Immobilized Biocatalysts Research Articles

Cell wall invertase immobilisation within gelatin gel

Yeast cell wall invertase (CWI) was immobilised within 10% gelatin hydrogel. The result of entrapment was the complete immobilisation of all the added CWI. The activity of immobilised biocatalyst was 93±3U/g, with activity yield of 30%. The optimum pH was in range of 4.5–5.0 for free and immobilised biocatalyst. The optimum temperature was 60°C for both free and gelatin immobilised CWI. Immobilised CWI was more stable than free CWI above optimum activity temperatures. The Km of free and immobilised CWI was 35.10±2.99mM and 71.45±3.23mM, respectively. The Vmax values were estimated as 8.23±0.24mM/min and 0.121±0.002mM/min, respectively. Immobilised CWI was tested in a batch reactor using 50% sucrose (w/v). After 70 consecutive cycles gelatin immobilised CWI retained 75% of its original activity.

Continuous hydrolysis of pullulan using covalently immobilized pullulanase in a packed bed reactor

Pullulanase covalently immobilized on Duolite XAD761 has been used for developing a continuous flow reactor for the hydrolysis of pullulan. Packed bed reactor containing 34.10 U of immobilized biocatalyst with feeding of pullulan solution (0.44%, w/v) at a flow rate of 5 mL h −1 supported maximum pullulan hydrolysis at 60 °C. The reactor was run continuously for 32 days and immobilized biocatalyst lost half of its original activity after 31 days of continuous operation at 60 °C. The volumetric productivity and yield of reducing sugars were 3.38 ± 0.02 g L −1 h −1 and 4.40 ± 0.01 mg mL −1, respectively during the beginning of the hydrolysis. The developed immobilized biocatalyst has shown good operational and mechanical stability and can be successfully used for the hydrolysis of pullulan in a continuous system. Literature survey reveals no report on continuous hydrolysis of pullulan using whole cells or immobilized enzyme.

Immobilization and Stabilization of Xylanase by Multipoint Covalent Attachment on Agarose and on Chitosan Supports

Xylanases have important applications in industry. Immobilization and stabilization of enzymes may allow their reuse in many cycles of the reaction, decreasing the process costs. This work proposes the use of a rational approach to obtain immobilized commercial xylanase biocatalysts with optimized features. Xylanase NS50014 from Novozymes was characterized and immobilized on glyoxyl-agarose, agarose-glutaraldehyde, and agarose-amino-epoxy support and on differently activated chitosan supports: glutaraldehyde-chitosan, glyoxyl-chitosan, and epoxy-chitosan. Two different chitosan matrices were tested. The best chitosan derivative was epoxy-chitosan-xylanase, which presented 100% of immobilization yield and 64% of recovered activity. No significant increase on the thermal stability was observed for all the chitosan-enzyme derivatives. Immobilization on glyoxyl-agarose showed low yield immobilization and stabilization degrees of the obtained derivative. The low concentration of lysine groups in the enzyme molecule could explain these poor results. The protein was then chemically modified with ethylenediamine and immobilized on glyoxyl-agarose. The new enzyme derivatives were 40-fold more stable than the soluble, aminated, and dialyzed enzyme (70 degrees C, pH 7), with 100% of immobilization yield. Therefore, the increase of the number of amine groups in the enzyme surface was confirmed to be a good strategy to improve the properties of immobilized xylanase.

Formation of cross-linked glucose oxidase aggregates in mesocellular foams

Enzyme immobilization into solid mesoporous inorganic supports is a promising strategy to enable their use in continuous-flow fixed-bed reactors. In this study, the formation of cross-linked enzyme aggregates (CLEAs) of glucose oxidase (GOx) in the pores of mesocellular foams (MCFs) was investigated. The enzymes can enter the ultra-large cavities connected through the smaller windows, where their agglomeration and cross-linking with glutardialdehyde (GA) will take place. After cross-linking, the diameter of the CLEAs is larger than the diameter of the pore entrance and, thus, the enzymes are trapped in the pores of the support. By varying the experimental parameters, the optimum conditions for the preparation of active and stable immobilized biocatalysts were determined with respect to the resulting activity and to the enzyme loading. It is found that the preparation is preferably performed at pH 5.0, with a time delay between GA and GOx addition of 2 h and a nGA/nGOx-ratio of more than 400.

Optimisation of lab-scale continuous alcohol-free beer production

In order to study the formation and conversion of the most important flavour compounds, the real wort used in alcohol-free beer fermentation was mimicked by a complex model medium containing glucose, yeast extract, and selected aldehydes. The fermentation experiments were carried out in a continuously operating gas-lift reactor with brewing yeast immobilised on spent grains (brewing by-product). During the continuous experiment, parameters such as oxygen supply, residence time (Rt), and temperature (T) were varied to find the optimal conditions for the alcohol-free beer production. The formation of ethanol, higher alcohols (HA), esters (ES), as well as the reduction of aldehydes and consumption of glucose were observed. The results suggest that the process parameters represent a powerful tool in controlling the degree of fermentation and flavour formation brought about by immobilised biocatalyst. Subsequently, the optimised process parameters were used to produce real alcohol-free beer during continuous fermentation. The final product was compared with batch fermented alcohol-free beers using the methods of instrumental and sensorial analysis.

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

AbstractThe lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (aw) of 0.97 was similar to that of commercial immobilised lipases at aw values lower than 0.5. Thus, the biocatalyst was further used at an aw of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (>80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.

Ethanol production using Saccharomyces cerevisiae cells immobilised on corn stem ground tissue

Cell immobilisation in alcoholic fermentation has been extensively studied during the past few decades because of its technical and economical advantages over those of free cell systems. A biocatalyst was prepared by immobilising a commercial Saccharomyces cerevisiae strain (baker yeast) on corn stem ground tissue for use in alcoholic fermentation. For this purpose, the yeast cells were submitted to the batch tests 'in situ' adsorption onto pieces of the corn stem ground tissue. Cells immobilisation was analysed by optical microscopy. It was determined that the addition of the corn stem ground tissue led to an increase of the pH value, total dissolved salts content, and sugar content in fermentation medium. The addition of 5 and 10g of the corn stem ground tissue per liter of medium, increased ethanol yield, decreased amount of residual sugar and the cells immobilisation was effective. Corn stem is one of the abundant, available, inexpensive, stable, reusable, nontoxic celulosic biomaterial with high porosity, which facilitates the transmission of substrates and products between carrier and medium. The prepared immobilised biocatalyst showed higher fermentation activity than free cells. The results indicate that corn stem might be an interesting support for yeast cell immobilisation, and also a cheap alternative recourse of mineral components with possibility of application for improving ethanol productivities.

Laccase from Pleurotus sajor-caju on functionalised SBA-15 mesoporous silica: Immobilisation and use for the oxidation of phenolic compounds

Laccase from Pleurotus sajor-caju was immobilised on functionalised SBA-15 mesoporous silica. The immobilisation process reached the equilibrium after about 100 min. In order to study the effect of loading ( L) on activity of the immobilised laccase, the adsorption isotherm was built and the activity of the corresponding immobilised biocatalysts was determined. The activity of the immobilised preparations reached a maximum at L = 217 kU g SBA-15 −1, whereas higher loadings gave rise to a less-efficient biocatalyst. The immobilised laccase was used for the oxidation of a mixture of four phenolic compounds (protocatechuic acid, ferulic acid, sinapic acid and caffeic acid) chosen among those present in olive mill wastewaters (OMWs). These compounds determine the phytotoxicity of OMWs. Different reaction rates were observed for the oxidation of the examined phenolic compounds. The biocatalyst was recycled and a conversion of 84 mol% at the 10th reuse and of about 60 mol% after the 14th reuse was obtained. In conclusion, the laccase immobilised on SBA-15 is a potential biocatalyst for bioremediation of OMWs, which is an important environmental problem in the regions around the Mediterranean Sea.

The adsorption of multimeric enzymes on very lowly activated supports involves more enzyme subunits: Stabilization of a glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports

Glutamate dehydrogenase (GDH) from Thermus thermophilus is a homotrimeric enzyme that tends to dissociate at acidic pH values. GDH is readily adsorbed on highly activated anionic exchangers (HAAE), but hardly adsorbed on lowly activated supports (LAAE) or on highly activated epoxy supports. When using amino-epoxy supports, GDH immobilized on HAAE-epoxy and more slowly on LAAE-epoxy supports. Both immobilized biocatalysts were incubated at pH 10 for different times to increase the multipoint covalent attachment. LAAE-epoxy-GDH was stable at pH 4 and 25 °C, the enzyme stability did not depend on the enzyme concentration and did not release any subunit to the supernatant, in opposition to the results obtained using HAAE-epoxy supports. The general application of this strategy to stabilize multimeric enzymes was verified by immobilizing a crude protein extract. It seems that proteins adsorb on LAAE by the larger region of their surface (that is the one that involves the highest number of enzyme subunits), since it is the only area large enough to permit a multipoint ionic exchange on this LAAE. On the contrary, using HAAE, some proteins may become adsorbed by clusters that were rich in anionic groups and located in a corner of the multimer, involving only some of the subunits in the enzyme immobilization. That way, a careful design of the design of the support permits to take full advantage of the immobilization on heterofunctional supports.

Accelerated Biocatalyst Stability Testing for Process Optimization

The deactivation of protein biocatalysts even at relatively low temperatures is one of the principal drawbacks to their use. To aid in the development of novel biocatalysts, we have derived an equation for both time- and temperature-dependent activity of the biocatalyst based on known concepts such as transition state theory and the Lumry-Eyring model. We then derived an analytical solution for the total turnover number (ttn), under isothermal operation, as a function of the catalytic constant kcat, the unfolding equilibrium constant K, and the intrinsic first-order deactivation rate constant(s) k(d,i). Employing an immobilized glucose isomerase biocatalyst in a CSTR and utilizing a linear temperature ramp beyond the Tm of the enzyme, we demonstrate an accelerated method for extracting the thermodynamic and kinetic constants describing the biocatalyst system. In addition, we demonstrate that the predicted biocatalyst behavior at different temperatures and reaction times is consistent with the experimental observations.

Comparative study of batch and continuous multi-stage fixed-bed tower (MFBT) bioreactor during wine-making using freeze-dried immobilized cells

A freeze-dried immobilized biocatalyst produced by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on gluten pellets and subsequent freeze-drying was used in a multistage fixed-bed tower (MFBT) bioreactor for batch and continuous wine-making. The MFBT bioreactor resulted in higher alcohol productivity compared to fermentations carried out in a packed bed (PB) bioreactor and showed an important operational stability and no decrease in activity, even at low fermentation temperature (5 °C) and after storage for 6 months at 4 °C. The production of amyl alcohols proved to be temperature dependent and was significantly reduced at low temperatures. Re-activation of the freeze-dried immobilized cells after storage for 6 months resulted in further decreased content of amyl alcohols. The SPME GC/MS analysis of volatile compounds revealed no significant differences in the wines produced by MFBT and PB bioreactors, while the preliminary sensory evaluation ascertained the overall improved quality of the produced wines. Potential industrial application of MFBT bioreactor is also assessed and discussed.

Immobilization of yeast cells in PVA particles for beer fermentation

In this study, the potential of application of non-aggressive LentiKat ® technique for brewer's yeast immobilization on polyvinyl alcohol was assessed. High cell loads of about 10 9 cells/ml were achieved by this procedure and immobilization procedure had no adverse effect on cell viability. The stability and activity of obtained immobilized biocatalyst was tested in the growth studies and fermentations. Immobilized cells exhibited high fermentation activity in both, laboratory and pilot-scale fermentations. In three successive gas-lift reactor fermentations the apparent attenuation of around 80% was reached after only 2 days, indicating good potential of immobilized cells for development of continuous primary beer fermentation. LentiKat ® particles showed high mechanical and fermentative stability, since they endured 30 days of operating time during 6-month period without significant change of cell activity, particle shape and particle size.

Immobilized biocatalysts for detoxification of neurotoxic organophosphorous compounds

New biocatalysts were developed using organophosphorus hydrolase (OPH, EC 3.1.8.1) with a polyhistidine tag at the N-terminus of the protein (His6-OPH). The use of His6-OPH together with previously developed approaches for the entrapment of cells into poly(vinyl alcohol) cryogels and covalent immobilization of enzymes into porous fabric materials, impregnated with chemically cross-linked chitosan sulphate gel, enabled dramatic improvement of catalytic characteristics against various organophosphorous compounds (OPCs; Paraoxon, Coumaphos, Methyl parathion, etc.). The polyhistidine tag of OPH was used to create a new immobilized biocatalyst using metal-chelating carriers, such as Ni2+-nitrilotriacetic acid-agarose and Co2+-iminodiacetic acid-polyacrylamide cryogel. The latter biocatalyst had high activity and stability for the continuous hydrolysis of OPCs.

Continuous removal of hydrogen peroxide with immobilised catalase for wastewater reuse

Hydrogen peroxide was continuously removed for wastewater reuse using an immobilised biocatalyst. A commercial catalase, which is an enzyme to decompose hydrogen peroxide to water and oxygen, was entrapped in chitosan beads. Hydrogen peroxide in aqueous solutions of varying pH, temperature and concentration was continuously removed through a reactor containing the catalase-entrapped chitosan beads at high efficiency for 24 h. Additional silicate coating of the chitosan beads resulted in significant improvements in the catalase performance under harsh conditions, which are often found in peroxide-based industrial processes. We expect that immobilisation of catalases can enhance their applicability for continuous degradation of hydrogen peroxide for wastewater reuse.

Transgalactosylation in a Water-Solvent Biphasic Reaction System with β-Galactosidase Displayed on the Surfaces of Bacillus subtilis Spores

The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that beta-galactosidase (beta-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 x 10(3) U/g (dry weight) of spores. The beta-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed beta-Gal, octyl-beta-D-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

AbstractA new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L(+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L−1) was reached under semi‐batch conditions. Potato starch (5–70 g L−1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry

Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

Design and characterisation of an enzyme system for inulin hydrolysis

The optimal conditions for inulin hydrolysis using a commercial inulinase preparation, either free or immobilised in activated Amberlite were established by factorial design and surface response methodology. The immobilised biocatalyst displayed highest activity at pH 5.5 and 50 °C, whereas the optimum pH for the free form was slightly more acidic (4.5), and the optimum temperature was a little higher (55 °C). The model system estimated optimal pH and temperature values of 5.4 and 52 °C for the immobilised system and 4.9 and 56 °C for the free system. Michaelis–Menten type kinetics adequately described both free and immobilised bioconversion systems, which were evaluated under the respective optimal pH and temperature conditions. The use of a non-linear regression method for the determination of the kinetic parameters provided a best fit to the experimental data, as compared to a conventional Lineweaver–Burk linearisation. The K m for inulin of the free biocatalyst was 153 g l −1 at 55 °C and pH 4.5, whereas the apparent K m for inulin of the immobilised biocatalyst was 108 g l −1 at pH 5.5 and 50 °C. The reutilisation of the immobilised biocatalyst throughout consecutive batches was evaluated. A significant decrease of enzyme activity was observed in the first two batches, after which the system exhibited significant stability. The low cost of the support, the stability of the immobilised biocatalyst towards pH and temperature and its high affinity for the substrate suggests its potential for inulin hydrolysis.

Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide

Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months.

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5–4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0–5.5-times higher compared to the immobilized form mainly due to the lower Km values. With constant 55–60% humidity the biocatalyst retained 77% and 67–70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.