Immobilization of laccase on itaconic acid grafted and Cu(II) ion chelated chitosan membrane for bioremediation of hazardous materials

BACKGROUND: Chitosan membranes were formed through a phase inversion technique and then cross‐linked with epichlorohydrin (CHX). Heterogeneous graft copolymerization of itaconic acid (IA) onto membrane was carried out with different monomer concentrations (CHX‐g‐p(IA)). The membrane properties such as equilibrium swelling ratio, porosity, and contact angle were measured, together with analysis by scanning electron microscopy (SEM), energy dispersive analysis of X‐rays (EDAX), atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy. RESULTS: The Cu(II) ion incorporated membranes (i.e. CHX‐g‐p(IA)‐Cu(II)) were used for reversible immobilization of laccase using CHX‐g‐p(IA) membrane as a control system. Maximum laccase adsorption capacities of the CHX‐g‐p(IA) and CHX‐g‐p(IA)‐Cu(II) membranes (with 9.7% grafting yield) were found to be 6.3 and 17.6 mg mL^?1 membrane at pH 4.0 and 6.0, respectively. The K_m value for immobilized laccase on CHX‐g‐p(IA)‐Cu(II) (4.16 × 10^?2 mmol L^?1) was 2.11‐fold higher than that of free enzyme (1.97 × 10^?2 mmol L^?1). Finally, the immobilized laccase was used in a batch system for degradation of three different dyes (Reactive Black 5, RB5; Cibacron Blue F3GA, CB; and Methyl Orange, MO). The immobilized laccase on CHX‐g‐p(IA)‐Cu(II) membrane was more effective for removal of MO dye than removal of CB and RB5 dyes. CONCLUSION: Flexibility of the enzyme immobilized grafted polymer chains is expected to provide easy reaction conditions without diffusion limitation for substrate dye molecules and their products. The support described, prepared from green chemicals, can be used for the immobilization of industrially important enzymes. Copyright © 2012 Society of Chemical Industry     

Continuous biotransformation of pyrogallol to purpurogallin using cross‐linked enzyme crystals of laccase as catalyst in a packed‐bed reactor

Cross‐linked enzyme crystals (CLEC) of laccase were prepared by crystallizing laccase with 75% (NH₄)₂SO₄ and cross‐linking using 1.5% glutaraldehyde. The cross‐linked enzyme crystals were further coated with 1 mmol L^?1 β‐cyclodextrin by lyophilization. The lyophilized enzyme crystals were used as such for the biotransformation of pyrogallol to purpurogallin in a packed‐bed reactor. The maximum conversion (76.28%) was obtained with 3 mmol L^?1 pyrogallol at a residence time of 7.1 s. The maximum productivity (269.03 g L^?1 h^?1) of purpurogallin was obtained with 5 mmol L^?1 pyrogallol at a residence time of 3.5 s. The productivity was found to be 261.14 g L^?1 h^?1 and 251.1 g L^?1 h^?1 when concentrations of 3 mmol L^?1 and 7 mmol L^?1 respectively were used. The reaction rate of purpurogallin synthesis was maximum (2241.94 mg purpurogallin mg^?1 CLEC h^?1) at a residence time of 3.5 s, when 5 mmol L^?1 pyrogallol was used as the substrate. The catalyst to product ratio calculated for the present biotransformation was 1:2241. The CLEC laccase had very high stability in reuse and even after 650 h of continuous use, the enzyme did not lose its activity. Copyright © 2006 Society of Chemical Industry     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

Preparation of chitosan‐coated magnetite nanoparticles and application for immobilization of laccase

In this study, immobilization of laccase (L) enzyme on magnetite (Fe₃O₄) nanoparticles was achieved, so that the immobilized enzyme could be used repeatedly. For this purpose, Fe₃O₄ nanoparticles were coated and functionalized with chitosan (CS) and laccase from Trametes versicolor was immobilized onto chitosan‐coated magnetic nanoparticles (Fe₃O₄‐CS) by adsorption or covalent binding after activating the hydroxyl groups of chitosan with carbodiimide (EDAC) or cyanuric chloride (CC). For chitosan‐coated magnetic nanoparticles, the thickness of CS layer was estimated as 1.0–4.8 nm by TEM, isoelectric point was detected as 6.86 by zeta (ζ)‐potential measurements, and the saturation magnetization was determined as 25.2 emu g^?1 by VSM, indicating that these nanoparticles were almost superparamagnetic. For free laccase and immobilized laccase systems, the optimum pH, temperature, and kinetic parameters were investigated; and the change of the activity against repeated use of the immobilized systems were examined. The results indicated that all immobilized systems retained more than 71% of their initial activity at the end of 30 batch uses. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Immobilization of Trametes versicolor laccase on different PGMA‐based polymeric microspheres using response surface methodology: Optimization of conditions

Enzymatic immobilization is a versatile alternative to improve enzyme stability and enable its reuse. In this study, the change in the immobilized enzyme properties due to the type of functional group on the carrier was evaluated. For that, monodisperse polymeric microspheres with two functional groups widely used in laccase immobilization—oxirane [poly(glycidyl methacrylate) (PGMA)] and hydrazide—were synthesized by dispersion polymerization to covalently immobilize the laccase Trametes versicolor. Using a response surface methodology, laccase immobilization was optimized for each microsphere type. As a result, laccase immobilization on PGMA carriers appears to broaden the pH and temperature ranges, storage stability, and reusability compared to free and hydrazide enzymes. These aforementioned characteristics indicate that PGMA microspheres could act as an ideal support for enzyme immobilization in biotechnological applications. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45249.     

Preparation of Cu(II)‐chelated poly(vinyl alcohol) nanofibrous membranes for catalase immobilization

Poly(vinyl alcohol) (PVA) nanofibers were formed by electrospinning. Metal chelated nanofibrous membranes were prepared by reaction between Cu(II) solution and nanofibers, and which were used as the matrix for catalases immobilization. The constants of Cu(II) adsorption and properties of immobilized catalases were studied in this work. The Cu(II) concentration was determined by atomic absorption spectrophotometer (AAS), the immobilized enzymes were confirmed by the Fourier transform infrared spectroscopy (FTIR), and the amounts of immobilized enzymes were determined by the method of Bradford on an ultraviolet spectrophotometer (UV). Adsorption of Cu(II) onto PVA nanofibers was studied by the Langmuir isothermal adsorption model. The maximum amount of coordinated Cu(II) (q_m) was 2.1 mmol g⁻¹ (dry fiber), and the binding constant (K_l) was 0.1166 L mmol⁻¹. The immobilized catalases showed better resistance to pH and temperature inactivation than that of free form, and the thermal and storage stabilities of immobilized catalases were higher than that of free catalases. Kinetic parameters were analyzed for both immobilized and free catalases. The value of V_max (8425.8 μmol mg⁻¹) for the immobilized catalases was smaller than that of the free catalases (10153.6 μmol mg⁻¹), while the K_m for the immobilized catalases were larger. It was also found that the immobilized catalases had a high affinity with substrate, which demonstrated that the potential of PVA‐Cu(II) chelated nanofibrous membranes applied to enzyme immobilization and biosensors. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

New generation polymeric nanospheres for catalase immobilization

Monosize poly(2‐hydroxyethyl methacrylate‐co‐N‐methacryloly‐L ‐histidinemethylester) [mon‐poly(HEMA‐MAH)] nanospheres were prepared via surfactant‐free emulsion polymerization method. L ‐Histidine groups of the mon‐poly(HEMA‐MAH) nanospheres were chelated with Fe³⁺ ions. Mon‐poly(HEMA‐MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, proton NMR, and scanning electron microscopy. Particle size of the mon‐poly(HEMA‐MAH) nanospheres was measured by Zeta Sizer. Elemental analysis of MAH for nitrogen was estimated as 0.94 mmol/g polymer. The catalase immobilized onto the mon‐poly(HEMA‐MAH)–Fe³⁺ nanospheres resulted in increasing the enzyme stability with time. Optimum operational temperature for both immobilized preparations was the same, and the temperature profiles of the immobilized preparations were significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed on the mon‐poly(HEMA‐MAH)–Fe³⁺ nanospheres without loss of adsorption capacity or enzymic activity. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Activity of Laccase Immobilized on TiO2-Montmorillonite Complexes

The TiO₂-montmorillonite (TiO₂-MMT) complex was prepared by blending TiO₂ sol and MMT with certain ratio, and its properties as an enzyme immobilization support were investigated. The pristine MMT and TiO₂-MMT calcined at 800 °C (TiO₂-MMT800) were used for comparison to better understand the immobilization mechanism. The structures of the pristine MMT, TiO₂-MMT, and TiO₂-MMT800 were examined by HR-TEM, XRD and BET. SEM was employed to study different morphologies before and after laccase immobilization. Activity and kinetic parameters of the immobilized laccase were also determined. It was found that the TiO₂ nanoparticles were successfully introduced into the MMT layer structure, and this intercalation enlarged the “d value” of two adjacent MMT layers and increased the surface area, while the calcination process led to a complete collapse of the MMT layers. SEM results showed that the clays were well coated with adsorbed enzymes. The study of laccase activity revealed that the optimum pH and temperature were pH = 3 and 60 °C, respectively. In addition, the storage stability for the immobilized laccase was satisfactory. The kinetic properties indicated that laccase immobilized on TiO₂-MMT complexes had a good affinity to the substrate. It has been proved that TiO₂-MMT complex is a good candidate for enzyme immobilization.     

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     

Trametes versicolor laccase immobilized poly(glycidyl methacrylate) based cryogels for phenol degradation from aqueous media

This study aims removal of phenols in wastewater by enzymatic oxidation method. In this study, Trametes versicolor laccase was covalently immobilized onto a cryogel matrix by the nucleophilic attack of amino groups of laccase to epoxy groups of matrix. Glycidyl methacrylate was chosen as functional monomer to prepare poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [p(HEMA‐co‐GMA)] cryogels. The enzyme immobilized matrix was characterized by FTIR, SEM, and swelling tests. The effect of pH, reaction time, temperature, substrate concentration, enzyme concentration, and storage period on immobilized enzyme activity was determined and compared with those of free enzyme. The model substrate was 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS). Lineweaver‐Burk plots were used to calculate K_m and V_m values. K_m values were 165.1 and 156.0 µM while V_m values were 55.2 µM min^?1 and 1.57 µM min^?1 for free and immobilized laccase, respectively. Immobilized enzyme was determined to retain 82.5% and 72.0% of the original activity, respectively, after 6 consecutive use and storage period of 4 weeks. The free enzyme retained only 24.0% of its original activity following the same storage period. Lastly, decomposition products resulting from enzymatic oxidation of a model phenolic compound (3,5‐dinitrosalicylic acid) in aqueous solution were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41981.     

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

Poly(N‐isopropylacrylamide‐co‐acrylic acid) (P(NIPAM‐co‐AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 ± 20 mg g^?1 and the activity yield of immobilized trypsin was 155% ± 3%. The maximum activity (V_max) and Michaelis constant (K_m) of immobilized enzyme were found to be 0.74 μM s^?1 and 0.54 mM, respectively. The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme‐immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43343.     

Performance of different immobilized‐cell systems to efficiently produce ethanol from whey: fluidized batch,packed‐bed and fluidized continuous bioreactors

BACKGROUND: The bioconversion of whey into ethanol by immobilized Kluyveromyces marxianus in packed‐bed and fluidized bioreactors is described. Both batch and continuous cultures were analyzed using three different strains of K. marxianus and the effect of the operating mode, temperature, and dilution rates (D) were investigated. RESULTS: All immobilized strains of K. marxianus (CBS 6556, CCT 4086, and CCT 2653) produced similar high yields of ethanol (0.44 ± 0.01 g EtOH g^?1 sugar). Significant variations of conversion efficiencies (66.1 to 83.3%) and ethanol productivities (0.78 to 0.96 g L^?1 h^?1) were observed in the experiments with strain K. marxianus CBS 6556 at different temperatures. High yields of ethanol were obtained in fluidized and packed‐bed bioreactors continuous cultures at different D (0.1 to 0.3 h^?1), with the highest productivity (3.5 g L^?1 h^?1) observed for D = 0.3 h^?1 in the fluidized bioreactor (87% of the maximal theoretical conversion), whereas the highest ethanol concentration in the streaming effluent (28 g L^?1) was obtained for D = 0.1 h^?1. Electronic micrographs of the gel beads showed efficient cell immobilization. CONCLUSION: Batch and continuous cultivations of immobilized K. marxianus in fluidized and packed‐bed bioreactors enable high yields and productivities of ethanol from whey. Copyright © 2012 Society of Chemical Industry     

Immobilization of Myceliophthora thermophila laccase on poly(glycidyl methacrylate) microspheres enhances the degradation of azinphos-methyl

Laccase enzymes are multicopper oxidases capable of oxidizing different compounds. However, to be able to use this biocatalyst for industrial applications, their immobilization is needed. The present work investigates the immobilization of Myceliophthora thermophila laccase (MtL) on monodisperse microspheres of poly(glycidyl methacrylate) (PGMA) to be used in azinphos-methyl degradation. The immobilization was optimized to achieve the highest activity of the immobilized enzyme. As result, the protein load obtained was 2.5 mg protein g⁻¹ carrier (35 U g⁻¹ of carrier). The immobilized enzymes showed a broadened pH and temperature range of optimum activity and significantly improved the storage and operational stability. Finally, the complete degradation of azinphos-methyl using immobilized laccase was achieved after 1 h of reaction. The collected data indicate that the immobilization of MtL on PGMA microspheres is an excellent alternative to improve biochemical properties in the enzyme and to allow their efficient use in pesticides degradation. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47417.     

Decolorisation of synthetic dyes by crude laccase from Rigidoporus lignosus W1

BACKGROUND: Effluents from the dyeing process containing dyes are difficult to degrade biologically. Therefore enzymatic dye decolorisation has received considerable attention recently. In this study the dye decolorisation potential of crude laccase from the white rot fungus Rigidoporus lignosus W1 was demonstrated on an anthraquinone dye, Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye, malachite green (MG). Effects of pH, temperature and ionic strength on laccase activity and decolorisation efficiency were investigated. RESULTS: Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE confirmed the decolorisation ability of the crude enzyme on RBBR and MG. A single laccase with a molecular mass of about 40 kDa appeared to be involved in the process. Efficient enzymatic decolorisation without redox mediator was achieved, with 39 mg L^?1 MG being removed within 2 h and 160 mg L^?1 RBBR within 1 h at 60 °C and pH 4.5. Although the laccase activity was inhibited in the presence of NaCl, it was renatured gradually in low concentrations of NaCl (<0.8 mol L^?1), resulting in unusual dye decolorisation kinetics. Surprisingly, unusual storage stability at alkaline pH was observed, with the laccase activity being enhanced 1.5–2‐fold after 3 h of incubation. CONCLUSION: Crude laccase with unusual storage stability from the fungus R. lignosus W1 showed excellent decolorisation ability on RBBR and MG without redox mediator. This laccase would seem to be a good candidate for application in dye decolorisation and textile effluent biotreatment. Copyright © 2008 Society of Chemical Industry     

One‐Pot Lipase Entrapment Within Silica Particles to Prepare a Stable and Reusable Biocatalyst for Transesterification

In order to enhance the reusability, Rhizomucor miehei lipase was entrapped in a single step within silica particles having an oleic acid core (RML@SiO₂). Characterization of RML@SiO₂ by scanning and transmission electron microscopy and Fourier transform infrared studies supported the lipase immobilization within silica particles. The immobilized enzyme was employed for transesterification of cottonseed oil with methanol and ethanol. Under the optimum reaction conditions of a methanol‐to‐oil molar ratio of 12:1 or ethanol‐to‐oil molar ratio of 15:1, stirring speed of 250 revolutions/min (flask radius = 3 cm), reaction temperature of 40 °C, and biocatalyst concentration of 5 wt% (with respect to oil), more than 98 % alkyl ester yield was achieved in 16 and 24 h of reaction duration in case of methanolysis and ethanolysis, respectively. The immobilized enzyme did not require any buffer solution or organic solvent for optimum activity; hence, the produced biodiesel and glycerol were free from metal ion or organic molecule contamination. The activation energies for the immobilized enzyme‐catalyzed ethanolysis and methanolysis were found to be 34.9 ± 1.6 and 19.7 ± 1.8 kJ mol^?1, respectively. The immobilized enzyme was recovered from the reaction mixture and reused in 12 successive runs without significant loss of activity. Additionally, RML@SiO₂ demonstrated better reusability as well as stability in comparison to the native enzyme as the former did not lose the activity even upon storage at room temperature (25–30 °C) over an 8‐month period.     

Towards the design of non-amyloidogenic proteins

Glucose oxidase was immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two methods: by covalent bonding through epichlorohydrin and by entrapment between pHEMA membranes. The highest immobilization efficiency was found to be 17.4% and 93.7% for the covalent bonding and entrapment, respectively. The K_m values were 5.9 mmol dm^?3, 8.8 mmol dm^?3 and 12.4 mmol dm^?3 for free, bound and entrapped enzyme, respectively. The V_max values were 0.071 mmol dm^?3 min^?1, 0.067 mmol dm^?3 min^?1 and 0.056 mmol dm^?3 min^?1 for free, bound and entrapped enzyme. When the medium was saturated with oxygen, K_m was not significantly altered but V_max was. The optimum pH values for the free, covalently-bound and entrapped enzyme were determined to be 5, 6, and 7, respectively. The optimum temperature was 30°C for free or covalently-bound enzyme but 35°C for entrapped enzyme. The deactivation constant for bound enzyme was determined as 1.7 × 10^?4 min^?1 and 6.9 × 10^?4 min^?1 for the entrapped enzyme.     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Study of a newly isolated thermophilic bacterium capable of Kuhemond heavy crude oil and dibenzothiophene biodesulfurization following 4S pathway at 60 °C

BACKGROUND: To meet stringent emission standards stipulated by regulatory agencies, the oil industry is required to bring down the sulfur content in fuels. As some compounds cannot be desulfurized by existing desulfurizing processes (such as hydrodesulfurization, HDS) biodesulfurization has become an interesting topic for researchers. Most of the isolated biodesulfurizing microorganisms are capable of desulfurization of refined products whose predominant sulfur species are dibenzothiophenes so biocatalyst development is still needed to desulfurize the spectrum of sulfur‐bearing compounds present in whole crude. RESULTS: The first desulfurizing bacterium active at 60 °C has been isolated, which reduces DBT concentration from 2 mmol L^?1 to 0.1 mmol L^?1 after 95 h, following the 4S pathway. Its DBT desulfurization pattern was represented by the Michaelis‐Menten equation. Various parameters such as V_max, K_m, µ_m, K_s and maximum specific DBT desulfurization rate were calculated which are 0.092 mmol L^?1 h^?1, 3.554 mmol L^?1, 0.157 h^?1, 3.722 mmol L^?1 and 0.192 mmol L^?1 DBT g^?1 DCW (dry cell weight) h^?1, respectively. It can desulfurize 50% of the sulfur content of Kuhemond heavy crude oil (KHC oil) with an initial sulfur content of 7.6%wt in 6 days. Its maximum specific desulfurization rate for KHC oil is equivalent to 0.005 g sulfur g^?1 DCW h^?1. The bacterium was isolated during a heavy crude oil biodesulfurization project initiated by PEDEC, a subsidiary of National Iranian Oil Company. CONCLUSION: The KHC oil sulfur removal efficiency of the bacterium is approximately five times that of BBRC‐9016 bacterium. It removes sulfur selectively without using sulfur‐containing compounds as its carbon source. By applying various media during its isolation, the probability of screening the correct microorganism is increased. Copyright © 2008 Society of Chemical Industry     

Modulated gluconic acid production from immobilized cells of Aspergillus niger ORS‐4.410 utilizing grape must

BACKGROUND: Gluconic acid (GA) production by immobilized cells of mutant Aspergillus niger ORS‐4.410 on polyurethane sponge (PUS) and calcium‐alginate (Ca‐alginate) was evaluated in repeated batches of solid state surface fermentation (SSF) and submerged fermentation (SmF) conditions, respectively, utilizing rectified grape must as carbon source. RESULTS: The passive immobilization of cells in fermentation medium solid support of having 0.4 cm³ cube size, 4% spore suspension, 0.6 g inoculum of PUS immobilized cells at 32 °C and 2.0 L min^?1 resulted in the maximum GA production (88.16 g L^?1) with a 92.8% yield, while the Ca‐alginate matrix with a 0.5 cm diameter bead size, 2–3% spore suspension, 15 g inoculum at 34 °C and 150 rpm agitation speed revealed 67.19 g L^?1 GA with a 85.2% yield. Repeated use of PUS showed higher levels of GA (110.94 g L^?1) in the third–fourth fermentation cycles with 95–98% yield and 22.50 g L^?1 d^?1 productivity under SSF that was 2.5‐fold higher than the productivity obtained from a typical fermentation cycle, and 54% greater than the productivity obtained with repetitive use of Ca‐alginate immobilized cells of A. niger under SmF. CONCLUSION: Using immobilized cells of A. niger in PUS, the rectified form of grape must can be utilized for GA production as an alternative source of carbohydrate by replacing the conventional fermentation conditions. Copyright © 2008 Society of Chemical Industry     

Catalytic potential of a poly(AAc‐co‐HPMA‐cl MBAm)‐matrix‐immobilized lipase from a thermotolerant Pseudomonas aeruginosa MTCC‐4713

A purified alkaline thermo‐tolerant lipase from Pseudomonas aeruginosa MTCC‐4713 was immobilized on a series of five noble weakly hydrophilic poly(AAc‐co‐HPMA‐cl MBAm) hydrogels. The hydrogel synthesized by copolymerizing acrylic acid and 2‐hydroxy propyl methacrylate in a ratio of 5 : 1 (HG_5:1 matrix) showed maximum binding efficiency for lipase (95.3%, specific activity 1.96 IU mg^?1 of protein). The HG_5:1 immobilized lipase was evaluated for its hydrolytic potential towards p‐NPP by studying the effect of various physical parameters and salt‐ions. The immobilized lipase was highly stable and retained ～92% of its original hydrolytic activity after fifth cycle of reuse for hydrolysis of p‐nitrophenyl palmitate at pH 7.5 and temperature 55°C. However, when the effect of pH and temperature was studied on free and bound lipase, the HG_5:1 immobilized lipase exhibited a shift in optima for pH and temperature from pH 7.5 and 55°C to 8.5 and 65°C in free and immobilized lipase, respectively. At 1 mM concentration, Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions promoted and Co²⁺ ions inhibited the hydrolytic activities of free as well as immobilized lipase. However, exposure of either free or immobilized lipase to any of these ions at 5 mM concentration strongly increased the hydrolysis of p‐NPP (by ～3–4 times) in comparison to the biocatalysts not exposed to any of the salt ions. The study concluded that HG_5:1 matrix efficiently immobilized lipase of P. aeruginosa MTCC‐4713, improved the stability of the immobilized biocatalyst towards a higher pH and temperature than the free enzyme and interacted with Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions to promote rapid hydrolysis of the substrate (p‐NPP). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4252–4259, 2006