Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.     

Preparation of nano-enzyme aggregates by crosslinking lipase with sodium tripolyphosphate

Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. The ionic cross-linking agent-sodium tripolyphosphate (TPP) was first used in preparing CLEAs. Aspergillus niger lipase was precipitated with ammonium sulfate and further cross-linked by TPP. The factors including enzyme concentration, pH of cross-linking medium, TPP dosage and cross-linking time were optimized. Maximum recovery activity (99.5 ± 0.634 %) and cross-linking yield (88.4 ± 0.46 %) can be obtained under the optimal process conditions, which can illustrate TPP had little effect on enzyme activity. CLEAs showed improved activity over broad pH and temperature range compared to the free enzyme. The thermal stability was obviously improved compared to free enzyme under the optimal temperature (40℃) and the half-life was 7.5-fold higher than that of free enzyme. Moreover, scanning electron microscopy (SEM) revealed that CLEAs had a cavity with porous structure and the particle size was 249 ± 3.98 nm. X-ray diffraction (XRD) showed the crystallinity of the CLEAs decreased. The changes in secondary structures of CLEAs revealed the increment in conformational rigidity. Such results suggested that the CLEAs has ideal application prospects.     

A smart bioconjugate of chymotrypsin

alpha-Chymotrypsin was immobilized on Eudragit S-100 via covalent coupling with 93% retention of proteolytic activity. The conjugate behaved as a smart biocatalyst and functioned as a pH-dependent reversibly soluble-insoluble biocatalyst. The pH optimum of chymotrypsin broadened on immobilization, and the immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme. The immobilized enzyme showed a slight shift in the temperature optimum and enhanced thermal stability retaining 70% of its original activity after 1 h of exposure to 40 degrees C as compared to the 25% residual activity for the free enzyme under identical conditions. K(m) and V(max) values did not change on immobilization. Also, the immobilized preparation was quite stable to reuse, it retained almost 85% of its original activity even after a fifth precipitation cycle. UV spectroscopy and circular dichroism were used to probe structural changes in the enzyme upon immobilization.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Cross-linking of horseradish peroxidase adsorbed on polycationic films: utilization for direct dye degradation

Horseradish peroxidase (HRP) was immobilized on the polyaniline (PANI) grafted polyacrylonitrile (PAN) films. The maximum HRP immobilization capacity of the PAN-g-PANI-3 film was 221?μg/cm(2). The HRP-immobilized PAN-g-PANI-3 film retained 79?% of the activity of the same quantity free enzyme. The HRP-immobilized PAN-g-PANI-3 film was operated for the decolorization of two different benzidine-based dyes in the presence of hydrogen peroxide. The maximum decolorization grade was obtained at pH 6.0 for both dyes. The HRP-immobilized PAN-g-PANI-3 film was very effective for removal of Direct Blue-53 compared to Direct Black-38 from aqueous solutions. The immobilized HRP exhibited high resistance to proteolysis by trypsin compared to the free counterpart. Immobilized HRP preserved 83?% of its original activity even after 8?weeks of storage at 4?°C, while the free enzyme lost its initial activity after 3?weeks of storage period.     

Development of a thiol‐ene based screening platform for enzyme immobilization demonstrated using horseradish peroxidase

Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster and more efficient screening, which we believe will result in a higher chance of discovery of optimal surface/enzyme interactions. The proposed system consists of a thiol‐functional microplate prepared through fast photochemical curing of an off‐stoichiometric thiol‐ene (OSTE) mixture. Surface functionalization by thiol‐ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT‐FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho‐HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed by fluorescence intensity (FI) measurements. Subsequently, Rho‐HRP activity was measured directly on the surface. The broad range of utilized surface chemistries permits direct correlation of enzymatic activity to the surface functionality and improves the determination of promising enzyme‐surface candidates. The results underline the high potential of this system as a screening platform for synergistic immobilization of enzymes onto thiol‐ene polymer surfaces. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1267–1277, 2017     

Development of Silica Gel-Supported Modified Macroporous Chitosan Membranes for Enzyme Immobilization and Their Characterization Analyses

The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Immobilized copper-ion affinity adsorbent based on a cross-linked β-cyclodextrin polymer for adsorption of Candida rugosa lipase

Lipase from Candida rugosa was immobilized on a β-cyclodextrin-based polymer by adsorption and subsequent cross-linking with epichlorohydrin (EP-CD). The ligand iminodiacetic acid (IDA) was then bonded with the cross-linked β-cyclodextrin (EP-CD-IDA). This affinity adsorbent was further chelated with Cu^{2 +} for the purpose of binding affinity and stability. The properties of the immobilized lipase were assayed and compared with those of the free enzyme. Results showed that 266 µg protein with an activity of 17.85 U was bound per gram of matrix, giving 188% of the specific activity of the free enzyme and a total recovered activity of 79.7% under the optimum conditions. The pH and thermal stabilities of lipase were improved after immobilization on the β-cyclodextrin-based polymer (EP-CD-IDA-Cu^{2 +}). In addition, experimental results indicated that the residual activity of the immobilized lipase was 50% after eight cycles of reuse.     

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of “starch” dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.     

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.     

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid‐based nanofiber membrane

In this study, polyacrylic acid‐based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid‐based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid‐based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Ca²⁺ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE‐based biosensors.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures]

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.     

Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on ---NH₂ groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m², respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Effects of pH and pore characters of mesoporous silicas on horseradish peroxidase immobilization

Effects of immobilization pH and pore characters of mesoporous silicas (MPSs), MCM-41, SBA-15, and MCF, were simultaneously investigated for the immobilization of horseradish peroxidase (HRP; EC 1.11.1.7). MCM-41 and SBA-15 were rod-like with respective average pore diameters of 32, and 54 Å, while that of MCF with spherical cell and frame structure was 148 Å. Moreover, the MPSs synthesized were of identical surface functional groups and similar contents of free silanol groups. At immobilization pH 6 and 8 almost 100% HRP loadings were obtained and insignificant leaching were observed for all types of supports at pH 6. However, MCF was found to give both the highest enzyme loading and leaching at pH 10. Maximum and minimum HRP activities were obtained at respective immobilization pH 8, and 6. Activities of immobilized HRP increased with support pore diameters in the order: MCM-41 < SBA-15 < MCF. HRP immobilized at pH 8 gave the highest storage stability (both at 4 °C and room temperature), and in opposition to pH 6. In addition, HRP immobilized in MCF was found to be the most stable under storage. The finding should be useful for the creation of biocatalysts and biosensors.