Immobilization of enzymes on spongy polyvinyl alcohol cryogels: the example of beta-galactosidase from Aspergillus oryzae.

The catalytic properties of a beta-galactosidase from Aspergillus oryzae, entrapped into a spongy polyvinyl alcohol cryogel, were studied. This polymeric matrix was selected because of its mild conditions of preparation and its stability, biocompatibility, structural strength and diffusive properties. The enzyme was entrapped, in high percentage, into cryogel sponges and its activity and kinetic parameters were determined and compared with those of the free enzyme, using as substrates o-nitrophenyl-beta-galactopyranoside (ONPG) or lactose. The immobilized enzyme showed a reduced activity with ONPG and lactose, probably because of substrate diffusion limitations through the matrix, but it was more stable to temperature, pH and ionic strength than the free enzyme. Lactose hydrolysis under continuous experimental conditions was performed using the matrix-enzyme cited above.     

Immobilization of pancreatic lipase on polyvinyl alcohol by cyanuric chloride

Porcine pancreatic lipase (EC 3.1.1.3) was covalently immobilized onto 2,4,6-trichloro-s-triazine (cyanuric chloride) activated polyvinyl alcohol (PVA). The influence of activating agent and enzyme concentration on the immobilization process were evaluated.Hydrolytic activities of free and immobilized enzyme were determined and the immobilization yield was estimated by measuring the quantity of protein, both in free enzyme solution and in washing solutions after immobilization. After the optimization of immobilization process, the physical and chemical characterization of immobilized enzyme was performed. Additionally, the thermal, pH, storage, and operational stability of the immobilized and free enzymes were tested. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme.     

Immobilization of Lactobacillus rhamnosus in polyvinyl alcohol/calcium alginate matrix for production of lactic acid

Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using “freezing–thawing” technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g⁻¹) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L⁻¹ h⁻¹, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L⁻¹ h⁻¹. This study suggested that the “freezing–thawing” technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.

     

Procedure for obtaining and catalytic properties of trypsin immobilized in cryogels of polyvinyl alcohol

Commercial preparations of trypsin, varying in activity, were immobilized in a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In an organic solvent environment, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-argininine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.     

Production of alcohol from Jerusalem artichokes by yeasts

Various yeasts such as several strains of Saccharomyces diastaticus, S. cerevisiae, and Kluyveromyces fregilis were investigated for their ability to ferment the carbohydrates from Jerusalem artichokes to alcohol. Juice extracted of the carbohydrates. Fermentation was also carried out with raw artichokes without prior juice extraction. Result indicate that this row material has good potential for fuel alcohol production by fermentation.     

Immobilization of Rhizopus oryzae in a modified polyvinyl alcohol gel for L(+)-lactic acid production

The production of L(+)-lactic acid (LA) by Rhizopus oryzae immobilized in polyvinyl alcohol (PVA) was investigated. To decrease diffusional resistance, we modified the PVA gel through the addition of sodium alginate and phosphate esterification. The production of L(+)-LA improved notably in the immobilized Rhizopus oryzae. Maximum L(+)-LA production (106.27 g/L), with a yield of 73.1 % and rate of 2.95 g/L·h, was obtained at a temperature of 38 °C, 6 % PVA, and 0.8 % sodium alginate. The immobilized R. oryzae was stable in 14 serial-batch cultures using non-growth medium. The immobilized beads also displayed good tolerance to low temperature and long-term storage at 4 °C with the preservation of biochemical properties.     

Swelling behavior of poly(vinyl alcohol) cryogels employed as matrices for cell immobilization

Poly(vinyl alcohol) cryogels are prepared from aqueous solutions of the polymer by freezing and thawing and are employed as matrices for cell immobilization. The swelling behavior of these macroporous gel carriers in pure water and in solutions of certain compounds (salts, amino acids, and glucose) was studied to elucidate the osmotic properties of the cryogels during long-term exposure to aqueous media. It was shown that after the initial sol fraction was washed out, the residual gel matrix possessed high stability even at extreme pH conditions (acid or alkali concentration up to 1.0 mol l⁻¹) or in the presence of strong chaotropic salts such as sodium rhodanide. Although the macroporous supermolecular structure of the carriers under consideration underwent certain changes as a result of aging processes during prolonged washing of the gel, the high porous morphology of the material was retained.     

Immobilization of beta-galactosidase on fibrous matrix by polyethyleneimine for production of galacto-oligosaccharides from lactose

The production of galacto-oligosaccharides (GOS) from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth was studied. A novel method of enzyme immobilization involving PEI-enzyme aggregate formation and growth of aggregates on individual fibrils of cotton cloth leading to multilayer immobilization of the enzyme was developed. A large amount of enzyme was immobilized (250 mg/g support) with about 90-95% efficiency. A maximum GOS production of 25-26% (w/w) was achieved at near 50% lactose conversion from 400 g/L of lactose at pH 4.5 and 40 degrees C. Tri- and tetrasaccharides were the major types of GOS formed, accounting for about 70% and 25% of the total GOS produced in the reactions, respectively. Temperature and pH affected not only the reaction rate but also GOS yield to some extend. A reaction pH of 6.0 increased GOS yield by as much as 10% compared with that of pH 4.5 while decreased the reaction rate of immobilized enzyme. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme under the same reaction conditions, suggesting diffusion limitation was negligible in the packed bed reactor and the enzyme carrier. Increase in the thermal stability of PEI-immobilized enzyme was also observed. The half-life for the immobilized enzyme on cotton cloth was close to 1 year at 40 degrees C and 21 days at 50 degrees C. Stable, continuous operation in a plug-flow reactor was demonstrated for about 3 days without any apparent problem. A maximum GOS production of 26% (w/w) of total sugars was attained at 50% lactose conversion with a feed containing 400 g/L of lactose at pH 4.5 and 40 degrees C. The corresponding reactor productivity was 6 kg/L/h, which is several-hundred-fold higher than those previously reported.     

Reject water treatment by improvement of whole cell anammox entrapment using polyvinyl alcohol/alginate gel

Reject water treatment performance was investigated by whole cell anammox sludge entrapped polyvinyl alcohol/sodium alginate gel in the stirred tank reactor (STR). The whole experiment was conducted through Phase 1 and Phase 2 in which synthetic wastewater and modified reject water were used as feeding medium, respectively. The anammox reactor demonstrated quick start-up after 22 days as well as stable and relatively high nitrogen removal rate of more than 8.0 kg-N m⁻³ day⁻¹ during the two both phases even under moderately low temperature of 25 ± 0.5°C during the last 2 months of Phase 2. The matured brownish red PVA beads had good characteristics with buoyant density of 1.10 g cm⁻³, settling velocity of 141 m h⁻¹ and diameter of 4 mm. The bacterial community was identified by 16S rDNA analysis revealing the concurrent existence of KSU-1 and new kind anammox bacterium Kumadai-I after changing influent from synthetic wastewater to reject water. It was speculated that Kumadai-I might play a role as “promotion” factor together with KSU-1 on high nitrogen removal rate. These results demonstrate the potential application of whole cell anammox entrapment by PVA/alginate gel for achieving stable and high-rate nitrogen removal from high ammonium with low C/N ratio contained wastewaters, such as reject water, digester liquor or landfill leachate.     

Immobilization of hog pancreas lipase in macroporous poly(vinyl alcohol)-cryogel carrier for the biocatalysis in water-poor media

Hog pancreas lipase was covalently attached to the beads of poly(vinyl alcohol)-cryogel – a macroporous hydrogel prepared by means of freeze-thaw technique. The immobilized biocatalyst thus obtained was examined in the reaction of enantioselective hydrolysis of the ethyl ester of N-benzylidene derivative of DL-phehylalanine in the medium of acetonitrile (contained 5 vol.% of water without any buffers). Eighty-three %-enantiomeric excess of the l-amino acid was reached after 144 h. Virtually the same result was obtained in the repeated use of the same immobilized biocatalyst after its 6-months-storing in a refrigerator.     

Immobilization of Anabaena azollae from Azolla filiculoides in polyvinyl foam for ammonia production in a photobioreactor system

Anabaena azollae (AS-DS), isolated from Azolla filiculoides and grown in nitrogen-free medium, was immobilized in 5-mm-cube polyvinyl foam pieces and incorporated into a photobioreactor system for the production of NH₃. NH₃ was produced continuously and in significant amounts. Benlate (methyl-1-butyl-carbamoyl)-2-benzimidazole carbamate at 5 ppm and l-methionine-d,l-sulphoximine at 50 m stimulated NH₃ production continuously for a period of 1 week.S. Kannaiyan is with the Biotechnology Unit, Department of Agricultural Microbiology, Tamilnadu Agricultural University, Coimbatore-641003, Tamilnadu, India; K.K. Rao and D.O. Hall are with the Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.     

Removal of flatulence-inducing sugars by using free and polyvinyl alcohol immobilized α-Galactosidase from Aspergillus oryzae

Consumption of soymilk and soybean derived foods has been hampered due to the presence of RFOs (raffinose family oligosaccharides). Soy-based foods free from RFOs have positive impact on their acceptance as protein rich food. α-Galactosidase was entrapped in PVA (polyvinyl alcohol) cross linked with boric acid. Immobilized enzyme showed shift in optimum pH of 0.4 units and the activity yield of the immobilized α-galactosidase was found to be 76%. Immobilized enzyme was used to reduce RFOs in soymilk. In batch reaction after 12 h incubation soluble and immobilized enzyme showed 92 and 83% reduction of RFOs in soymilk, respectively. In repeated batch experiments immobilized enzyme showed 64% of its hydrolyzing activity after 6th cycle. PVA-immobilized α-galactosidase in fluidized bed reactor showed highest reduction (92%) of RFOs at a flow rate of 30 mL/h. The results of this study are interesting for their use in food processing industry.     

Nanofibers from blends of polyvinyl alcohol and polyhydroxy butyrate as potential scaffold material for tissue engineering of skin

Nanofibers were prepared by electrospinning from pure polyvinyl alcohol (PVA), polyhydroxybutyrate (PHB), and their blends. Miscibility and morphology of both polymers in the nanofiber blends were studied by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC), revealing that PVA and PHB were miscible with good compatibility. DSC also revealed suppression of crystallinity of PHB in the blend nanofibers with increasing proportion of PVA. The hydrolytic degradation of PHB was accelerated with increasing PVA fraction. Cell culture experiments with a human keratinocyte cell line (HaCaT) and dermal fibroblast on the electrospun PHB and PVA/PHB blend nanofibers showed maximum adhesion and proliferation on pure PHB. However, the addition of 5 wt % PVA to PHB inhibited growth of HaCaT cells but not of fibroblasts. On the contrary, adhesion and proliferation of HaCaT cells were promoted on PVA/PHB (50/50) fibers, which inhibited growth of fibroblasts.     

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs

Summary The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3-azido-3-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mm) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent K _m and V _max revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest. Offprint requests to: J. Magdalou     

锦鸡儿属灌木阻止干旱草地沙漠化生态过程

纵观全球沙漠化的研究进展,从宏观上研究沙漠化现状和后果的报道较多,对沙漠化内在过程的综合研究较少,灌木在阻止干旱区草原沙漠化中的生态作用缺乏研究。研究假设：在干旱地区,灌木的存在提高土壤营养水平,提升土壤保水能力,促进林下植物和土壤生物群落,进而减轻风蚀和固结土壤,这些相互联系的生态过程共同阻止了草原沙漠化。为了验证上述假设,我们以内蒙古阿拉善地区的荒漠化草原为样地,研究锦鸡儿属灌木的固土能力、提高土壤保水能力、改善土壤营养能力和促进生物群落能力,从生物、土壤、水分和沙物质运动等多过程来探讨锦鸡儿属灌木在阻止草原沙漠化中的作用。目的是弄清灌木阻止干旱草地沙漠化的生态过程。在阿拉善荒漠化草原区,锦鸡儿属灌木盖度大约为12.40%。在100 m²的土地上现有灌木固土2.01 m³,固土效率为167 cm³/g鲜重,每年灌木生长可以增加固土能力0.264 m³/100 m²土地。锦鸡儿属灌木提高了土壤湿度(灌丛效应SE=0.120)和土壤对雨水的保持能力(SE 0.155),改善了土...     

Immobilization of amyloglucosidase from SSF of Aspergillus niger by crosslinked enzyme aggregate onto magnetic nanoparticles using minimum amount of carrier and characterizations

Immobilizations of enzymes are done for operational stability, recovery and re-use of the enzymes and easy separation of products. Amyloglucosidase (AMG) obtained from solid state fermentation (SSF) of Aspergillus niger was directly immobilized by novel technique of crosslinked enzyme aggregate onto magnetic nanoparticles. AMG was covalently linked to the magnetic nanoparticle (MNP) to form a monolayer of AMG (MNP–AMG), followed by crosslinked aggregates with free AMG (which was not immobilized) to yield MNP with high enzyme loading (MNP–AMG_n). Under optimized conditions, very high recovery (92.8%) of enzyme activity was obtained in MNP–AMG_n using 14 times less carrier compared to the quantity of carrier required by conventional method. MNP–AMG_n showed enhanced affinity for substrate, thermal stability, storage stability and reusability.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Immobilization of laccase by encapsulation in a sol-gel matrix and its characterization and use for the removal of estrogens

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol-gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2-22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5-17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10-30% and tolerance to different inactivating agents (NaN(3) , ZnCl(2) , CoCl(2) , CaCl(2) , methanol, and acetone) was 20-40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed-bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10-15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady-state conditions. In addition, a 63% decrease in estrogenic activity was detected.     

Conjugation of heparin into carboxylated pullulan derivatives as an extracellular matrix for endothelial cell culture

A carboxylated pullulan, for use as a structural material for a number of tissue engineering applications, was synthesized and conjugated with heparin. By immobilization of heparin to pullulan, endothelial cells (ECs) attached on the heparin-conjugated pullulan were more aggregated than when attached to other pullulan derivatives. Attachments were 50, 45, 49, and 90% for a polystyrene dish, pullulan acetate, carboxylated pullulan, and heparin-conjugated pullulan, respectively. Heparin-conjugated pullulan inhibited the proliferation of smooth muscle cells (SMCs) in vitro. Heparin-conjugated pullulan material can thus be used for the proliferation of vascular ECs and to inhibit the proliferation of SMCs.