Biocatalyst Beads Research Articles

Entrapment of enzyme aggregates in chitosan beads for aroma release in white wines

Glycosidases are enzymes involved in the cascade reactions leading to the release of aromatic compounds in white wines. However, the use of commercial soluble glycosidases is facing difficulties due to their fast inactivation, poor reaction control, low efficiency of enzyme use, and the presence of catalyst residues in the product. Co-immobilization as cross-linked enzyme aggregates (combi-CLEAs) is a sound alternative allowing the immobilization of enzymes in their own protein matrix, yielding highly stable and active biocatalysts. Notwithstanding, their micrometer sized particles limit their application in industrial processes. To overcome this, combi-CLEAs of β-D-glucosidase (βG) and α-L-arabinofuranosidase (ARA) were entrapped in polymeric chitosan beads. The effect of crosslinking reagents and crosslinking time on the specific activity and stability of combi-CLEAs was studied, and the best conditions for the entrapment of the combi-CLEAs in polymeric chitosan beads were determined varying the concentration of the chitosan solution and the pH of the gelation agent solution. The resulting biocatalyst beads (average diameter 1.24 mm), retained full activity after 91 days of incubation under winemaking conditions, having specific activities of 0.91 and 0.88 international units of activity per gram for βG and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines.

A new approach for describing and solving the reversible Briggs‐Haldane mechanism using immobilized enzyme

Recently immobilized enzymes have been widely used in industrial processes due to their outstanding advantages, such as high stability and recyclability; however, their kinetic behaviour is generally controlled by mass diffusion effects. Thus, in order to improve these enzymatic processes, a clear discernment between the kinetic and diffusion mechanisms that control the production of the metabolite require investigation. In practice, it is typical to establish apparent kinetics for immobilized enzyme operations, and the validity of the apparent kinetics is restricted to the studied cases. In this work, a new approach for mathematically describing the kinetic and diffusion mechanics in an immobilized biocatalyst bead is established, in which the fraction of residual enzymatic activity is included, and is defined as a measure of the active and available enzymes in the bead porous network. In addition, the diffusion and kinetic mechanisms are described by the effective diffusion coefficient and the free enzyme kinetics, since the porous network of the bead is assumed as the bioreaction volume. Therefore, free enzyme kinetics were determined from glucose to fructose bioconversion using a stirred tank reactor with free glucose‐isomerase, in which substrate and enzyme concentrations and temperature were varied. The fraction of residual enzymatic activity () and the effective diffusion coefficient () were obtained from the isomerization of glucose to fructose using a stirred tank reactor with immobilized glucose‐isomerase in calcium alginate beads at different substrate and enzyme concentrations. Finally, simulations were carried out to establish the bioreaction solid‐phase characteristics that most significantly influence productivity.

An Innovative Biocatalyst for Continuous 2G Ethanol Production from Xylo-Oligomers by Saccharomyces cerevisiae through Simultaneous Hydrolysis, Isomerization, and Fermentation (SHIF)

Many approaches have been considered aimed at ethanol production from the hemicellulosic fraction of biomass. However, the industrial implementation of this process has been hindered by some bottlenecks, one of the most important being the ease of contamination of the bioreactor by bacteria that metabolize xylose. This work focuses on overcoming this problem through the fermentation of xylulose (the xylose isomer) by native Saccharomyces cerevisiae using xylo-oligomers as substrate. A new concept of biocatalyst is proposed, containing xylanases and xylose isomerase (XI) covalently immobilized on chitosan, and co-encapsulated with industrial baker’s yeast in Ca-alginate gel spherical particles. Xylo-oligomers are hydrolyzed, xylose is isomerized, and finally xylulose is fermented to ethanol, all taking place simultaneously, in a process called simultaneous hydrolysis, isomerization, and fermentation (SHIF). Among several tested xylanases, Multifect CX XL A03139 was selected to compose the biocatalyst bead. Influences of pH, Ca2+, and Mg2+ concentrations on the isomerization step were assessed. Experiments of SHIF using birchwood xylan resulted in an ethanol yield of 0.39 g/g, (76% of the theoretical), selectivity of 3.12 gethanol/gxylitol, and ethanol productivity of 0.26 g/L/h.

Towards a green enantiomeric esterification of R/S-ketoprofen: A theoretical and experimental investigation

Methanol, ethanol, 1- and 2-propanol were used as reactants and solvents in the esterification of R/S-ketoprofen catalyzed with Novozym® 435. The interaction of the alcohols with Novozym® 435 was studied at a molecular level through various spectroscopic techniques and molecular modeling. The results evidenced the dissolution of the polymeric support, loss of active protein, strong adsorption of the alcohols, modification of the secondary structure of the protein and smoothing of the inner structure of the biocatalyst's beads upon extended contact with the alcohols. Nevertheless, none of those drawbacks influences the specific activity and enantiomeric excess toward S(+)-enantiomer that remain unaltered upon extended contact with ethanol, 1- and 2-propanol as acyl acceptors. However, theoretical calculations demonstrated that methanol introduces steric and electronic hindrance within the step of the coordination of the R/S-ketoprofen with the catalytic triad.

Simulation of Enzyme Catalysis in Calcium Alginate Beads

In the present study, a general mathematical model for a fixed bed–immobilized enzymereactor was developedto simulate the process of diffusion and reaction inside the biocatalyst particle. The modeling and simulation of starch hydrolysis using immobilized α–amylase was used as a model for this study. Corn starch hydrolysis was carried out at constant pH of 5.5 and temperature of 50°C. The substrate flowrate was ranging from 0.2 – 5.0 ml/min, substrate initial concentrations 1 to 100 g/L. α–amylase was immobilized on to calcium alginate hydro-gel beads of 2mm average diameter.In this work Michaelis–Menten kinetics has been considered. The effect of substrate flow rate (i.e. residence time) and initial concentration on intra-particle diffusion has been taking into consideration. The performance of the system is found to be affected by the substrate flow rate and initial concentrations. The reaction is controlled by the reaction rate. The model equation was a non-linear second order differential equation simulated based on the experimental data for steady state condition. The simulation was achieved numerically using FINITE ELEMENTS in MATLABSoftware package. The simulated results give satisfactory results for substrate and product concentration profile within the biocatalyst bead.

Investigation of the stability of Novozym® 435 in the production of biodiesel

The effect of the composition of the feedstock (free fatty acids and methanol), the presence or absence of moisture and the contact with biodiesel on the catalytic, enzymatic and physical stability of Novozym® 435 during enzymatic biodiesel production was studied. The continuous removal of moisture leads to the deactivation of the biocatalyst and favours the degradation of the polymethylmethacrylate (PMMA) that constitutes the support of the lipase B of Candida antarctica (CALB). Nevertheless, the presence of PMMA along with the active protein was detected in the reaction media regardless of the operating conditions. Surprisingly, biodiesel (without the presence of methanol) is able to diffuse inside the biocatalyst's beads producing its swelling, modifying the internal texture and also dissolving the polymeric matrix.

Simulation of Enzyme Catalysis in Calcium Alginate Beads

A general mathematical model for a fixed bed immobilized enzyme reactor was developed to simulate the process of diffusion and reaction inside the biocatalyst particle. The modeling and simulation of starch hydrolysis using immobilized α-amylase were used as a model for this study. Corn starch hydrolysis was carried out at a constant pH of 5.5 and temperature of 50°C. The substrate flow rate was ranging from 0.2 to 5.0 mL/min, substrate initial concentrations 1 to 100 g/L. α-amylase was immobilized on to calcium alginate hydrogel beads of 2 mm average diameter. In this work Michaelis-Menten kinetics have been considered. The effect of substrate flow rate (i.e., residence time) and initial concentration on intraparticle diffusion have been taken into consideration. The performance of the system is found to be affected by the substrate flow rate and initial concentrations. The reaction is controlled by the reaction rate. The model equation was a nonlinear second order differential equation simulated based on the experimental data for steady state condition. The simulation was achieved numerically using FINITE ELEMENTS in MATLAB software package. The simulated results give satisfactory results for substrate and product concentration profiles within the biocatalyst bead.

Esterification of R/S-ketoprofen with 2-propanol as reactant and solvent catalyzed by Novozym® 435 at selected conditions

Abstract The enzymatic esterification of R/S-ketoprofen with 2-propanol catalyzed with the commercial biocatalyst Novozym® 435 is addressed in this investigation. The low reaction rate registered in this reaction was investigated in terms of the effect of the alcohol on the physicochemical–enzymatic stability of the biocatalyst and the interaction of the substrates with the catalytic triad at a molecular level. The effect of contacting 2-propanol:H2O mixture on Novozym® 435 was investigated at 45 °C for an extended period of time (8 days). The mixture dissolves the polymethylmethacrylate (PMMA) that constitutes the support of the Candida antarctica B lipase (CALB). Additionally, the alcohol diffuses into the biocatalyst's beads remaining strongly adsorbed (the alcohol desorption is evidenced only upon heating at 187 °C) and altering the inner texture of the biocatalyst's beads. Additionally, 2-propanol modifies the secondary structure of the enzyme by decreasing the β-sheet contribution and increasing the β-turn structure. The molecular modeling of the interaction of R/S-ketoprofen and 2-propanol with the catalytic triad of the lipase provides evidences that the secondary alcohol exerts an important steric hindrance for the reaction to proceed.

Stereo-selective conversion of mandelonitrile to (R)-(−)-mandelic acid using immobilized cells of recombinant Escherichia coli

Immobilized cells of a recombinant Escherichia coli expressing nitrilase from Pseudomonas putida were used to catalyze the hydrolysis of mandelonitrile (2-hydroxy-2-phenylacetonitrile) to (R)-(−)-mandelic acid. The cells had been immobilized by entrapment in an alginate matrix. Conditions for the hydrolysis reaction were optimized in shake flasks and in a packed bed reactor. In shake flasks the best conditions for the reaction were a temperature of 40 °C, pH 8, biocatalyst bead diameter of 4.3 mm, sodium alginate concentration in the gel matrix of 2 % (w/v, g/100 mL), a cell dry mass concentration in the bead matrix of 20 mg/mL, an initial substrate concentration of 50 mM and a reaction time of 60 min. Under these conditions, the conversion of mandelonitrile was nearly 95 %. In the packed bed reactor, a feed flow rate of 20 mL/h at a substrate concentration of 200 mM proved to be the best at 40 °C, pH 8, using 4.3 mm beads (2 % w/v sodium alginate in the gel matrix, 20 mg dry cell concentration per mL of gel matrix). This feed flow rate corresponded to a residence time of 0.975 h in the packed bed.

Investigation of the causes of deactivation–degradation of the commercial biocatalyst Novozym® 435 in ethanol and ethanol–aqueous media

Abstract The effect of contacting neat ethanol and an ethanol: H 2 O mixture on Novozym ® 435 was investigated at room temperature and 45 °C, during various periods of time of interaction with the alcohol (from 40 min to 8 days) and at different biocatalyst: ethanol (ethanol/water) ratios. The alcohol dissolves the polymethylmethacrylate (PMMA) that constitutes the support of the Candida antarctica B lipase (CALB) regardless of the conditions investigated and diffuses into the biocatalyst's beads remaining strongly adsorbed (the desorption of the alcohol is evidenced only upon heating at 150 °C). The diffusion of the alcohol alters the inner texture of the beads generating channels and increasing the roughness of the polymeric material. Additionally, the ethanol (with or without water added) modifies the secondary structure of the enzyme by decreasing the α-helix contributions and increasing the β-sheet structure.

Investigation on the development of sturdy bioactive hydrogel beads

AbstractBiocatalytic hydrogel beads, which retain higher activity, expand, and contract with changes in pH, having biocompatibility, are developed. Composite spherical beads of chitosan having a diameter of 1–2 mm were prepared by ionic gelation using sodium tripolyphosphate (TPP). Above 3% TPP, the activity of the enzyme decreases. The mechanical strength of the chitosan–TPP beads was further improved by the addition of clay or cassava starch granules. The immobilization of protease (fungal, Aspergillus) was done with glutaraldehyde crosslinking. The chitosan–starch hydrogel beads showed significant increase in firmness and stiffness when compared with chitosan–clay beads. The swelling studies show that the particles expand at pH 1.2 and contract at pH 7.4. The activity retention of the immobilized protease was as high as 70% and exhibited a high pH and lower temperature optima than the free enzyme. Chitosan–starch hydrogel beads exhibited degradation peaks at about 90–110°C in TGA analysis. The biocatalyst beads retained 85% of the original catalytic activity even after eight cycles of repeat use. The freeze‐dried beads has good storage stability and can be used either as artificial bioreactor systems in detergent or in therapeutic formulations © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Kinetics and diffusion studies in urease-alginate biocatalyst beads

Urease was immobilized with calcium alginate by entrapment method in the form of spherical beads and stored in Tris/acetate buffer (pH 7.3) at <TEX>$4^{\circ}C$</TEX>. Urease immobilized at different concentration of alginate beads (3%, 4% and 5%) showed higher apparent <TEX>$K_{m}$</TEX> values than the soluble urease. Furthermore, <TEX>$K_{m}$</TEX> has been shown to be corelated with effective diffusion coefficient (De) at different concentration of alginate gel. The present study showed that diffusion and reaction contribute to control the overall rate.

Continuous biotransformation of glycopeptide antibiotic A40926 in a cascade of three airlift bioreactors using immobilized Actinoplanes teichomyceticus cells

Immobilized cells of Actinoplanes teichomyceticus ATCC 31121 were used to selectively cleave the acyl group of A40926 yielding the deacylated form of the molecule. The feasibility of this particular biotransformation in a series of three perfectly mixed airlift bioreactors with immobilized cells was examined. A continuously operated airlift cascade was designed using a model for a series of reactors with immobilized biocatalyst beads obeying Michaelis–Menten kinetics. In independent experimental runs the cascade bioreactor system was operated continuously for 56 days with an overall conversion of 99%. Model estimates for reactor volumes and relative conversions were found to be in a good agreement with the experimental results.

Glucose production from maltodextrins employing a thermophilic immobilized cell biocatalyst in a packed-bed reactor

This research is focused on the development of an immobilized cell biocatalyst applicable for the conversion of maltodextrins into glucose at high temperatures. In particular, Escherichia coli BL21 (DE3) cells expressing α-glucosidase from Sulfolobus solfataricus MT4 were entrapped in a Ca-alginate polysaccharidic matrix obtaining over 70% immobilization yield. Compared to the free α-glucosidase, the immobilized cells showed a shift of optimal pH towards alkaline (from 5.0 to 5.5) and the same increasing activity up to 100 °C. These biocatalyst beads were packed into a tubular reactor, which was exploited for the biotransformation of a commercial dextrin mixture at industrial concentrations (30–40%, w/v) into glucose at 75 °C, achieving up to 98% conversion. The corresponding glucose productivity was about 1.75 g h −1 g biocatalyst −1.

Biocatalytic Hydrolysis of 3‐Hydroxyalkanenitriles to 3‐Hydroxyalkanoic Acids

AbstractMicrobial catalysts having a combination of nitrile hydratase and amidase activities had a significantly‐higher specific activity for hydrolysis of 3‐hydroxyalkanenitriles than microbial nitrilase catalysts. Comamonas testosteroni 22–1, Dietzia sp. ADL1 and Comamonas testosteroni 5‐MGAM‐4D nitrile hydratase/amidase biocatalysts each hydrolyzed 3‐hydroxyvaleronitrile to 3‐hydroxyvaleric acid (as the ammonium salt) in 99–100% yields, but in consecutive batch reactions with catalyst recycle, alginate‐immobilized C. testosteroni 5‐MGAM‐4D had superior enzyme stability and volumetric productivity. In a series of 85 consecutive batch reactions with biocatalyst recycle for the production of 1.0 M 3‐hydroxyvaleric acid, the recovered nitrile hydratase and amidase activities in the final reaction were 29% and 40%, respectively, of the initial activities. The catalyst productivity for this series of reactions was 670 g 3‐hydroxyvaleric acid/g dry cell weight (50 g 3‐hydroxyvaleric acid/g biocatalyst bead), and the volumetric productivity of the initial reaction in the series was 44 g 3‐HVA/L/h. Similar results were obtained with alginate‐immobilized C. testosteroni 5‐MGAM‐4D for the hydrolysis of 3‐hydroxybutryonitrile and 3‐hydroxypropionitrile to the corresponding 3‐hydroxyalkanoic acid ammonium salts.

Deacylation of antibiotic A40926 by immobilized Actinoplanes teichomyceticus cells in an internal-loop air-lift bioreactor

A40926 is a natural glycopeptide antibiotic. It is a precursor of dalbavancin, a semi-synthetic second-generation glycopeptide in clinical development for the treatment of β-lactam-resistant Staphyloccous aureus and Staphylococcus epidermidis infections. Deacylation of A40926, producing an intermediate useful for the synthesis of novel derivatives, can be carried out by the action of Actinoplanes teichomyceticus cells. Kinetics parameters for this particular deacylation reaction were obtained by fitting the experimental data to a Michaelis–Menten kinetic model. The feasibility of A40926 deacylation by immobilized A. teichomyceticus cells in an air-lift bioreactor was examined. A single continuously operated air-lift reactor (ALR) was evaluated by a model describing diffusion and reaction in the biocatalyst beads. Model estimates were in a good agreement with experimental results. The system was operated continuously for 21 days with overall conversion above 60%.

Trehalose production at high temperature exploiting an immobilized cell bioreactor.

The enzymatic production of trehalose from dextrins was studied as a series reaction in a packed bed reactor containing immobilized recombinant Escherichia coli cells, expressing either the Sulfolobus solfataricus (strain MT4) trehalosyl-dextrin forming enzyme (TDFE) or the trehalose-forming enzyme (TFE). The cells, subjected to thermal treatments to increase cell permeability and to inactivate the unwanted host proteins, were entrapped separately or together in a calcium alginate polymeric matrix. The biocatalyst beads were used to pack a tubular glass reactor that was operated in a recycle mode. The performances of a bioreactor containing alternate layers of EcTFE and EcTDFE alginate beads were evaluated and compared with the performance of the co-immobilized biocatalysts. The latter showed a superior throughput, therefore the bioreactor packed with the co-entrapped biocatalysts was tested for the production of trehalose from concentrated dextrin solutions (10%-30% w/v) and a conversion up to 90% was obtained. This conversion corresponded to a production of 127 g trehalose h(-1) kg(-1) of biocatalyst. The results obtained suggest that the bioprocess described may be of interest in the development of a large-scale industrial process for trehalose production at high temperature.

Bacillus subtilis cells immobilised in PVA-cryogels

Bacillus subtilis viable cells were immobilised in PVA-cryogel beads using the ‘freezing-thawing’ method in a two-phase (water–oil) system. Conditions providing both high thermal and mechanical stability and suitable porosity of the carrier were optimised. For monitoring and analysis of changes inside the biocatalyst beads, and for determination of diffusive properties of the carrier, an image analysis was applied. It was revealed that bacterial spores, sodium alginate and bacterial cellulose accelerated hardening of the cryogels and modified their porosity. Proteins (haemoglobin, azoalbumin, azocasein) penetrated beads of the cryogel.

Processing efficiency of immobilized non‐growing bacteria: Biocatalytic modeling and experimental analysis

AbstractModeling methods used to optimize the biocatalytic efficiency of freely suspended cells have been applied to non‐growing microbial cells entrapped within a macro‐porous carrier. The catalytic rate, which is dependent on cell concentration inside the biocatalyst beads, coincided with catalytic parameters for freely suspended cells. Immobilized non‐growing cell systems could be optimized utilizing the characteristics of freely suspended cells without requiring extensive experimentation to define catalytic behaviour inside the biocatalyst. A dynamic diffusion–reaction model was developed and validated using experimental data for thiodiglycol degradation by Alcaligenes xylosoxidans subsp. xylosoxidans immobilized within macro‐porous poly(vinyl alcohol) cryogel in a completely mixed batch bioreactor.

Production of ethanol from starch by co-immobilizedZymomonas mobilis-glucoamylase in a fluidized-bed reactor

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilized Zymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. The Z. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of kappa-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35 degrees C and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.