Immobilization of mortierella vinacea cells by radiation polymerization

Immobilization of Mortierella vinacea cells, which contain active α-galactosidase, by radiation polymerization at low temperatures was studied. The durability of the enzymatic activity of the immobilized cells was examined by repeating the batch enzyme reaction. The enzymatic activities of the immobilized cells obtained with hydrophilic monomers was affected by the concentrations of the cells and monomer in which optimum conditions were observed. The enzymatic activity of the immobilized cells obtained with hydrophilic monomer was compared to that of hydrophobic monomers. Michaelis constants of the immobilized cells varied with monomer concentration. The effect of addition of porous solid substances on the immobilization of the cells was studied.     

Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability

In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine.     

Immobilization of microbial cells in membrane form by radiation-induced cast-polymerization

A method of cell immobilization by radiation-induced cast-polymerization is reported. Immobilization parameters which influence the enzymatic activity of immobilized cell membranes have been studied in batch enzyme reactions. These parameters are monomer concentration, membrane thickness and additive concentration. The Michaelis constant of immobilized cell membranes depends on the membrane thickness and additive concentration. The heat and pH stability of immobilized cell membranes was studied.     

一种选择性拆分布洛芬对映体的固定化脂肪酶

为了提高扩展青霉TS414脂肪酶(PEL)对布洛芬的拆分效率,建立了适于非水相中选择性拆分(R,S)-布洛芬的固定化方法. 结果表明,固定化介质的类型、冻干pH和外加水量等因素对固定化PEL酶促拆分(R,S)-布洛芬有较大影响. 在冻干pH为9.0、外加水量为0、以大孔吸附树脂AB-8为固定化载体的体系中,40℃反应30 h后,拆分反应的转化率可达47%,对映体过量值eeP可达98.75%. AB-8固定化后,PEL在有机相反应体系中的分散性得到了明显改善,大幅度提高了酶促拆分反应的效率;大孔吸附树脂AB-8固定化PEL具有较高的操作稳定性,连续10批拆分反应的平均转化率在47%以上,eeP值均稳定在98%以上.     

Study on the activity and stability of urease immobilized onto nanoporous alumina membranes

Nanoporous alumina membranes were employed as substrate materials for urease immobilization. Anodic porous alumina was prepared by the two-step anodization of high purity aluminum. By controlling anodization conditions, the nanoporous structure with desired dimension was obtained. Urease immobilization onto nanoporous alumina membranes was performed by four different protocols. Effect of pore diameter, pore length and immobilization methods on the activity and stability of immobilized enzyme was discussed in detail. The results show that the enzymes immobilized onto porous alumina with big pore diameter possess high activity and poor stability as compared to small pore diameter. The effect of pore length is complicated, the activity of enzyme increases with the increasing pore length for big pore size; while for correspondingly small pore size, enzymatic activity slightly depends on pore length. The immobilization methods have a slight effect on enzymatic activity, whereas enzyme immobilization by chitosan coating and reticulation with glutaraldehyde exhibits a good long-term stability as compared to that only via physical adsorption.     

Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran

Highly porous activated carbon (HPAC) was used as carrier matrix for immobilization of acid protease (AP). Immobilization of acid protease on mesoporous activated carbon (AP-HPAC) performs as best enzyme carrier. At pH 6.0, 250 mg acid protease g⁻¹ HPAC was immobilized. The optimum temperature for both free and immobilized AP activities were 50 °C. After incubation at 50 °C, the immobilized AP maintained about 50% of its initial activity, while the free enzyme was completely inactivated. When testing the reusability of AP-HPAC combination immobilized system, a significant catalytic efficiency was maintained along more than five consecutive reaction cycles. The highly porous nature of the carbon permits significant higher loadings of enzyme, which results in a higher enzyme-support strength and increased stability. The changes in the AP, HPAC and AP-HPAC were confirmed by Fourier Transform Infrared spectroscopy (FT-IR). Furthermore, scanning electron microscopy (SEM) allowed us to observe that the morphology of the surface of HPAC and the AP-HPAC.     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

Kinetic Studies of Site-Specifically and Randomly Immobilized Alkaline Phosphatase on Functionalized Membranes

A comparison of enzyme activities has been made between a site-specifically immobilized and a randomly immobilized bacterial alkaline phosphatase (BAP) on macroporous membranes. An octapeptide tag (FLAG™) was attached at the N-terminus of alkaline phosphatase by recombinant DNA techniques (gene fusion) to yield BAP that is modified in a site-directed fashion (SDBAP). The corresponding antibody (antiFLAG™) was immobilized on an aldehyde-modified polyethersulfone (MPS) membrane via protein A. Immobilization of SDBAP on this membrane result in a membrane–protein A–antiFLAG–SDBAP linkage. This site-specifically immobilized enzyme demonstrated a relative activity (RA), defined as the ratio of immobilized activity (V_max) to the corresponding homogeneous enzymatic activity, of 85% as compared with the randomly immobilized BAP which had an RA of 0·8%. BAP, when chemically conjugated to the FLAG peptide and immobilized via antiFLAG and protein A on the MPS membrane, showed an RA of only 1·9%, demonstrating the effectiveness of site-directed immobilization. SDBAP was also immobilized on the MPS membrane in the absence of protein A. In this case, the RA dropped to 22%, further explaining the effectiveness of ordered immobilizations as compared with random immobilizations. The ratio of immobilized enzyme activity to the activity in the absence of added phosphate inhibitor for the immobilized BAP was three-fold higher than the corresponding homogeneous ratio, showing a reduction in product inhibition for the immobilized enzyme. © 1997 SCI.     

Efficient Immobilization of Lecitase in Gelatin Hydrogel and Degumming of Rice Bran Oil Using a Spinning Basket Reactor

Immobilization of Lecitase (Phospholipase A1) in gelatin hydrogel and its stability is studied with a view to utilizing the immobilized enzyme for degumming rice bran oil. Excellent retention of enzyme activity (>80%) is observed in hydrogel containing 43.5% gelatin crosslinked with glutaraldehyde. Compared to the free enzyme which has a broad pH-activity profile (6.5–8.0), the activity of the immobilized enzyme is strongly dependent on pH and has a pH-optimum of pH 7.5. The optimum temperature of enzyme activity increases from 37 to 50 °C. Compared to the free enzyme which loses all its activity in 72 h at 50 °C, the immobilized enzyme retains its activity in full. The immobilized enzyme has been used efficiently in a spinning basket bioreactor for the degumming of rice bran oil with 6 recycles without loss of enzyme activity. The phosphorus content of the oil decreases from 400 ppm to 50–70 ppm in each cycle. After charcoal treatment and dewaxing, a second enzymatic treatment brings down the phosphorus content to <5 ppm.     

酸性脲酶的固定化研究

以明胶为包埋材料,戊二醛为交联剂固定化粪产碱菌所产的酸性脲酶。对酸性脲酶的固定化条件(包括明胶含量、戊二醛浓度、吸附时间、交联时间和粗酶液用量)及酶学性质(温度和pH值)进行了研究。结果表明,固定化酶的最适宜条件为：明胶的质量分数15％,戊二醛质量分数0.3％,吸附时间4 h,交联时间20 min,粗酶液用量4 mL。...     

Esterification of lauric acid using lipase immobilized in the micropores of a hollow-fiber membrane

A porous anion-exchange hollow-fiber membrane was prepared by radiation-induced graft polymerization and chemical modification to immobilize lipase for enzymatic reaction in an organic solvent. The amount of anion-exchange group introduced to the porous hollow-fiber membrane was 2.5 mol/kg_fiber. A lipase solution was allowed to permeate through the porous anion-exchange hollow-fiber membrane, and lipase molecules that adsorbed onto the grafted polymer brush were cross-linked with glutaraldehyde. The lipase was immobilized at a density of 0.14 kg_lipase/kg_fiber, which was equivalent to a degree of multilayer binding of 20. Esterification was carried out by passing a solution of lauric acid and benzyl alcohol in anhydrous issoctane through the lipase-immobilized membrane, and lipase activity was determined. A reaction percentage of 50% was achieved at space velocity 68 h⁻¹. The maximum immobilized lipase and native lipase activities were 8.9 and 0.38 mol/(h·kg_lipase), respectively. Thus, the activity of the immobilized lipase was 23.4 times higher than that of the native lipase.     

Protease immobilization onto porous chitosan beads

Water-insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N-benzyl-L -arginine ethyl ester (BAEE), but rather low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized proteases on the porous chitosan beads are estimated. The apparent K_m values were larger for immobilized proteases than for the free ones, while V_m values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Immobilization of Yarrowia lipolytica Lipase on Bamboo Charcoal to Resolve (R,S)‐Phenylethanol in Organic Medium

Immobilization of lipase on bamboo charcoal was confirmed by spectra of FTIR, UV‐Vis, and XPS. Under optimal conditions, the immobilized lipase retains 87 % of the hydrolytic activity of the free lipase. Structural changes of the immobilized lipase were analyzed by circular dichroism and FTIR spectra, indicating that the secondary structure of the lipase was preserved well after immobilization. For catalyzing the enantioselective reaction in heptane, the catalysis efficiency of the immobilized lipase is 1.4 times that of free lipase. Immobilized lipase maintains the selectivity of free lipase.     

Papain immobilization onto porous poly(λ-methyl L-glutamate) beads

Water-insoluble papain was prepared by immobilizing papain onto the surface of porous poly(λ-methyl L -glutamate) (PMLG) beads with and without spacer. The mode of the immobilization between papain and porous PMLG beads was covalent fixation. The relative activity and the stability of the immobilized papain was investigated. The retained activity of the papain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N-benzyl L -arginine ethyl ester (BAEE), compared with that of the peptide binding method. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized papain on the PMLG beads were estimated. The apparent K_m was larger for immobilized papain than for the free enzyme, while V_m was smaller for the immobilized papain. The thermal stability of the covalently immobilized papain was higher than that of the free papain. The initial enzymatic activity of the covalently immobilized papain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Improved catalytic activity by catalase immobilization using γ‐cyclodextrin and electrospun PCL nanofibers

Nanofibrous structures are promising for biocatalyst immobilization due to their large surface area which facilitates the enzyme attachment, stability, ease of separation, and fine porous structure. There is limited research available on the change in enzyme activity following interaction with cyclodextrin. In this study, catalase enzyme was immobilized into nanofibrous structures by various techniques, with and without γ‐CD addition, and the enzymatic activity of catalase was evaluated. In addition, catalase‐γ‐CD complex containing PEO polymer solution was electrospun in between PCL nanofibrous layers as a newly developed technique. The enzyme immobilized nanofibrous structures were characterized by SEM, XRD, and FT‐IR analysis methods. Among all the activity tests, best enzyme activity was recorded with catalase‐γ‐CD physical mixture encapsulated PCL nanofibrous layers. Moreover, the test results indicated that the use of cyclodextrin in immobilization process considerably improves the catalytic activity of the enzyme. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44404.     

纳米羟基磷灰石颗粒固定化漆酶的研究木

利用羟基磷灰石对漆酶进行了固定化研究,确定了羟基磷灰石对漆酶固定的最适条件,并研究了不同温度下游离漆酶与固定化漆酶活力的差异,同时也对游离漆酶与固定化漆酶的Km值进行了研究.结果表明:0.08 g羟基磷灰石固定1 mL粗酶液,当固定化过程pH为6,交联剂戊二醛浓度为6%,固定化时间为1 h时,酶活力达到最大.当温度在3...     

A new approach for describing and solving the reversible Briggs-Haldane mechanism using immobilized enzyme

Recently immobilized enzymes have been widely used in industrial processes due to their outstanding advantages, such as high stability and recyclability; however, their kinetic behaviour is generally controlled by mass diffusion effects. Thus, in order to improve these enzymatic processes, a clear discernment between the kinetic and diffusion mechanisms that control the production of the metabolite require investigation. In practice, it is typical to establish apparent kinetics for immobilized enzyme operations, and the validity of the apparent kinetics is restricted to the studied cases. In this work, a new approach for mathematically describing the kinetic and diffusion mechanics in an immobilized biocatalyst bead is established, in which the fraction of residual enzymatic activity is included, and is defined as a measure of the active and available enzymes in the bead porous network. In addition, the diffusion and kinetic mechanisms are described by the effective diffusion coefficient and the free enzyme kinetics, since the porous network of the bead is assumed as the bioreaction volume. Therefore, free enzyme kinetics were determined from glucose to fructose bioconversion using a stirred tank reactor with free glucose-isomerase, in which substrate and enzyme concentrations and temperature were varied. The fraction of residual enzymatic activity () and the effective diffusion coefficient () were obtained from the isomerization of glucose to fructose using a stirred tank reactor with immobilized glucose-isomerase in calcium alginate beads at different substrate and enzyme concentrations. Finally, simulations were carried out to establish the bioreaction solid-phase characteristics that most significantly influence productivity.     

Photocatalytic removal of NOx over immobilized BiFeO<Subscript>3</Subscript> nanoparticles and effect of operational parameters

Perovskite type BiFeO₃ (BFO) was synthesized by sol-gel auto-combustion method. Synthesized BFO was immobilized on the micro slides glass plates by sol-gel dip-coating method. The sample was characterized by XRD, FESEM, UV-Vis DRS, and BET techniques. The XRD pattern confirmed the perovskite structure, and from the Debye-Scherrer equation the average crystalline size was calculated as 19 nm. The FE-SEM images of prepared BFO showed porous structure with low agglomeration. The band gap energy was calculated about 2.13 eV, and the specific surface area (SSA) of prepared BFO nanostructure was obtained 55.1m² g^?1. The photocatalytic activity of prepared pure and immobilized BFO was investigated in the removal of NOx under UV irradiation, in the batch photoreactor. The effects of operational parameters such as initial concentration of NOx, light intensity and amount of coated photocatalyst, under identical conditions, were investigated. The results showed that the highest conversion of NOx was obtained as 35.83% in the 5 ppm of NOx with 1.2 g immobilized BFO and under 15 W illumination lamp.     

Bisacryloylpiperazine as vinylating agent for enzyme immobilization by graft-copolymerization onto polysaccharides

A method of enzyme immobilization by graft-copolymerization onto polysaccharides is reported. Bisacryloylpiperazine has been used as a vinylating reagent and the reaction product with several enzymes (HRP, GOD, Am, ChT, Cel) was copolymerized onto different matrices (cellulose, Sepharose, Sephadex, starch). Immobilization parameters which influence the copolymer activity have been studied for the insolubilization of horseradish peroxidase onto cellulose. These parameters are pH, time, and temperature of bisacryloylpiperazine enzyme activation reaction. Under the best immobilization conditions copolymer activity linearly depends on enzyme concentration. Enzyme coupling efficiency depends on the type of enzyme and it ranges from 7 to 20%. The most important characteristics of these immobilized enzyme systems were tested and compared with those of similar systems obtained by glycidylmethacrylate enzyme activation (stability in continuous washing, kinetic characteristics, and storage, thermal, and operational stability). Immobilized enzyme graft copolymers have kinetic behaviour very close to that of the free enzymes. Diffusion is not seriously limited because immobilization reaction does not alter the enzymatic activity. By means of bisacryloylpiperazine it was possible to immobilize chymotrypsin with better results than those previously obtained, particularly coupling efficiency and long term continuous working.     

Cinnamic ester of D‐sorbitol for immobilization of mushroom tyrosinase

Mushroom tyrosinase was immobilized by adsorption onto the totally cinnamoylated derivative of D ‐sorbitol. The polymerization and cross‐linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of tyrosinase on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The pH value, enzyme concentration and immobilization time were all important parameters affecting immobilization efficiency; also, enzyme immobilization efficiency correlated well with the tyrosinase isoelectric point. The immobilized enzyme showed an optimum measuring pH of 3.5 and greater activity at acid and neutral pH values than the soluble enzyme. The optimal reaction temperature was 35 °C and the temperature profile was broader than that of the free enzyme or of the enzyme immobilized on other supports. The apparent Michaelis constant of mushroom tyrosinase immobilized on the SOTCN derivative acting on 4‐tert‐butylcatechol (TBC) was 0.40 ± 0.02 mmol dm^?3, which was lower than for the soluble enzyme, suggesting that the affinity of this enzyme for this substrate was greater when immobilized than when in solution. Immobilization stabilized the enzyme and made it less susceptible to activity loss during storage at pH values in the range 4–5.5, and the suicide inactivation of the immobilized tyrosinase was null or negligible in a reaction medium with 4‐tert‐butylcatechol at a concentration of 0.4 mmol dm^?3. The results show that cinnamic carbohydrate esters of D ‐sorbitol are an appropriate support for tyrosinase immobilization and could be of use for several tyrosinase applications. Copyright © 2005 Society of Chemical Industry