Immobilization ofPhanerochaete chrysosporium on porous polyurethane particles with application to biodegradation of 2-chlorophenol

Porous polyurethane particles were prepared and used for the immobilization of white rot fungusPhanerochaete chrysosporium. The immobilized cells were employed for the production of lignin peroxidase. Polyurethane immobilized spores, or mycelial pellets ofPhanerochaete chrysosporium as well as freely suspended mycelial pellets of fungus were used as biocatalyst for the degradation of 2-chlorophenol. The polyurethane carriers appear to be superior to the other carriers already used for the immobilization of fungus.     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.     

Influence of a supplementary carbon source on biodegradation of pyridine by freely suspended and immobilizedPimelobacter sp.

The effect of the presence of supplementary glucose or acetate on the growth and pyridine-degrading activity of freely suspended and calcium-alginate-immobilizedPimelobacter sp. was investigated. Although the supplementary carbon sources could be degraded simultaneously with pyridine,Pimelobacter sp. exhibited a preference for pyridine over supplementary carbon sources. Thus, the pyridine-degrading activity of the freely suspended cells was not decreased significantly by the addition of either glucose (1.5–6 mM) or acetate (6–24 mM) to the pyridine (6–24 mM). In the semi-continuous immobilized cell culture, immobilized cells also exhibited a preference for pyridine over supplementary carbon sources and did not switch their substrate preference throughout the culture. Owing to a high cell concentration, the volumetric pyridine degradation rate at 24 mM pyridine in the immobilized cell culture was approximately six times higher than that in the freely suspended cell culture. Furthermore, the immobilized cells could be reused 16 times without losing their pyridine-degrading activity during the culture period tested. Taken together, the use of immobilizedPimelobacter sp. for the degradation of pyridine is quite feasible because of the preference for pyridine over supplementary carbon sources, the high volumetric pyridine degradation rate, and the reusability of immobilized cells.     

Biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp.

Freely suspended and Ca-alginate-immobilized cells of Pimelobacter sp. were used for degradation of pyridine. When the pyridine concentration was up to 2 g l^–1, freely suspended cells completely degraded pyridine regardless of the initial cell concentrations used. However, when the pyridine concentration increased to 4 g l^–1, the initial cell concentration in freely suspended cell culture should be higher than 1.5 g dry cell weight l^–1 for complete degradation of pyridine. In addition, a freely suspended cell culture with a high initial cell concentration resulted in a high volumetric pyridine-degradation rate, suggesting the potential use of immobilized cells for pyridine-degradation. When the immobilized cells were used for pyridine-degradation, neither specific pyridine-degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine-degradation rate in the range 0.082–0.129 g l^–1 hr^–1 could be achieved by the immobilized cells because of the high cell concentration. Furthermore, when the immobilized cells were reused in degrading pyridine at a concentration of 2–4 g l^–1 they did not lose their pyridine-degrading activity for 2 weeks. Taken together, the data obtained here showed the feasibility of using immobilized cells for pyridine-degradation.     

Immobilization of Acetobacter sp. CCTCC M209061 for efficient asymmetric reduction of ketones and biocatalyst recycling

ABSTRACT: BACKGROUND: The bacterium Acetobacter sp. CCTCC M209061 is a promising whole-cell biocatalyst with exclusive anti-Prelog stereoselectivity for the reduction of prochiral ketones that can be used to make valuable chiral alcohols such as (R)-4-(trimethylsilyl)-3-butyn-2-ol. Although it has promising catalytic properties, its stability and reusability are relatively poor compared to other biocatalysts. Hence, we explored various materials for immobilizing the active cells, in order to improve the operational stability of biocatalyst. RESULTS: It was found that Ca-alginate give the best immobilized biocatalyst, which was then coated with chitosan to further improve its mechanical strength and swelling-resistance properties. Conditions were optimized for formation of reusable immobilized beads which can be used for repeated batch asymmetric reduction of 4[prime]-chloroacetophenone. The optimized immobilized biocatalyst was very promising, with a specific activity of 85% that of the free-cell biocatalyst (34.66 mumol/min/g dw of cells for immobilized catalyst vs 40.54 mumol/min/g for free cells in the asymmetric reduction of 4[prime]-chloroacetophenone). The immobilized cells showed better thermal stability, pH stability, solvent tolerance and storability compared with free cells. After 25 cycles reaction, the immobilized beads still retained >50% catalytic activity, which was 3.5 times higher than degree of retention of activity by free cells reused in a similar way. The cells could be recultured in the beads to regain full activity and perform a further 25 cycles of the reduction reaction. The external mass transfer resistances were negligible as deduced from Damkohler modulus Da < <1, and internal mass transfer restriction affected the reduction action but was not the principal rate-controlling step according to effectiveness factors eta < 1 and Thiele modulus 0.3<[empty set] <1. CONCLUSIONS: Ca-alginate coated with chitosan is a highly effective material for immobilization of Acetobacter sp. CCTCC M209061 cells for repeated use in the asymmetric reduction of ketones. Only a small cost in terms of the slightly lower catalytic activity compared to free cells could give highly practicable immobilized biocatalyst.     

Culture conditions for intracellular lipase production by Rhizopus chinensis and its immobilization within biomass support particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells.It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity.The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells.The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Bioconversion using immobilized recombinant flocculent yeast cells carrying a fused enzyme gene in an `intelligent' bioreactor

Immobilized recombinant cells of the flocculent yeast Saccharomyces diastaticus carrying an expression plasmid for a fused enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 reductase were used in the bioconversion reaction from acetanilide (AA) to p-acetaminophene (p-AAP). Immobilization of the strain within reticulated polyurethane foam biomass support particles (BSPs) was effected passively in situ in a fluidized-bed bioreactor using `draw and fill' operation. In repeated batch reactions both the final product concentration and the production rate were notably improved compared with the results obtained using freely suspended cells without BSPs. Cells immobilized within BSPs exhibited a significantly high level of expression of the fused enzyme. In addition, a high proportion of plasmid-carrying cells was maintained among the immobilized cells, in contrast to a much lower proportion among freely suspended cells released from the BSPs. Since the bioreactor became packed with highly expressing cells immobilized within BSPs as a consequence of spontaneous screening, it was termed an `intelligent' bioreactor, and is believed to offer significant potential for the further development of efficient production processes.     

Methane hydroxylation by <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b: Monitoring the biocatalyst activity for methanol production optimization in an innovative membrane bioreactor

A quasi-total loss of the bacterial hydroxylating activity was identified to be responsible for methanol production stop. Different strategies acting on the reaction mixture were implemented to apprehend the biocatalyst behavior in view to extend methanol production. Activity monitoring showed first that sodium formate addition did not maintain the biocatalyst activity and even disrupted bacterial equilibrium when added into the reaction mixture with still active biocatalysts. Reaction medium renewals had no influence on methanol production and highlighted a limited hydroxylating potential of the biocatalyst while addition of fresh biocatalysts in the reaction mixture resulted in methanol consumption. Finally, performing hydroxylation directly in the native bacterial culture appeared as a way to enhance methanol production by both release of intracellular methanol accumulated in the cells during cultivation and effective production by methane hydroxylation.     

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.     

Comparison of the aspartase activity and its stability in Escherichia coli cells immobilized on polyacrylamide gel and carrageenan

The conditions for immobilization of Escherichia coli cells (Soviet strain 85) on the natural polysaccharide carrier carrageenan (Soviet-made) were investigated and kinetic regularities of the aspartase reaction catalysed by immobilized in carrageenan cells of E. coli 85 were established. The conditions for retaining a high aspartase activity and stability of biocatalysts based on the E. coli 85 cells immobilized in PAAG and carrageenan were determined using full-loaded tanks for continuous synthesis of L-aspartic acid. The time-stable aspartase activity of the biocatalyst can be increased by treating the beads of the catalyst with bifunctional reagents (hexamethylenediamine, glutaraldehyde), the most active catalyst for the biotechnological synthesis of L-aspartic acid being obtained when carrageenan is used.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Immobilized Escherichia coli BL21 as a catalyst for the synthesis of adenine and hypoxanthine nucleosides

Different supports, such as alginate, agar, agarose, and polyacrylamide, were used to immobilize Escherichia coli BL 21 by entrapment techniques. The transglycosylation reaction involved in the synthesis of adenosine from uridine and adenine was chosen as a model system to study the characteristics of these biocatalysts. Whole cells immobilized on agarose proved to be optimal and could be used up to 30 times without significant loss of activity. This biocatalyst was further employed to test its ability in the synthesis of other adenine and hypoxanthine nucleosides. Ribo-, 2'-deoxyribo-, and arabinonucleosides could be prepared in high yields starting from the corresponding pyrimidine nucleosides and purine bases. Similar product yields were obtained with both free and immobilized cells, though, in the latter case, a longer reaction time was necessary.     

Production of 6-aminopenicillanic acid by immobilized Pleurotus ostreatus

Summary 6-Aminopenicillanic acid from penicillin V is produced by Pleurotus ostreatus immobilized by entrapment in a chitosan matrix. In these carriers the cell concentration increases after network formation by irreversible shrinking of the biocatalyst. Specific activity of the biocatalyst for the hydrolysis reaction is 1,31 mol.min^-1. (g wet weight of catalyst)^-1 corresponding to a relative activity of 38%. Catalytic half-life of immobilized Pl. ostreatus is 25 days compared to 2.5 days for free suspended cells.This paper is part of the Dissertation of Michael Kluge, Technical University Braunschweig 1981.     

Preparation of L-aspartic acid by means of immobilized Alcaligenes metalcaligenes cells

Two types of biocatalysts based on immobilized cells of Alcaligenes metalcaligenes exhibiting aspartate ammonia-lyase activity (EC 4.3.1.1) were developed for the enzymic preparation of L-aspartic acid from ammonium fumarate. The first type of the biocatalyst consists in individual covalently crosslinked and permeabilized cells(I), while the second type is represented by cell aggregates (II). For the above preparation, biocatalyst I can be used only discontinuously in a mixed reactor. After termination of the reaction between individual cycles of its use, the biocatalyst is returned to the reactor in the form of a highly concentrated cell suspension or paste. Biocatalyst II can be used discontinuously or continuously in a fixed-bed column of the catalyst. The effects of pH, substrate concentration and temperature on the reaction velocity and effectivity of enzymic conversion was investigated. Optimal parameters of the reaction are as follows: pH 8.5, initial substrate concentration, 1.35 mol/L, temperature for discontinuous process, 37 degrees C, and temperature for continuous process, 25 degrees C. Under these conditions the enzymic conversion of substrate to product is quantitative. Under optimal toring conditions, the specific activity of both catalysts does not change within a period of one year. The operational half-life of the biocatalyst II during continuous use in a fixed-bed column of the catalyst under standard reaction conditions depends on the quality of the substrate. The discontinuous preparation of L-asparatic acid with the aid of biocatalyst I and continuous preparation of this product with the aid of biocatalyst II have been verified under pilot-plant conditions.     

Response surface methodology-based optimization of glucose acylation bio-catalyzed by immobilized lipase

This paper describes in detail the selection and optimization of immobilized lipases for enhanced regioselective acylation of glucose into glucose monolaurate (GlcML). Initially, nature of biocatalyst, immobilization approach, reaction media, glucose, and lauric acid concentration were screened out. Finally, lipases from Rhizopus arrhizus immobilized on dead mycelia were investigated under various reaction conditions (Temperature, shaking speed, enzyme dose, and water content) following a fully rotatable central composite design (FRCCD) to optimize the activity of lipases. The immobilized lipases-based biocatalysts in the presence of polar solvents (tertiary alcohols) and higher concentrations of substrates i.e. glucose and lauric acid (100 and 300?mmol?L^?1, respectively) offered conversion rate of 1.5 mmolmin^?1?L^?1. Moreover, optimization of reaction conditions revealed that 162.5 lipase units/100mL at 31.25?°C, 3% water content, and 105?RPM shaking speed enhanced the conversion rate by 0.5 mmolmin^?1?L^?1 rendering the reaction more economical. Hence, lipases-based immobilized biocatalysts may provide an intelligent and green choice for commercial scale synthesis of GlcML for food and pharmaceutical industries.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.     

Design of a nonuniformly distributed biocatalyst

The properties of a nonuniformly distributed biocatalyst, where the active enzymes are immobilized on the exterior or the interior portions o a solid support, are compared with those of a conventional biocatalyst which is uniformly distributed in a spherical geometry. To investigate the performance of nonuniformly distributed biocatalysts their effectiveness factors are computed and compared for six different enzyme distribution configurations: one-half core, one-half shell, one-third center space, one-third middle annulus, one-third outer shell, and the uniformly distributed. According to the results of numerical analysis, the biocatalyst performance of the exterior "shell" configuration is always far more effective for the immobilized enzymes with positive order reaction kinetics such as Michaelis-Menten and competitive product inhibition. However, in the case of negative order enzymatic reaction kinetics such as substrate inhibition, the interior "core" configuration of the biocatalyst can render far greater enzyme utilization efficiency.     

Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases

Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).     

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 K_m 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.