A study of ethanol production of yeast cells immobolized with polymer carrier produced by radiation polymerization

Polymer carriers, poly(hydroxyethyl acrylate(HEA)-methoxy polyethylene glycol methylacrylate (M-23G)) and poly (hydroxyethyl accrylate(HEA)-glycidyl methlacrylate(GMA)) using for immobilization of yeast cells were prepared by radiation polymerization at low temperature. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol productivity of immobilized yeast cells with these carriers was related to the monomer composition of polymers and the optimum monomer composition was 20% : 10% in poly(HEA-M-23G) and 17%: 6% in poly(HEA-GMA). In this case, the ethanol productivity of immobilized yeast cells was 29mg/ml/h which was about 4 times that of cells in free system. The relationship between the activity of immobilized yeast cells and the water content of polymer carrier were also discussed.     

pH-sensitive chitosan films for baker's yeast immobilization

Dried baker’s yeast cells were immobilized on a chitosan film, which is a natural polymer. Prepared chitosan films were treated with glutaraldehyde to facilitate the immobilization of the cells. The effects of the amount of glutaraldehyde, incubation time, pH, and temperature on immobilization were investigated. The amount of glutaraldehyde was chosen to be 0.01% (weight). The highest amount of yeast immobilization was obtained with 5 h incubation. It was determined that optimum temperature for immobilization is 25°C, and the optimum pH for immobilization is 6. Immobilized cells were allowed to stand for 3 d in distilled water and buffer solution (pH 6) to investigate the desorption, but no desorption was found. The maximum immobilization capacities were found to be 90 μg protein cm⁻² film in optimum conditions.     

Growth of yeast cells immobilized with porous swelling carriers produced by radiation polymerization

A new method of immobilization of yeast cells for the advantageous growth of cells inside and on the surface of a polymer carrier by physical adsorption is proposed. Porous and swellable polymer carriers were prepared by radiation-induced polymerization at low temperature. These polymer carriers were incubated with yeast cells at 30°C under aerobic conditions. Yeast cells were adsorbed on the surface of polymer carriers and subsequently infiltrated the polymer carriers by multiplication. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.     

Use of insoluble yeast beta-glucan as a support for immobilization of Candida rugosa lipase

In the present study, insoluble yeast beta-glucan (IYG) has been explored as a support matrix for enzyme immobilization. IYG contains mainly beta-(1-3) linkages along with some intra- or inter-molecular branches of beta-(1-6) linkages with large number of free hydroxyl groups. Epichlorohydrin was used to convert these free hydroxyl groups into activated epoxy groups that are capable of forming covalent linkages with various groups of enzyme molecule. The epoxy-activated IYG was evaluated for immobilization of Candida rugosa lipase (CRL). Post-immobilization treatment of 5% glutaraldehyde was given in order to achieve stable and irreversible binding of enzyme on the support. The resultant biocatalytic IYG support expressed lipase activity of 8136.7 U/g and 59.6% activity yield. There was 51.05% retention of synthetic activity after six repeated esterification cycles, indicating its stability and reusability in non-aqueous medium. Moreover, the immobilized lipase gave the storage half-life of about 285 days (at 4 degrees C).     

Bioreduction of quinone derivatives by immobilized Baker''''s yeast in hexane

Immobilization techniques and biocatalytic transformations performed in organic media are new developing methods for organic reactions. Baker's yeast was immobilized on the alginate supports. This preparation contained about 20% of dry yeast cells. The immobilized Baker's yeast were found to be very effective in the reduction of o-benzoquinone, p-benzoquinones, naphthoquinones, and anthraquinones in hexane.     

Ethanol Production Using Immobilized <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in Lyophilized Cellulose Gel

A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12–216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.     

Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

Polymer-Supported Tetrabromooxomolybdate(V): A New Type of Catalyst for Oxidation by t-Butyl Hydroperoxide

Tetrabromooxomolybdate(V) was immobilized in alkylammonium cation-type polymers obtained by the reaction of poly(p-chloromethyl-styrene-co-divinylbenzene-co-styrene) (abbreviated CMS) with amines and derived from poly(p-vinylpyridine) and poly(p-vinylpyridine-co-divinylbenzene). These immobilized polymers were active catalysts for the oxidation of alcohols and epoxidation of olefins with t-butyl hydroperoxide (abbreviated t-BuOOH). Among these polymers, we could find a polymer catalyst showing specificity, which was obtained by immobilization of tetrabromooxomolybdate(V) in the polymer derived from the reaction of CMS with trimethylamine. This immobilized polymer does not catalyze epoxidation of olefins but catalyzes oxidation of alcohols with t-BuOOH. Ammonium tetrabromooxomolybdate(V) complex was stabilized by the immobilization in the polymers, and it was found that the reactivity of the active group is due to the microenvironment supplied by the polymer chain.     

Eradication of Gram‐Positive and Gram‐Negative Bacteria by Photosensitizers Immobilized in Polystyrene

Immobilization of photosensitizers in polymers opens prospects for their continuous and reusable application. Methylene blue (MB) and Rose Bengal were immobilized in polystyrene by mixing solutions of the photosensitizers in chloroform with a polymer solution, followed by air evaporation of the solvent. This procedure yielded 15–140 μm polymer films with a porous surface structure. The method chosen for immobilization ensured 99% enclosure of the photosensitizer in the polymer. The antimicrobial activity of the immobilized photosensitizers was tested against Gram‐positive and Gram‐negative bacteria. It was found that both immobilized photosensitizers exhibited high antimicrobial properties, and caused by a 1.5–3 log₁₀ reduction in the bacterial concentrations to their total eradication. The bactericidal effect of the immobilized photosensitizers depended on the cell concentration and on the illumination conditions. Scanning electron microscopy was used to prove that immobilized photosensitizers excited by white light caused irreversible damage to microbial cells. Photosensitizers immobilized on a solid phase can be applied for continuous disinfection of wastewater bacteria.     

Wet and Dry Forms of Bacterial Cellulose Synthetized by Different Strains of <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis> as Carriers for Yeast Immobilization

The present study aimed to explore and describe the properties of bacterial cellulose (BC) membranes obtained from three different strains of Gluconacetobacter xylinus for 72, 120, and 168 h, used as a carrier support for the immobilization of Saccharomyces cerevisiae. The experiments also included the analysis of glucose consumption and alcohol production during the fermentation process displayed by yeasts immobilized on the BC surface. The results of the present study demonstrate that the number of immobilized yeast cells is dependent on the type of cellulose-synthesizing strain, cellulose form, and duration of its synthesis. The BC in the form of wet membranes obtained after 3 days of synthesis displayed the most favorable properties as a carrier for yeast immobilization. The immobilization of yeast cells on BC, regardless of its form, increased the amount of the produced alcohol as compared to free cells. The yeast cells immobilized in BC were able to multiply on its surface during the fermentation process.     

Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization

The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated. In particular, the effects of surface modification of the beads through hydrophilic polymers on the amount protein immobilized and on the extent of retained activity after immobilization were adressed. Furthermore, immobilization at unmodified and hydrophilized beads from aqueous solution was compared to that from a water-in-oil microemulsion. It was found that the amount trypsin immobilized at the unmodified GMA-GDMA beads was significantly higher than that at hydrophilized GMA-GDMA beads. However, also the extent of specific activity loss after immobilization was larger for the unmodified than for the hydrophilized beads. Despite the latter, however, the total activity displayed by the hydrophilized beads was comparable to the unmodified beads at best. On the other hand, by peforming the immobilization from the microemulsion a high immobilization yield can be reached even for the hydrophilized beads, which also results in a higher degree of retained activity in the latter case than obtained for immobilization at the unmodified beads. Using this approach therefore resulted in the highest total activity of the trypsin-activated GMA-GDMA beads.     

Platinum nanocatalysts immobilized on latex supports

Several latex dispersions of different hydrophobicity were investigated with respect to their ability to adsorb platinum nanoparticles that had been reduced in their presence. Two reduction methods were tested, specifically the slower method of refluxing the alcoholic solutions and the more rapid method of reaction with KBH₄. The immobilization of the metal particles and their nanosize dimensions were demonstrated by transmission electron microscopy, and their catalytic activity was tested by the hydrogenation of cyclohexene as a model reaction. Some additional immobilized platinum nanoparticles were prepared in the presence of various protective polymers. This can lead to various advantages with respect to, for instance, the stability and the catalytic properties of these materials. Even in the presence of such additional protective polymers, the platinum nanoparticles remained immobilized for some of the hydrophobic latexes both before and after catalytic hydrogenations. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35 : 1207–1216, 1997     

Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H(2)O(2) with a linear range of 5.0×10(-6) to 9.3×10(-4) mol L(-1).     

Immobilized baker's yeast reduction in fluorous media

The first example of immobilized bakers' yeast mediated reduction of ketones in fluorous media is described. The use of fluorous media permits simple work-up and reuse of the solvent without any purification.     

Fabrication and anti-fouling properties of photochemically and thermally immobilized poly(ethylene oxide) and low molecular weight poly(ethylene glycol) thin films

Poly(ethylene oxide) (PEO) and low molecular weight poly(ethylene glycol) (PEG) were covalently immobilized on silicon wafers and gold films by way of the CH insertion reaction of perfluorophenyl azides (PFPAs) by either photolysis or thermolysis. The immobilization does not require chemical derivatization of PEO or PEG, and polymers of different molecular weights were successfully attached to the substrate to give uniform films. Microarrays were also generated by printing polymer solutions on PFPA-functionalized wafer or Au slides followed by light activation. For low molecular weight PEG, the immobilization was highly dependent on the quality of the film deposited on the substrate. While the spin-coated and printed PEG showed poor immobilization efficiency, thermal treatment of the PEG melt on PFPA-functionalized surfaces resulted in excellent film quality, giving, for example, a grafting density of 9.2×10(-4)?(-2) and an average distance between grafted chains of 33? for PEG 20,000. The anti-fouling property of the films was evaluated by fluorescence microscopy and surface plasmon resonance imaging (SPRi). Low protein adsorption was observed on thermally-immobilized PEG whereas the photoimmobilized PEG showed increased protein adsorption. In addition, protein arrays were created using polystyrene (PS) and PEG based on the differential protein adsorption of the two polymers.     

Comparison of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase Immobilization Yield of Entrapment,Adsorption, and Covalent Bond Techniques

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 degrees C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 degrees C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage.     

Electrochemical grafting by reduction of 4-aminoethylbenzenediazonium salt: Application to the immobilization of (bio)molecules

We propose in this study a simple and rapid way to produce stable amino-derivatized conductive surfaces for the subsequent immobilization of (bio)molecules. This was achieved through the use of (4-aminoethyl)benzenediazonium salt (AEBD), which was immobilized on glassy carbon and gold electrodes by its electrochemical reduction. The presence of terminal grafted amino functions was evidenced with XPS by analyzing N1s core level. Besides this conventional surface characterisation, an electrochemical strategy is proposed here to evidence the presence of immobilized amines, in which the chemical reactivity of amines towards 2,4,6-trinitrobenzenesulfonic acid (TNBS) is used. Surface-bound TNBS served as an electrochemical marker and was detected by cyclic voltammetry. Additionally, pre-modified gold electrodes with amino functions can be derivatized with biomolecules such as glutathione (GSH). Glutathione attachment was evidenced by studying the electrochemical behaviour of ferri/ferrocyanide redox before and after its immobilization. The functionalized electrodes were then used for the detection of copper ions in neutral aqueous solutions.     

Immobilization of concanavalin A to glucose‐containing polymers

Sol‐gel reversible hydrogels sensitive to environmental glucose concentration were prepared using concanavalin A (Con A) and glucose‐containing polymers. Since the components of the hydrogels in the sol state can be released to the environment through pores of the dialysis membrane, it was necessary to immobilize Con A to the glucose‐containing polymers. Con A was immobilized by two different approaches. First, vinyl groups were introduced to Con A so that it can participate in the vinyl polymerization of allyl glucoside. Second, glucose‐containing polymers containing chemically reactive pendant groups were synthesized so that Con A could be immobilized to the preformed polymers. Both approaches resulted in effective immobilization of Con A to the glucose‐containing polymers, but the second method appeared to be better in terms of maintaining the bioactivity of Con A.     

猪胰脂肪酶的固定化及其在有机相中催化丁酸甲酯和正丁醇的酯交换反应研究

考察了大孔苯乙烯－二乙烯苯交联共聚物的交联度、致孔剂量及胺化试剂对载体固定化猪胰脂肪酶的影响。选择出一种最佳载体对猪胰脂肪酶进行固定化，对比了自由酶和固定化酶在有机相中催化丁酸甲酯和正丁醇的酯交换反应。结果表明，酶经固定化后催化反应活力比自由酶提高近１倍。     

Simultaneous purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis on a hydrophobic support

Purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis could be simultaneously accomplished by hydrophobic interaction on Phenyl Sepharose CL-4B in the presence of 50 mM pyrophosphate buffer (pH 8.5). The presence of a high salt concentration of 2M, which is generally required for the hydrophobic interactions, was not essential for the hydrophobic immobilization. The enzyme in free as well as immobilized form was optimally active between pH 7.0 and 9.0. The immobilized preparation could be reused in a batch process for the conversion of d-amino acids to α-keto acids. When the activity of the preparation dropped below practical limits, the gel could be regenerated by water wash and recharged with fresh crude extract from yeast.