Immobilized Biocatalysts Research Articles

Engineered phenylalanine dehydrogenase in organic solvents: homogeneous and biphasic enzymatic reactions

The use of engineered phenylalanine dehydrogenase N145A supported on Celite for the reductive amination of phenylpyruvic acid in homogeneous and biphasic aqueous-organic solvents is reported. The results indicate that the immobilised biocatalyst is remarkably robust, even in the presence of high concentrations of polar or non-polar organic solvents such as acetone, methanol, n-hexane, toluene and methylene chloride. Cofactor regeneration with alcohol dehydrogenase from Saccharomyces cerevisiae and ethanol was successfully explored. Application to the non-natural poorly water-soluble 2-oxo acid p-NO(2)-phenylpyruvic acid was successfully performed, resulting in the biocatalytic synthesis of p-NO(2)-phenylalanine. In all cases 100% stereoselectivity for the production of the amino acid was retained.

Chrysotile as a support for the immobilisation of Mycobacterium sp. NRRL B-3805 cells for the bioconversion of β-sitosterol in an organic–aqueous two-liquid phase system

The use of chrysotile, a plentiful and low-cost inorganic material, as an effective immobilisation matrix for mycobacterial cells was evaluated. The side-chain cleavage of β-sitosterol to 4-androstane-3,17-dione (AD), in an organic–aqueous two-liquid phase system, was chosen as model system. Bis(2-ethylhexyl) phthalate (BEHP), a biocompatible organic solvent, was used as organic phase. The biotransformation was carried out with either growing or previously grown cells. Mycobacterial cells were shown to adsorb onto chrysotile in both aqueous buffered system and in organic–aqueous two-liquid phase systems. Cells tend to aggregate among the chrysotile fibres during and after growth. The formed chrysotile/cells complex retained catalytic activity under bioconversion conditions for 1 week, at least. No mass transfer hindrances were observed when the immobilised form of the biocatalyst was used, as compared to the free form. However, among the several approaches to produce the most efficient immobilised biocatalyst, the use of previously grown cells in contact with chrysotile led to the highest product yields as well as highest bioconversion rates. The feasibility of this approach was further evidenced since it allowed the implementation of a continuous mode of operation. A constant product yield of 50% was observed for a 1-week period with this experimental set-up. This work clearly highlights the ability of chrysotile to provide an efficient immobilisation matrix for whole cells to be used as biocatalysts in organic–aqueous two-liquid phase bioconversion systems.

Preparation of enantiopure ( S)-ketoprofen by immobilized Candidarugosa lipase in packed bed reactor

Various materials have been used as supports for the immobilization of a Candida rugosa lipase (Lipase OF). The enzyme adsorbed on silica gel showed the maximum activity recovery (37%), and was thus used to construct a packed bed reactor for the continuous production of ( S)-ketoprofen. The performance of the bioreactor containing 4.0 g immobilized biocatalyst was evaluated as a function of parameters such as flow rate, the ratio of height to diameter (H/D) and substrate concentration. The space velocity (SV) of the reactor showed a linear increase when the flow rate was increased in the range of 5.9 (SV = 0.9 h −1)–25 ml h −1 (SV = 3.9 h −1) and leveled out at values for flow rate between 25 (SV = 3.9 h −1) and 64 ml h −1 (SV = 10.2 h −1). The initial rate remained the same when the H/D was varied from 2.9 to 38.6. The initial rate rose in a linear fashion as the substrate concentration was increased from 5 to 20 mg ml −1 and remained almost at the same level when the substrate concentration was between 20 and 50 mg ml −1. Using a packed bed bioreactor, optically pure ( S)-ketoprofen (>99% ee (enantiomeric excess)) was produced with a conversion of 30% and productivity of 1.5 mg g −1 biocatalyst h −1.

Design of immobilised dextransucrase for fluidised bed application

Immobilisation of dextransucrase from Leuconostoc mesenteroides NRRL B-512F in alginate is optimised for applications in a fluidised bed reactor with high concentrated sugar solutions, in order to allow a continuous formation of defined oligosaccharides as prebiotic isomalto-oligosaccharides. Efficient design of fluidised bed immobilised biocatalyst in high density solutions requires particles with elevated density, high effectiveness and both thermal and mechanical stability. Inert silica flour/sand (Mikrosil 300) as supplement turned out to be best suited for increasing the density up to 1400 kg m −3 of the alginate beads and generating a stable expanded bed without diffusional restrictions. Kinetic investigations demonstrate that low effectiveness of immobilised enzyme due to close association to dextranpolymers (dextran content of enzyme preparation >90%) is compensated by reducing the particle size and/or by decreasing the dextran content. A low dextran content (5%) is sufficient to immobilise and stabilise the enzyme, thus diffusional limitation is reduced essentially while operational stability is maintained. Fluidisation behaviour and bed expansion proved to be appropriate for the intended application. Both calculated and measured expansion coefficients showed good agreement for different conditions.

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.

Biospecific immobilisation of mannan-modified α-amylase on Concanavalin A Sepharose

Bacillus subtilis α-amylase was chemically modified with yeast mannan, yielding a conjugate containing 1.2 mol of polysaccharide per mol of protein and retaining 60% of the original amylolytic activity. This neoglycoenzyme was immobilised on Concanavalin A Sepharose via a biospecific lectin-carbohydrate recognition mechanism. The immobilised biocatalyst retained high amylolytic activity and its optimum temperature was 10°C higher than the native enzyme. Thermal stability was increased by 12°C for α-amylase after glycosidation-immobilisation.

Immobilization of yeast on dried raisin berries for use in dry white wine-making

Cells of a commercial Saccharomyces cerevisiae strain ( Uvaferme 299) were immobilized on dried raisin berries ( Sultanina variety) to produce an immobilized biocatalyst for use in dry white wine-making. The immobilised biocatalyst was found to be suitable for wine making at ambient temperatures (15–25 °C). The wines produced had low volatile acidities and low methanol and acetaldehyde contents, while volatile by-products showed no statistically significant differences from wines produced by free cells. The immobilized cell system had a good operational stability for more than 4 months. Sensory evaluation revealed differences between wines produced by immobilized and free cells.

Purine nucleoside synthesis from uridine using immobilised Enterobacter gergoviae CECT 875 whole cells

Biocatalysed purine nucleoside synthesis was carried out using immobilised Enterobacter gergoviae CECT 875. Similar yields (80–95%) in adenosine were obtained with both free and immobilised cells though in the last case a long reaction time was necessary. The immobilised cells can be reused at least for more than 30 times without significant loss of enzymatic activity. The immobilised biocatalyst in agarose is active in the synthesis of unnatural nucleosides.

DOE awards chemical makers funding for biomass conversion

Crosslinked, cryostructured monoliths prepared from Lactobacillus reuteri cells were evaluated as potential immobilized whole-cell biocatalyst for conversion of glycerol, to potentially important chemicals for the biobased industry, i.e. 3-hydroxypropionaldehyde (3HPA), 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO). Glutaraldehyde, oxidized dextran and activated polyethyleneimine/modified polyvinyl alcohol (PEI/PVA) were evaluated as crosslinkers; the latter gave highly stable preparations with maintained viability and biocatalytic activity. Scanning electron microscopy of the PEI/PVA monoliths showed high density of crosslinked cells with wide channels allowing liquid flow through. Flux analysis of the propanediol-utilization pathway, incorporating glycerol/diol dehydratase, propionaldehyde dehydrogenase, 1,3PDO oxidoreductase, phosphotransacylase, and propionate kinase, for conversion of glycerol to the three chemicals showed that the maximum specific reaction rates were −562.6, 281.4, 62.4 and 50.5 mg/gCDW h for glycerol consumption, and 3HPA (extracellular), 3HP and 1,3PDO production, respectively. Under optimal conditions using monolith operated as continuous plug flow reactor, 19.7 g/L 3HPA was produced as complex with carbohydrazide at a rate of 9.1 g/Lh and a yield of 77 mol%. Using fed-batch operation, 1,3PDO and 3HP were co-produced in equimolar amounts with a yield of 91 mol%. The monoliths embedded in plastic carriers showed high mechanical stability under different modes in a miniaturized plug flow reactor.

Low-temperature brewing by freeze-dried immobilized cells.

We propose a novel biocatalyst in brewing. A cryotolerant strain of Saccharomyces cerevisiae was immobilized on delignified cellulosic material followed by freeze-drying of the immobilized cells without the use of any cryoprotectant. The freeze-dried immobilized biocatalyst was used in repeated-batch fermentation of wort and showed reduced fermentation time and increased productivities as compared with free freeze-dried cells (FFDCs). It also demonstrated suitability for low-temperature brewing (5 and 0 degrees C). The fermentation time in repeated-batch fermentations at 15 degrees C was 1.5-2 d for a period of 13 mo, showing a high operational stability of the system. At 0 degrees C the freeze-dried immobilized biocatalyst showed a 2- to 3.5-fold decrease in fermentation time in comparison with FFDCs. Polyphenol contents, bitterness, and diacetyl concentration were lower in beers produced by freeze-dried immobilized cells as compared with FFDCs. At 0 degrees C polyphenols were 40% lower than at 15 degrees C. Higher alcohols were reduced and ethyl acetate increased in comparison with FFDCs. Amyl alcohols at 0 degrees C were lower than half of their content at 15 degrees C, while ethyl acetate was 31 mg/L at 0 degrees C and 18 mg/L at 15 degrees C. These data justify the improved aroma and taste of beers produced by freeze-dried immobilized biocatalyst mainly at low temperatures.

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

AbstractPenicillin V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy‐activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55 °C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10‐fold increase in its half‐life at 40 °C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation.© 2001 Society of Chemical Industry

Design of an automated bioreactor system to screen immobilised whole cell catalysts

An automated CSTR system has been developed to study the stability of immobilised whole cell biocatalysts for ammonium acrylate production. Two potentially useful amidase-active rhodococci have been identified: One possesses an amidase of much higher activity than the other but of much lower stability. The activity and stability of each biocatalyst over a range of temperatures has been determined and used to compare the productivity of the biocatalysts in a bioconversion.

Production and Characterisation of Cross-Linked Enzyme Crystals (Clecs®) for Application as Process Scale Biocatalysts

Cross-linked enzyme crystals (CLECs®) are a novel form of immobilised biocatalyst designed for application in large-scale biotransformation processes. In this work we review the production and characterisation of CLECs® prepared from three enzymes (yeast alcohol dehydrogenase I (YADHI), Candida rugosa lipase and α-chymotrypsin) over a range of crystallisation and cross-linking conditions. Optimisation and control of the crystallisation process, with respect to crystal form and enzyme activity yield, was facilitated by the use of triangular crystallisation diagrams which allowed three parameters (e.g. protein concentration, precipitant concentration and pH) to be varied simultaneously. These diagrams showed regions, or 'crystallisation windows', in which particular crystal forms or optimal activity recoveries (up to 87%) could be obtained. They also identified conditions for reproducible scale-up of the lipase crystallisation from 0.5 to 500 mL scale.In order to evaluate the suitability of a particular batch of CLECs® for large-scale use, a hierarchy of standard tests is proposed. This is designed to expose key properties of the CLECs® relative to each other, and the free enzyme, and to minimise the number of experiments necessary to evaluate each batch of biocatalyst. In general, the CLECs® of each enzyme were found to be more resistant to harsh environmental conditions, such as extremes of temperature and pH and the presence of solvents or proteases, than the free enzymes. Cross-linking of the crystals with glutaraldehyde also yielded mechanically robust catalysts that could withstand the various forces associated with shear in agitated vessels and particle compression in repeated dead-end filtration cycles. The hierarchy of tests proposed here clearly indicated that many of the above properties were also dependent on both the crystal form and size, and the concentration of cross-linking reagent used. Accurate control of the crystallisation conditions used for CLEC® production is therefore vital as this will influence the suitability of the CLECs® for their end use.

Lipase immobilisation: an equilibrium study of lipases immobilised on hydrophobic and hydrophilic/hydrophobic supports

This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm 3). Activity, efficiency and operational stability of the immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol. Equilibrium data were modelled by the Langmuir, Freundlich and Redlich–Peterson formulae. It was found that Lipolase 100L/Accurel, PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled Redlich–Peterson behaviour. Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support 45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871. Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase 100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively.

Site-protected fixation and immobilization of Escherichia coli cells displaying surface-anchored beta-lactamase.

Bacteria displaying heterologous receptors or enzymes on their surface hold great potential as whole-cell adsorbents and biocatalysts, respectively. For industrial applications, such surface-engineered cells need to be killed and chemically fixed to prevent disintegration and leakage of the displayed proteins under process conditions. It is also highly desirable to couple the chemically stabilized cells onto a solid support matrix for additional mechanical stability, flexibility in reactor choice, and easy separation from processed medium. Recently, we described the development of a readily scalable methodology for cell killing, fixation, and outer membrane stabilization via glutaraldehyde fixation followed by secondary crosslinking (Freeman, A., Abramov, S. and Georgiou, G. 1996. Biotechnol. Bioeng. 52: 625-630). Glutaraldehyde treatment was also found, however, to reduce the specific activity of a model enzyme, beta-lactamase displayed on the surface of E. coli. Here, we show that crosslinking carried out in the presence of beta-lactamase inhibitors, namely phenyl boronic acid or sodium borate, protects the active site from chemical modification resulting in up to threefold higher specific activities without affecting the cell-stabilizing effect of the glutaraldehyde treatment. To prepare an immobilized whole cell biocatalyst, residual unreacted surface aldehyde groups were employed to immobilize covalently the fixed bacteria onto chitosan-coated cellulose powder. The binding of the bacteria onto chitosan-coated cellulose was quantitative up to cell loading of 83 mg dry cell weight/g of support. Cell immobilization did not introduce mass transfer limitations and created only a modest reduction in Vmax. Thus, chemical crosslinking, affected in presence of reversible active-site inhibitors and coupled with cell immobilization on chitosan-coated cellulose represents a widely useful methodology for the process application of recombinant bacteria displaying surface-anchored heterologous proteins.

Investigation of catalytic properties of immobilized enzymes and cells by flow microcalorimetry.

The investigation of catalytic properties of immobilized biocatalysts (IMB) is a time-consuming and not-always-simple procedure, requiring a simple and accurate method of enzyme-activity measurement. In comparison with generally-used techniques, flow microcalorimetry (FMC) has proven to be a very practical and versatile technique for direct monitoring of the course of enzyme reactions. The principal advantage of FMC is integration of the enzyme reaction and its monitoring in one step. This review summarizes the information needed for the complete kinetic or catalytic characterization of the IMB by FMC, without the requirement of any independent analytical method. The optimal experimental procedure is proposed. Examples of experimental studies on immobilized biocatalysts using the FMC are provided. The method is applicable to purified enzymes as well as to enzymes fixed in cells.

Sorption isotherms and kinetics in the primary biodegradation of anionic surfactants by immobilized bacteria: I. Pseudomonas C12B

The surfactant-degrading biocatalyst Pseudomonas C12B was immobilized by covalent linking on silanized inorganic supports and by physical entrapment of cells within reticulated polyurethane foam. Both immobilized biocatalysts have been shown to be appropriate for the effective primary biodegradation of the anionic surfactants sodium dodecyl sulphate (SDS), dodecylbenzene sulphonic acid (DBS), dioctyl sulphosuccinate (DOSS) and dihexyl sulphosuccinate (DHSS). The overall surfactant removal from water by cells entrapped in reticulated polyurethane foam exhibits a biphasic process, a rapid sorption step of the surfactant onto the cell-loaded support and the intrinsic primary biodegradation slower step, both acting cooperatively. The optimization of variables for the adsorption and the biodegradation processes (flow rate, particle size, substrate concentration) have been studied. Sorption isotherms for the surfactants on reticulated polyurethane foam have been established as type II of the Brunauer, Deming, Deming and Teller (BDDT) classification. The kinetics of the primary biodegradation of SDS by cells covalent linked on sepiolite treated with 3-aminopropyl triethoxysilane (APTS) were found to be first-order. In this case, surfactant adsorption does not exist.

Cloning, expression and immobilization of glutamate racemase from Lactobacillus fermenti for the production of D-glutamate from L-glutamate

A new immobilized biocatalyst for the racemization of L-glutamate on a preparative scale was developed. The gene encoding the glutamate racemase from Lactobacillus fermenti has been isolated by PCR amplification from its chromosomal DNA and overexpressed in Escherichia coli under the control of lac promoter. The recombinant enzyme (25-30% of total proteins) was rapidly immobilized on highly activated glyoxyl-agarose gels. The immobilized enzyme retained up to 80% of catalytic activity. In fact, 14 g of biocatalyst containing 20 mg of immobilized protein were able to racemize 90 mg of L-glutamic acid in less than 30 minutes.

Ethanol production at 45°C by alginate-immobilized Kluyveromyces marxianus IMB3 during growth on lactose-containing media

The thermotolerant yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and this was used in batch-fed reactor systems to convert lactose (4 g/l) to ethanol. Production of ethanol by the free and immobilized biocatalyst in the presence and absence of Mn2+ was compared. In systems containing the free microorganism in the presence and absence of Mn2+, ethanol increased to a maximum of 8 g/l within 40 hours with no significant difference in production by both systems. Ethanol production by the immobilized system in the absence of Mn2+ increased to a maximum of 13 g/l within 40 hours and then decreased to 9 g/l within 80 hours. Ethanol production by the immobilized system in the presence of Mn2+ increased to 14 g/l within 60 hours and this decreased to 13 g/l at 80 hours. When all systems were re-fed at 80 hours, ethanol production by systems containing the free biocatalyst increased to a maximum of 3 g/l while the immobilized system in the presence of Mn2+ increased to a maximum of 12 g/l. Subsequent experiments involving re-feeding the system at shorter time intervals demonstrated that ethanol production by the immobilized system on lactose-containing media at 45 °C was far superior to ethanol production by the free biocatalyst.

Bioreactor with a semi-fixed packing: anaerobic lactose to lactic acid fermentation

The bioreactor with a semi-fixed packing consists of frames with stretched “Raschell”-type sacks on them. This construction enables the attainment of a larger interfacial area of the biofilm carrier per unit reactor volume as well as the easy removal of the exausted biomass at certain biofilm thickness. In the present paper the batch anaerobic conversion of lactose to lactic acid in this reactor with immobilized bacteria Lactobacillus casei is studied. The dilution rate of the feeding substrate solution was 1 mm/s. Comparison of our experimental results with data, obtained for free cells and other construction of bioreactors with immobilized cells is made. It is shown that the used immobilised biocatalyst is superior to free bacterial cells, or attached to the inner pores of polyurethane foam.