Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers

Extracellular laccases produced by three different wood-rotting fungi, Cerrena unicolor, Heterobasidion annosum and Trametes versicolor, were immobilized via covalent bonds formation on DEAE-Granocel 500, CM-Granocel 500, and acrylic carriers. Out of the tested carriers, only the DEAE-Granocel 500, which was activated by divinyl sulphone appeared to be a suitable matrix for the expression of enzymic activity. Only one laccase of all the tested enzymes produced by C. unicolor showed the best binding to the carrier and a satisfactory enzymic activity. The immobilized laccase exhibited the highest enzymic activity at pH 5.2 and it was more resistant to thermal denaturation than the native enzyme. At 90 °C, it retained 75% activity compared to the free enzyme. It was also more stable during storage at 4 °C: after 4 months the immobilized laccase retained 98% of initial activity. Immobilized C. unicolor laccase was active in 10–60% concentration of methanol, acetone, isopropanol or acetonitrile. The best enzymic activity was observed in 20% solution of acetonitrile in buffer.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Activity and storage stability of immobilized glucose oxidase onto magnesium silicate

Glucose oxidase (GOD) was covalently immobilized onto florisil (magnesium silicate) carrier via glutaraldehyde. Immobilization conditions were optimized: the amount of initial GOD per grams of carrier as 5 mg, pH as 5.5, immobilization time as 120 min and temperature as 10 °C. Under the optimized reaction conditions activities of free and immobilized GOD were measured. Free and immobilized GOD samples were characterized with their kinetic parameters, and thermal and storage stabilities. K_M and V_max values were 68.2 mM and 435 U mg GOD⁻¹ for free and 259 mM and 217 U mg GOD⁻¹ for immobilized enzymes, respectively. Operational stability of the immobilized enzyme was also determined by using a stirred batch type column reactor. Immobilized GOD was retained 40% of its initial activity after 50 reuses. Storage stabilities of the immobilized GOD samples stored in the mediums with different relative humidity in the range of 0–100% were investigated during 2 months. The highest storage stability was determined for the samples stored in the medium of 60% relative humidity. Increased relative humidity from 0% to 60% caused increased storage stability of immobilized GODs, however, further increase in relative humidity from 80% to 100% caused a significant decrease in storage stability of samples.     

Glucose biosensor based on nano-SiO2 and "unprotected" Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Fast separation of bromelain by polyacrylic acid-bound iron oxide magnetic nanoparticles

The adsorption of bromelain from an aqueous solution by polyacrylic acid (PAA)-bound iron oxide magnetic nanoparticles was studied. The magnetic composite nanoparticles were shown to be efficient for the separation of bromelain. Except at pH <3, the adsorption of bromelain increased with the decrease in solution pH and reached almost 100% at pH 3–5. The adsorbed bromelain could be desorbed by the addition of KCl and complete desorption was achieved at pH 7 when [KCl]>0.6 M. The adsorption behaviour followed the Langmuir isotherm with a maximum adsorption amount of 0.476 mg/mg and a Langmuir adsorption equilibrium constant of 58.4 ml/mg at pH 4 and 0.1 M phosphate. In addition, it was notable that both the adsorption and desorption of bromelain were quite fast and could be completed in about 1 min due to the absence of internal diffusion resistance. Bromelain retained 87.4% activity after adsorption/desorption.     

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Hydrodynamic studies on chitosans in aqueous solution

Three commercial chitosans with a degree of acetylation of 25–30% were studied by light scattering (static and dynamic), analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), and capillary viscometry in 0.02 M acetate buffer/0.1 M NaCl, pH 4.5. The molecular masses obtained by sedimentation equilibrium measurements or sedimentation and diffusion coefficients according to the Svedberg equation agreed well or fairly well with those from static light scattering whereas the molecular masses calculated via the Scheraga–Mandelkern equation were found too low by almost 50%. The various Mark–Houwink type relationships suggested a nearly free-draining flexible worm-like chain. A prolate ellipsoid of revolution with an axial ratio a/b25 was shown to be a hydrodynamically equivalent body of the flexible worm-like chain that had been derived from static light scattering. The findings illustrate the fact that a hydrodynamically strongly asymmetric shape need not mean a strongly elongated shape of the molecules in reality.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Synthesis and characterization of cross-linked malonylchitosan microspheres for controlled release of acyclovir

The purpose of the present study was to obtain a polymeric system for delayed release of the drug acyclovir (ACV), which can be used for treatment of Herpes simplex and Varicella Zoster. The gelled chitosan (GCT) microspheres were obtained by coacervation-phase separation. They were treated with malonic acid to obtain malonylchitosan (MLCT) microspheres, which were characterized by, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (¹³C NMR), elemental analysis (CHN), thermogravimetric analysis (TG/DTG) and scanning electron microscopy (SEM). The drug was encapsulated in MLCT microspheres by a contact adsorption technique, and the final formulation (MLCT-ACV), was analyzed for loading efficiency, degree of swelling and in vitro release profiles. The results obtained support the N-substitution of malonyl groups in the MLCT microspheres. The loading efficiency increased with impregnation time and a major amount of drug was encapsulated after 24 h. The swelling rate was higher in acid pH. The median release time was 5.5 h in pH 1.2 and 6.8. The mechanism involved in release was non-Fickian (0.43 < n < 0.85, n = 0.8474) and Super Case II kinetics (n > 1, n = 1.0491) at pH 1.2 and 6.8, respectively.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Studies on the properties of immobilized urokinase: effects of pH and temperature

Human urokinase was immobilized on an ethylene vinyl acetate copolymer surface. Soluble urokinase showed its maximum activity at pH 8.5, while the immobilized enzyme was most active at pH 9.0. Apparently, the shift in optimal pH was due to the polyanionic nature of the carrier surface on which the enzyme was immobilized. Optimal temperatures of soluble urokinase and immobilized enzyme were identical, i.e., 37 degrees C. The stability of immobilized enzyme against thermal degradation was several times higher than that of the soluble enzyme. Its stability at higher temperatures is one of the main reasons for the clinical use of immobilized urokinase as an antithrombotic material.     

Novel magnetic microspheres of P (GMA-b-HEMA): preparation, lipase immobilization and enzymatic activity in two phases

Magnetic oleic-acid-coated Fe?O? nanoparticles were first introduced into 1, 1-diphenylethylene (DPE)-controlled radical polymerization system to prepare superparamagnetic microspheres for enzyme immobilization by two steps of polymerization. In the presence of DPE, glycidyl methacrylate, 2-hydroxyethyl methacrylate and methacryloxyethyl trimethyl ammonium chloride with charge were selected as copolymering monomers based on their reactive functional group and excellent biocompatibility which were suitable for immobilization of Candida rugosa lipase (CRL). The resulting magnetic microspheres were characterized by means of scanning electron microscope, Fourier transform infrared spectrum, thermogravimetric analysis and vibrating sample magnetometry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE analysis was also conducted to demonstrate whether CRL is covalently immobilized or only physically adsorbed. The results indicated that the polymerization was successfully carried out, and lipase was immobilized on the magnetic microspheres through ionic adsorption and covalent binding under mild conditions. The immobilized lipase exhibited high activity recovery (69.7%), better resistance to pH and temperature inactivation in aqueous phase, as well as superior reusability in nonaqueous phase. The data showed that the resulting carrier could hold an amphiphilic property.     

Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.