5 days incubation of this immobilized lipase in n-hexane at 50 °C, 84% of initial activity was remained, while the residual activity of the lipase immobilized without ILs was 28%.     

Influence of the alkyl-substituted silane precursor on sol–gel encapsulated lipase activity

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of three precursor types (tetraethoxysilane (TEOS), methyltrimethoxysilane (MTMS) and polydimethylsilane (PDMS)) in the presence and absence of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as additives. Silica and their derivatives were characterised with regard to mean pore diameter, specific surface area, pore size distribution (BET method), weight loss upon heating thermogramivemetric analysis (TGA), chemical composition Fourier transform infrared spectroscopy (FT-IR), and catalytic activities. Immobilisation yields based on the recovered lipase activity vary from 3.02 to 31.98% and the highest efficiency was attained when lipase was encapsulated using TEOS in the presence of the PEG. Further information was obtained by testing the derivatives in esterification reactions and a different reactivity profile was found. Better performance was obtained with derivatives containing lipase encapsulated within gels prepared with MTMS as precursor in the presence of PEG. This lipase preparation exhibits increased esterification activity (155 μmol g⁻¹ min⁻¹), up to of three times greater than that prepared with TEOS (52 μmol g⁻¹ min⁻¹), and almost twice that prepared with MTMS/PDMS (89 μmol g⁻¹ min⁻¹) as precursors.     

Critical exponents for sol–gel transition in aqueous alginate solutions induced by cupric cations

The sol–gel transition in aqueous alginate solutions of four alginate samples having different molecular weights (M_W) and M/G ratios induced by cupric cations was monitored by rheology measurements. The gel point f_gel and the relaxation critical exponent n were determined using the Winter’s criterion over the alginate concentration C_Alg of 1–4 wt%. The scaling for the zero shear viscosity η₀ before the gel point and the equilibrium modulus G_e after the gel point was established against the relative distance ε from the gel point at the concentration of C_Alg = 1 wt%, giving the critical exponents k and z. The results indicated that f_gel was almost independent of the alginate concentration and became higher for the sample with lower molecular weight. The critical exponent n decreased with the increase in C_Alg for these four Cu-alginate samples and the fractal dimension d_f estimated from n suggested a denser structure in the critical gel with high G content. The critical exponent n evaluated from k and z agreed well with n determined from the Winter’s criterion.     

Effects of relative humidity on enzyme activity immobilized in sol–gel-derived silica nanocomposites

Enzyme immobilization has attracted great interest in biotechnology processes. Herein we report the immobilization of urease from Canavalia ensiformis (jack bean) in sol–gel-derived silica nanocomposites. Urease activity, differential scanning calorimetry (DSC), nitrogen and water adsorption isotherms were used to characterize the effect of storage at various relative humidities on enzyme activity immobilized in sol–gel-derived silica nanocomposites. In this study, the nanocomposites consist of tetraethoxysilane, as inorganic silicate precursor, in combination with glycerol or trehalose as organic additives. Entrapped urease was more stable for all the formulations aged with a relative humidity of 80%. However, significant differences (p < 0.05) in enzyme activity recovered at this relative humidity were observed between samples with different formulations, reflecting the effect of additives during the immobilization process. The applications of biocompatible sol–gel-derived matrices can be further extended and utilized in the development of biosensors with immobilized biomolecules that can be used for long time periods by taking into account different factors, among which the storage relative humidity has permitted to greatly improve the stability of the immobilized urease.     

Influence of the sol–gel chemistry on the activity of a lipase encapsulated in a silica aerogel

Silica aerogels have been shown to be efficient encapsulation media for the lipase of Burkholderia cepacia. The present study has focused on the encapsulation of this lipase in an aerogel made from 80% tetramethoxysilane (TMOS) and 20% methyltrimethoxylsilane (MTMS), dried by the supercritical CO₂ method. By varying different parameters in the synthesis chemistry of such materials, the structure and texture of the resulting gels can be significantly affected. The aim of the present study was to examine the possible existence of correlations between modifications of the gel’s synthesis procedure, and the catalytic activity of the gel-encapsulated lipase in the esterification reaction of lauric acid with 1-octanol. The synthesis parameters studied included aging of the wet gels in different solvents, variation of the solvent used during gel synthesis, variation in the molar ratio of hydrolysis water to silicon precursor and replacement of MTMS by another alkoxide. The biocatalytic activity was found to depend significantly on these different treatments. The results were analyzed in the light of the gel texture and structure characterization, respectively analyzed by nitrogen adsorption isotherms and ²⁹Si, ¹H and ¹³C NMR. These results suggest that the main role of the aerogel is to maintain the enzyme dispersed as it would be in an aqueous solution, even though it is used in an organic solvent where the lyophilized enzyme is insoluble. The nature of the gel surface groups, in particular their capability to modify the substrates concentration inside the gel, close to the active site of the enzyme, by comparison with the concentration in the solvent outside the gel, seem to have a more secondary effect.     

Rheological and calorimetric study of the sol–gel transition of κ-carrageenan

Rheological and DSC techniques were used to study the effect of κ-carrageenan and KCl concentrations, 0–300 mM, on the sol–gel transition as well as on the linear viscoelasticity, at 25 °C, of the resulting gels. In heating and cooling DSC tests, the peak temperature was taken as the sol–gel transition point. In rheological tests, sol–gel transitions were determined from the variation of dynamic moduli with frequency and temperature, the independence of the phase angle on frequency and the evolution with temperature of dynamic moduli on cooling and heating at constant frequency and strain. Transition temperatures from DSC and rheology were in good agreement among them and with those previously reported. The three procedures yielded similar results, but the transition temperatures were more easily determined through the independence of the phase angle on frequency. Frequency sweeps showed gel behavior with stiffness increasing with polysaccharide and salt concentration. Below 100 mM KCl, G′ increased notably, whereas higher concentrations produced only marginal increases.     

An eco-friendly – novel approach for attaining wrinkle – free/soft-hand cotton fabric

A novel approach for upgrading both the wrinkle free and softness properties of cotton fabrics without adversely affecting their strength properties using an eco-friendly finishing regimes was investigated. Factors affecting the performance properties of the finished substrate such as pre-treatment, i.e., carboxymethylation (CMC) or ionic-crosslinking, post-treatment with amino functional silicone softener and its concentration, degree of carboxymethylation as well as thermofixation conditions were studied. The obtained results revealed that post-treatment with the amino based silicone micro emulsion (SiE) up to 30 g/L at pH 4 to a wet pickup of 100% followed by drying at 100 °C for 5 min and curing at 170 °C for 3 min results in a remarkable improvement in fabric resiliency (expressed as dry and wet wrinkle recovery angles), as well as in softness degree, without seriously affecting its retained strength. Improvement extent of the aforementioned properties is governed by the nature of the pre-treatment steps. Fixation of the amino-functional silicone softener onto/or within the modified cellulose structure is accompanied by a formation of semi-inter and/or intra-penetrated network (semi-IPN) thereby enhancing both the extent of crosslinking and networking as well as providing very high softness. FTIR analysis proved the formation of Si–O–Si–cellulose complex. Scanning electron micrograph shows that cotton, CMC and ionic crosslinked cotton fabrics treated with SiE shows higher surface smoothness and considerable reduction in protruding loose fibers, ditches and grooves compared with the untreated one.     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

纤维素固相化木瓜蛋白酶

 本文用叠氮法制备了纤维素固相化木瓜蛋白酶(简称CMCP)。与相应酶液水解酪蛋白的反应相比,它表现出较低的酶活性,较高的最适pH值和较高的稳定性。CMCP的比活回收率约为24％,最适pH值向碱性范围移动约为0.5个单位。CMCP经60℃热处理,持续3h活性无明显下降,在4℃下保存127天,活性只下降了40％左右。对这些参数,本文都根据CMCP的结构特点进行了分析。 CMCP柱还表现出明显的对啤酒的防浊能力。过柱的啤酒,氨基酸的含量大大增加。     

壳聚糖固定化木瓜蛋白酶的研究

以自制的壳聚糖作为载体,用戊二醛作交联剂,优化了固定化条件,研制成壳聚糖固定化木瓜蛋白酶。其活性回收率达到42—53％,操作半衰期达到一个月以上,对热、乙醇以及尿素的稳定性有很大的提高,Km值为0.67×10~2mg/mL,最适温度65—70℃,最适pH8.0,能使啤酒中的蛋白质浓度从56.5mg/L减少到2.7mg/L,可以消除啤酒的低温混浊现象。     

Immobilization of cellulases on the reversibly soluble polymer Eudragit S‐100 for cotton treatment

Cellulases can penetrate into the fiber, causing tensile strength loss of the cellulosic fibers or fabrics. To minimize the tensile strength loss, we have immobilized cellulases on Eudragit S‐100. The characteristics of covalent Eudragit cellulase were evaluated using gel filtration analysis and UV spectra. Gel filtration analysis revealed that the cellulases were covalently bound to the polymer. Covalent Eudragit cellulase was loaded with the enzyme of about 40% and had a relative activity about 80% at a Eudragit S‐100 concentration of 15 g/L. When cellulase is bound to the polymer, the solubility profile becomes similar to the one of Eudragit. In addition, the effects of the enzyme on the cotton yarns and fabric using cellulases have been investigated. Native and immobilized cellulases caused improvements in whiteness and wrinkle recovery angle of the fabric in comparison to the control samples. The bending stiffness results show that native and immobilized cellulase treated cotton fabric has an improved softness than the control samples. It was found that using the immobilized cellulase reduced the weight and tensile strength, because the hydrolytic attack is only limited to the surfaces of cotton fibers.     

Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents

Lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia) was successfully immobilized in sol–gels under low methanol conditions using lyophilization in order to dry the gel. The enzyme was also cross-linked with glutaraldehyde to CLEAs without any additives. These immobilized enzyme preparations were employed for the highly enantioselective acylations of 1-phenylethanol (1), 1-(2-furyl)ethanol (2) and N-acylated 1-amino-2-phenylethanol (3) with vinyl acetate in organic solvents. Enzymatic hydrolysis of the obtained ester product was observed as a side reaction of the acylation of 3 in the presence of lipase PS powder. Hydrolysis was suppressed when the immobilized preparations of lipase PS were used.     

Synthesis,characterization and sol–gel entrapment of a crown ether‐styryl fluoroionophore

The synthesis and initial evaluation of a new dye‐functionalized crown‐ether, 2‐[2‐(2,3,5,6,8,9,11,12,14,15‐decahydro‐1,4,7,10,13,16‐benzohexaoxacyclooctadecin)ethenyl]‐3‐methyl benzothiazolium iodide (denoted BSD), are reported. This molecule contains a benzyl 18‐crown‐6 moiety as the ionophore and a benzothiazolium to spectrally transduce ion binding. Binding of K⁺ to BSD in methanol causes shifts in the both absorbance and fluorescence emission maxima, as well as changes in the molar absorptivity and the emission intensity. Apparent dissociation constants (K_d) in the range 30–65 µ m were measured. In water and neutral buffer, K_d values were approximately 1 m m . BSD was entrapped in sol–gel films composed of methyltriethoxysilane (MTES) and tetraethylorthosilicate (TEOS) with retention of its spectral properties and minimal leaching. K⁺ binding to BSD in sol–gel films immersed in pH 7.4 buffer causes significant fluorescence quenching, with an apparent response time of approximately 2 min and an apparent K_d of 1.5 m m . Copyright © 2009 John Wiley & Sons, Ltd.     

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied. Silica gels were used directly and characterized by mercuric porosimetry. Agitation rates (100–740 rpm) and particles size (35–75 to 250–500 μm) of silica gels did not affect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limiting factor of the trypsin adsorption onto the mesopores structure of silica gels. The adsorption of trypsin was determined as a function of their initial concentration and multilayer formed at high trypsin concentration.     

Thermodynamic analysis of sol–gel transition of gelatin in terms of water activity in various solutions

Sol–gel transition of gelatin was analyzed as a multisite stoichiometric reaction of a gelatin molecule with water and solute molecules. The equilibrium sol–gel transition temperature, T_t, was estimated from the average of gelation and melting temperature measured by differential scanning calorimetry. From T_t and the melting enthalpy, ΔH_sol, the equilibrium sol‐to‐gel ratio was estimated by the van't Hoff equation. The reciprocal form of the Wyman–Tanford equation, which describes the sol‐to‐gel ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on the sol–gel transition of gelatin was clearly explained and the contributions of hydration and solute binding to gelatin molecules were separately discussed in sol–gel transition. The general solution for the free energy for gel‐stabilization in various solutions was obtained as a simple function of solute concentration. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 685–691, 2015.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

Effect of urea on solution behavior and heat-induced gelationof chitosan-β-glycerophosphate

In this study, we have investigated the effect of urea on the physicochemical (pH and conductivity) and rheological properties of the chitosan-β-GP system in order to assess the main polymer–polymer interactions at low and high temperature. The pH of the solutions was slightly increased due to the consumption of H⁺ in solution through the hydrolysis of urea. Furthermore, the addition of urea considerably decreased the conductivity, and therefore the ionic strength of the solutions, and this effect was more important at high temperature. It indicated that urea strongly affects polymer–polymer interactions by weakening hydrogen bonds at low temperature, but in addition can hinder hydrophobic effect at high temperature since the reduction of ionic strength results in less screening of electrostatic repulsion between protonated glucosamine groups. At 15 °C, the addition of urea to chitosan-β-GP solutions decreased their elasticity, shortened relaxation times and simplified the relaxation process due to the disruption of hydrogen bonds. Heat-induced gelation of the chitosan system in the presence of urea showed higher gelation temperature (T_gel) in non-isothermal tests and longer gelation time (t_gel) in isothermal conditions. The activation energy for gelation also increased with increasing urea concentration. We concluded that the detrimental effect of urea on the gelation process was mainly related to a decrease in polymer-polymer hydrophobic effect, as shown by the decrease in conductivity.