Expanding Application of Immobilized <Emphasis Type="Italic">Candida Antarctica</Emphasis> Lipase B: A Green Enzyme Catalyst for Knoevenagel Condensation Reaction

We propose a green synthesis of benzopyran compound based on immobilized lipase. Firstly, by using a combined electrospun membrane and biocompatible chitosan, we show that the chitosan-functionalized electrospun PMA-co-PAA fibrous membrane can provide effective anchoring sites for immobilization of the Candida antarctica lipase B (CALB), and a maximum loading amount of CALB (152 mg/g) can be achieved. Then, the resulting membrane displays enhanced stability in catalyzing Knoevenagel condensation reaction of salicylaldehyde with active methylene compound in mixed aqueousorganic media. The catalytic activity and stability of the CALB on chitosan-modified fiber is better than its non-immobilized counterpart. Furthermore, this immobilize CALB catalyst still retained about 45 % of the initial activity after 5 cycles, which potentially had implications for the design of “green” enzyme catalyst for sustainable organic synthesis.     

Chitosan Activated with Genipin: A Nontoxic Natural Carrier for Tannase Immobilization and Its Application in Enhancing Biological Activities of Tea Extract

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract.     

Immobilization of Mucor miehei esterase on core-shell magnetic beads via adsorption and covalent binding: Application in esters synthesis

In this work, poly(hydroxyethyl methacrylate/glycidylmethacrylate), p(HPMA-GMA), magnetic beads were prepared via suspension polymerization, and grafted with poly(methacrylic acid), p(MMAc) via redox polymerization. Esterase from M. miehei was immobilized onto core-shell magnetic beads with two different methods (i.e., adsorption and covalent binding). For adsorption, the effects of pH and initial esterase concentration on the immobilization efficiency via adsorption have been studied. The maximum adsorption capacity of the magnetic beads for esterase was found to be 27.6 mg/g magnetic beads at pH 7.0. The covalent immobilization of esterase via carbodiimide coupling on mp(HEMA-GMA)-g-p(MAAc) beads was found to be the more effective than the direct immobilization of the enzyme on the mp(HEMA-GMA) beads. The covalently immobilized esterase on the mp(HEMA-GMA)-g-p(MAAc) beads exhibited higher activity compared to the adsorbed esterase on the same magnetic beads. Tributyrin hydrolysis properties of the free and both immobilized esterase preparations were determined under different experimental conditions. The operational, thermal and storage stabilities of the immobilized esterase preparations were increased compared with the free esterase. Finally, esterification reactions have been performed to produce citronellyl-esters (namely: acetate, butyrate and propionate) in n-hexane medium using covalently immobilized esterase. The immobilized esterase was effectively reused in successive batch runs in n-hexane medium for citronellyl-butyrate synthesis, and only 23 % activity was lost after 6 cycles.     

Cationized albumin-biocoatings for the immobilization of lipid vesicles

Tethered lipid membranes or immobilized lipid vesicles are frequently used as biomimetic systems. In this article, the authors presented a suitable method for efficient immobilization of lipid vesicles onto a broad range of surfaces, enabling analysis by quantitative methods even under rigid, mechanical conditions-bare surfaces such as hydrophilic glass surfaces as well as hydrophobic polymer slides or metal surfaces such as gold. The immobilization of vesicles was based on the electrostatic interaction of zwitterionic or negatively charged lipid vesicles with two types of cationic chemically modified bovine serum albumin (cBSA) blood plasma proteins (cBSA-113 and cBSA-147). Quantitative analysis of protein adsorption was performed as the cBSA coatings were characterized by atomic force microscopy, surface zeta potential measurement, fluorescence microscopy, and surface plasmon spectroscopy, revealing a maximal surface coverage 270-280?ng/cm(2) for 0.02 mg/ml cBSA on gold. Small unilamellar vesicles as well as giant unilamellar vesicles (GUVs) were readily immobilized (～15?min) on cBSA coated surfaces. GUVs with 5-10 mol% negatively charged 1,2,-dipalmitoyl-sn-glycero-3-phosphoglycerol remained stable in liquid for at least 5 weeks.     

Sericin-fixed filk fiber as an immobilization support of enzyme

In this study, we attempted to evaluate a novel use of sericin-fixed silk fiber (SFx) as an immobilization support of enzyme. Sericin was fixed on the silk fiber using glutaraldehyde as a fixation reagent. After 6 hours of fixation, the degree of fixation increases linearly with linear decrease of the amount of bound α-chymotrypsin (CT). This suggests that the increase of the degree of fixation is due to the further crosslinking of free aldehyde groups on the surface of sericin-fixed silk fiber (SFx). Even though perfect fixation was not achieved, sericin did not dissolve seriously and could be removed by further washing. The specific activity did not differ significantly after 6 hours of fixation. The activity of immobilized CT on SFx decreased to its half after 6 hours of incubation at 50°C. However, it retained 78% of initial activity even after 1 hour of treatment with 100% ethanol. As a result, the SFx could be used as an immobilization support of enzyme in non-aqueous media at ambient temperature.     

Immobilization of Urease onto Modified Egg Shell Membrane through Cross Linking

Background:Immobilization is an approach in industry to improve stability and reusability of urease. The efficiency of this technique depends on the type of membrane and the method of stabilization. Methods:The PEI-modified egg shell membrane was used to immobilize urease by absorption and glutaraldehyde cross-linking methods. The membranes were characterized by FTIR and AFM, and Nessler method was applied to measure the kinetic of the immobilized enzymes. Finally, the storage stability (6 °C for 21 days) and reusability (until enzyme activity reached to zero) of the immobilized enzymes were investigated. Results:Based on FTIR, three new peaks were observed in both the absorption- (at 1389.7, 1230.8, and 1074.2 cm^-1) and the cross-linking (at 1615-1690, 1392.7, 1450 cm^-1) immobilized enzymes. The surface roughness of the native membrane was altered after PEI treatment and enzyme immobilization. The optimal pH of cross-linking immobilized enzymes was shifted to a more neutral pH, while it was alkaline in adsorption-immobilized and free enzymes. The reaction time decreased in all immobilized enzymes (100 min for free enzyme vs. 60 and 30 min after immobilizing by adsorption and cross-linking methods, respectively). The optimal temperature for all enzymes was 70 °C and they had a higher K_m and a lower V_max than free enzyme. The stability and reusability of urease were improved by both methods. Conclusion:Our findings propose these approaches as promising ways to enhance the urease efficiency for its applications in industries and medicines. Key Words: Egg shell, Immobilization, Polyethylenimine, Urease     

Immobilization of gelatin onto poly(glycidyl methacrylate)-grafted polycaprolactone substrates for improved cell-material interactions

To enhance the cytocompatibility of polycaprolactone (PCL), cell-adhesive gelatin is covalently immobilized onto the PCL film surface via two surface-modified approaches: a conventional chemical immobilization process and a surface-initiated atom transfer radical polymerization (ATRP) process. Kinetics studies reveal that the polymer chain growth from the PCL film using the ATRP process is formed in a controlled manner, and that the amount of immobilized gelatin increases with an increasing concentration of epoxide groups on the grafted P(GMA) brushes. In vitro cell adhesion and proliferation studies demonstrate that cell affinity and growth are significantly improved by the immobilization of gelatin on PCL film surfaces, and that this improvement is positively correlated to the amount of covalently immobilized gelatin. With the versatility of the ATRP process and tunable grafting efficacy of gelatin, this study offers a suitable methodology for the functionalization of biodegradable polyesters scaffolds to improve cell-material interactions.     

Preparation of amidoxime-modified polyacrylonitrile nanofibers immobilized with laccase for dye degradation

A novel approach to preparing multifunctional composite nanofibrous membrane was developed. Polyacrylonitrile (PAN) nanofibrous membrane was fabricated by electrospinning and then the nitrile groups in PAN copolymer was chemically modified to obtain amidoxime modified PAN (AOPAN) nanofiber membrane which was further used as a functional support for laccase immobilization. During the process of reactive dye degradation catalyzed by the AOPAN nanofiber membrane immobilized with laccase, metal ion adsorption occurred at the same time. The chemical modification was confirmed by Fourier transform spectroscopy (FTIR). Scanning electron microscope (SEM) was employed to investigate the surface morphologies of the electrospun nanofibers before and after laccase immobilization. The effects of environmental factors on laccase activity were studied in detail. It was found that the optimum pH and temperature for the activity of immobilized laccase was 3.5 and 50 °C. The relative activity retention of the immobilized laccase decreased dramatically during the initial four repeated uses. After 20 days’ storage, the activity retention of immobilized laccase was still high above 60 %. It has also proved that laccase immobilized on AOPAN nanofiber membrane performed well in dye degradation and metal ion adsorption.     

Manufacturing polymethyl methacrylate nanofibers as a support for enzyme immobilization

Nanofibers have a great potential for enzyme immobilization application due to their large surface area to volume ratio besides their porous structure. In this work, we produce polymethyl methacrylate (PMMA) nanofibers via electrospinning method in dimethylformamide (DMF) as solvent. Thereafter, we employ a chemical method on final PMMA nanofiberous web to covalently immobilize acetylcholinesterase (AChE) enzyme on membrane surface. Morphology and tensile properties of nanofibers are studied as first steps of characterization to make sure of obtaining a properly stable membrane for enzyme carrying application. Thereafter, the stability and activity of immobilized enzymes as two main characteristic parameters are tested and reported for different applications such as biosensor manufacturing.     

Covalent immobilization of urease to modified ethyl cellulose

Graft polymerization technology is a good way to modify polymers. New functional groups are added to polymer structure using graft polymerization. Enzyme immobilization could be done from these added functional groups. Ethyl cellulose was selected as a support for enzyme immobilization and no many studies has been conducted about it. It is insoluble in water and suitable for reuse. In this study, methacrylic acid was graft polymerized to ethyl cellulose using benzophenone. In graft polymerization studies, optimum amounts of methacrylic acid and benzophenone were determined as 60 mmol and 0.6 g, respectively. Percentage of graft polymerization was maximum in toluene as solvent and optimum graft polymerization time was found as 3 hours. The graft polymerization percentage was 225.7 % at optimum conditions. This value was very good for UV-induced graft polymerization technique. FT-IR spectra of ethyl cellulose and methacrylic acid graft polymerized ethyl cellulose showed that graft polymerization was carried out successfully. -COOH groups were added to ethyl cellulose structure after graft polymerization. Then, urease was immobilized to methacrylic acid graft polymerized ethyl cellulose using 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide as the condensing agent which accelerates the reaction between -COOH from methacrylic acid graft polymerized ethyl cellulose and -NH2 from urease. Optimization studies were also performed for the immobilization of urease. Optimized values for urease immobilization; optimum amount of 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide was found as 5 mg, temperature was determined as 37 °C, 2 hours were selected as optimum time, pH and amount of urease were found to be pH 7 and 10 mg, respectively. Remained activity of immobilized urease was 1.74 % before optimization studies. After optimization of immobilization studies, this ratio has increased to 29.85 %. The immobilized urease activity was increased 17.2 times.     

新型固体碱催化大豆油制备生物柴油的工艺研究

制备了新型固体碱催化剂KNO3/AlSBA-15,并以此催化大豆油与甲醇酯交换反应制备生物柴油,对其工艺条件进行了优化.结果表明:醇油物质的量比为12∶1,催化剂用量为大豆油质量的3％,反应温度65C,反应时间4h,生物柴油的产率可达81％以上.该催化剂对酯交换制备生物柴油具有较高的催化活性和良好的重复使用性.     

Nano immobilization of enzyme to improvement of biofuel cell electrode's function

This study centers on development of the enzyme presentation as a cathode catalyst. Laccase enzyme is immobilized at nano meter scale in quaternary ammonium bromide salt treated Nafion polymers and assorted with high surface area carbon powders that are then deposited onto a solid support to create the cathode electrode. Optimization of the synthesis circumstances resulted in notably enhanced performance toward oxygen reduction reaction.     

分子筛负载杂多酸催化大豆油制备生物柴油

采用等体积浸渍法制备了负载型催化剂PW/MCM-41,并以此催化大豆油与甲醇酯交换反应制备生物柴油.考察了磷钨酸负载量和催化剂焙烧温度对催化剂催化活性的影响,以及醇油物质的量比、催化剂用量、反应时间和反应温度对生物柴油产率的影响.结果表明:磷钨酸负载量为30%、焙烧温度为300℃时,催化剂活性最高.酯交换反应的最佳条件...     

Evaluating the potential of wine-making residues and corn cobs as support materials for cell immobilization for ethanol production

Three wine-making residues (grape seeds, skins and stems), and corn cobs were evaluated as support material for immobilization of Saccharomyces cerevisiae and the ethanol production by the immobilized cells was assessed. The main objective of this study was to find an abundant and low cost material suitable for the cells immobilization and able to be used in a next step of wine production by immobilized yeast cells. The four natural materials were used as support in two different forms: untreated, and treated by a sequence of acid and basic reactions. Untreated grape skin and corn cobs provided the highest cell immobilization results (25.1 and 22.2 mg cells/g support, respectively). The maximum ethanol production yield (about 0.50 g/g) was also obtained when the cells were immobilized in these untreated materials. It was also found that the support materials released nutrients to the medium, which favored the yeast development and the ethanol production. The use of immobilized cells systems under agitated conditions gave ethanol yields similar to those obtained by the static fermentations, but the immobilized cell concentration was significantly lower. In brief, static fermentation with cells immobilized on grape skins or corn cobs appear to be an interesting alternative for use on wine-making. The use of grape skins, particularly, which is a by-product of the wine elaboration, could be of larger interest to obtain an integrated wine production process with by-product reuse.     

Study on the stability and reusability of Glutamate Dehydrogenase immobilized on bacterial cellulose nanofiber

In this study, a simple method with improved properties for Glutamate Dehydrogenase immobilization is proposed by using bacterial cellulosic nanofiberas as a novel and inexpensive biomatrix. Acetobacter xylinum ATCC 10245 has been chosen and cultivated for synthesis of this nanofiber with the diameter of 30-70 nm. Thereafter, Glutamate Dehydrogenase was immobilized into the nanofiber pores in two steps: 1) enzyme adsorption and 2) crosslinking by glutaraldehyde. Optimum pH for the activity of immobilized enzyme and optimum temperature was reported in this work. Also the relative activity of immobilized enzyme was about 38 % in comparison to the enzyme activity of the first day after about two months. Moreover after reusing of the enzyme for 9 cycles, the immobilized enzyme retained over 83 % of its initial activity. Glutamate Dehydrogenase is immobilized on bacterial cellulosic nanofiber for the first time which can affect on facilitation of Glutamate production. Also can be a good method for having a stable and robust immobilized Glutamate Dehydrogenase that can withstand harsh operating conditions in processes which are common in these kind of reactions.     

Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan⁻¹) and specific activity (U mg protein⁻¹), leading to the preservation of more than 90% of the initial total activity (U mL⁻¹). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6–7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.     

<Emphasis Type="Italic">In situ</Emphasis> Cross-linked Chitosan Composite Superfine Fiber via Electrospinning and Thermal Treatment for Supporting Palladium Catalyst

Uniform chitosan fibers (CS/PEO) with diameter of 398±76 nm were prepared by electrospinning with merely 5 wt.% of poly(ethylene oxide) (PEO) loading, and then annealed at elevated temperature without the use of additional crosslinker to improve the thermostability and solvent resistance. Swelling test shows that the CS/PEO composite fibers annealed at 200 oC were stable in 50 wt.% acetic acid aqueous solution. The mechanical strength test shows that the annealing temperature can affect the tensile strength of CS/PEO composite fiber mat. The cross-linked CS/PEO composite fibers provide a useful platform for the immobilization of palladium catalyst to catalyze the Mizoroki-Heck reactions of aromatic halides with olefins. Moreover, these CS/PEO composite fibers could be post modified with special ligands to chelate palladium species efficiently to further improve the catalytic activity and stability.     

A promising approach for bio-finishing of cotton using immobilized acid-cellulase

In this research, cellulases were immobilized on Eudragit S-100 to minimize the tensile strength loss of cotton fabric caused by the enzymatic hydrolysis. About 76 % of the enzyme activity and 81 % of the amount of protein were recovered after the immobilization process, and the immobilized cellulase exhibited good reuse ability. The immobilized cellulase had the better adsorptive performance on cotton than the free cellulase. In addition, the results revealed that the catalytic efficiency of the immobilized cellulase on cotton was degradation, perhaps because the diffusion of the enlarged cellulase molecules is significantly inhibited in the interior of the cotton fiber. Moreover, the cotton fabric treated with the immobilized cellulase showed less weight and strength losses. SEM pictures further indicated that the cotton fabric treated with the immobilized cellulase suffered less damage.     

Biological nitrate removal using sugar-industry wastes

Biological denitrification experiment was conducted using sugar-industry wastes, namely final molasses as a carbon source and bagasse charcoal pellets as supporting media for denitrifying bacteria. We employed an upflow fixed-bed reactor filled with the pellets and biofilm attached onto them. This was fed with potassium-nitrate and dilute-molasses solutions. Total nitrogen removals of more than 85% were achieved at influent carbon–nitrogen (C/N) ratios between 2 and 4, and hydraulic residence times of more than 0.8 h. This demonstrated that final molasses could be used as an alternative carbon source. On the other hand, final molasses also contained some organic/ammonium nitrogen and refractory organic matter including colors, both of which were difficult to remove with the reactor. Accordingly, at higher C/N ratios, these substances caused major increases in effluent total-nitrogen and organic-carbon concentrations. Therefore, an optimum C/N ratio was found to be around 2.     

Synthesis of electrospun carbon nanofiber/WO<Subscript>3</Subscript> supported Pt,Pt/Pd and Pt/Ru catalysts for fuel cells

In the present study, nano-sized Pt/WO₃-carbon nanofiber, Pt-Pd/WO₃-carbon nanofiber and Pt-Ru/WO₃-carbon nanofiber electrocatalysts were synthesized and the performance of prepared catalysts were compared with catalysts coated carbon black for the oxygen reduction reaction (ORR). The morphology and structure of prepared catalysts were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. The SEM images showed that the catalyst nanoparticles were well dispersed on the both carbon nanofiber and carbon black supports. Electrochemical measurements including linear sweep voltammetry (LSV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA) tests were applied to investigate the potential of the fabricated electrodes on the ORR. The results demonstrated that the catalysts based on carbon nanofibers showed a significant increase of activity toward the ORR. Also, the Pt/Pd coated carbon nanofibrous electrode showed the highest electrochemical activity.