Studies of L-phenylalanine production by immobilised aminoacylase in stabilized calcium alginate beads

Aminoacylase I (EC 3.5. 1.14) was immobilised by entrapment in uncoated calcium alginate beads and calcium alginate beads coated with chitosan, polyethyleneimine and polyethyleneimine-glutaraldehyde for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The operational stability, thermal stability, effects of pH and temperature and kinetic constants, K_m and V_max values of free and immobilised enzymes were studied. Scanning electron micrographs revealed differences in the structures of the surfaces of coated and uncoated beads. Chitosan-coated calcium alginate beads was found to be the best among the immobilised systems studied. The activity and the optimum temperature of immobilised aminoacylase were higher than those of the free enzyme. In addition, the beads showed stable activity under operational conditions. The immobilised aminoacylase lost about 20% of its initial activity after ten cycles of reuse. Polyethyleneimine and polyethyleneimine-glutaraldehyde treatments were also found to enhance the operational stability of the enzyme but its activity was greatly reduced.     

Biosorption of heavy metals from aqueous solution using modified activated carbon: comparison of linear and nonlinear methods

BACKGROUND: Biosorption of heavy metals from aqueous solution by modified activated carbon with Phanerochaete chrysosporium immobilised in Ca‐alginate beads was investigated using a batch system and comparison of linear and nonlinear methods. RESULTS: The amount of Cu(II), Zn(II) and Pb(II) ion sorption by the beads was as follows: activated carbon with P. chrysosporium immobilised in Ca‐alginate beads (ACFCA) (193.4, 181.8, 136.6 mg g^?1) > activated carbon immobilised in Ca‐alginate beads (ACCA) (174.8, 162.0, 130.7 mg g^?1) > P. chrysosporium (F) (148.8, 125.6, 120.4 mg g^?1) > activated carbon (AC) (138.8, 112.3, 109.3 mg g^?1) > plain Ca‐alginate beads (PCA) (125.4, 105.2, 98.2 mg g^?1). The widely used Langmuir and Freundlich isotherm models were utilised to describe the biosorption equilibrium process. CONCLUSION: The results of this study suggest that the immobilisation of modified activated carbon with P. chrysosporium in Ca‐alginate beads is suitable for a batch system. The isotherm parameters were estimated using linear and nonlinear regression analyses. The surface charge density of the biosorbents varied with the pH of the medium; the maximum biosorption of heavy metal ions on the biosorbents was obtained when the pH was between 5.6 and 7.4. Copyright © 2008 Society of Chemical Industry     

Immobilization of hybridoma cells in chitosan alginate beads

A new method for immobilizing hybridoma cells using chitosan-stabilized calcium alginate beads was developed. The ionotropic gelation of chitosan and calcium with alginate resulted in the formation of highly cross-linked, porous beads that were mechanically and chemically stable in phosphate buffered medium. Hybridomas entrapped in these beads were cultured semi-continuously using periodic medium exchange. Viable population densities in the order of 5 × I0⁷ cells/mL were attained within the beads and up to two-fold increases in volumetric monoclonal antibody (MAb) productivity over batch suspension cultures were observed. Oxygen mass transfer limitations within the chitosan-alginate beads were evaluated by considering biokinetics and diffusive transport. Model equations were developed and used to evaluate the effect of bead diameter on the contained cells. The predictions were consistent with experimental observations of maximum viable population densities attained in beads of various size.     

Development of silver nanoparticles‐loaded calcium alginate beads embedded in gelatin scaffolds for use as wound dressings

Silver nanoparticles (AgNPs)‐loaded calcium alginate beads embedded in gelatin scaffolds were developed to sustain and maintain the release of silver (Ag⁺) ions over an extended time period. The UV irradiation technique was used to reduce Ag⁺ ions in alginate solution to AgNPs. The average sizes of AgNPs ranged between ca 20 and ca 22 nm. The AgNPs‐loaded calcium alginate beads were prepared by electrospraying of a sodium alginate solution containing AgNPs into calcium chloride (CaCl₂) solution. The AgNPs‐loaded calcium alginate beads were then embedded into gelatin scaffolds. The release characteristics of Ag⁺ ions from both the AgNPs‐loaded calcium alginate beads and the AgNPs‐loaded calcium alginate beads embedded in gelatin scaffolds were determined in either deionized water or phosphate buffer solution at 37 °C for 7 days. Moreover, the AgNPs‐loaded calcium alginate beads embedded in gelatin scaffolds were tested for their antibacterial activity and cytotoxicity. © 2014 Society of Chemical Industry     

Amylolytic enzymes produced by Bacillus amyloliquefaciens MIR-41 in batch and continuous culture

Bacillus amyloliquefaciens MIR-41 has been isolated from natural sources and characterized using statistical methods. The strain showed three types of glucanase activities: α-amylase, α-glucosidase and pullulanase in defined medium and starch as carbon source. Extracellular activity of α-amylase was approximately three times higher in continuous culture than in batch. Maximal productivity was found at a dilution rate of 0·16 h^?1. Alpha-glucosidase was expressed as a cell-bound enzyme in batch culture, but in continuous culture was mostly extracellular. Pullulanase was cell-bound in all cases. The expression of the enzymes was dependent upon the system and the dilution rate used. No cell lysis was detected in the chemostat as compared with batch cultures.     

Stabilisation of β-glucosidase entrapped in alginate and polyacrylamide gels towards thermal and proteolytic deactivation

β-D -Glucosidase was immobilised by entrapment in two different matrices (calcium alginate and polyacrylamide gels), in order to compare how the immobilisation could stabilise the enzyme towards thermal and proteolytic deactivation. While the enzyme trapped in polyacrylamide gel showed an optimum temperature for activity at 10°C lower than that of the free enzyme, the optimal temperature after immobilisation in alginate beads was not altered (60°C). The immobilisation of enzyme in alginate beads caused a larger increase in the thermal stability than the entrapment in polyacrylamide gels. The stabilisation factors obtained as 55, 60 and 65°C for β-glucosidase immobilised in alginate and polyacrylamide gels were 2·03, 3·06, 2·19 and 2·04, 0·35, 1·01, respectively. In contrast, the β-glucosidase immobilised in polyacrylamide gels was more resist-ant in proteolysis than that trapped in alginate beads. © 1998 Society of Chemical Industry     

Regeneration of NADH in a bioreactor using yeast cells immobilised in alginate fibre. II: Detailed results

Saccharomyces cerevisiae cells were immobilised in calcium alginate fibres and used in a reactor as a source of alcohol dehydrogenase for the NAD⁺ to NADH reaction. Kinetic parameters were established for both the free enzyme and the fibre reactor. Detailed calculations for the free enzyme studies established the superiority of the Elmore–Kingston–Shields computer calculation of the initial rates. There was little difference between the Cornish-Bowden and the Hanes methods for subsequent generation of the kinetic parameters. Initial reaction rates that were obtained for the free enzyme gave kinetic parameters which were 65–80% below those obtained in the bioreactor. It is apparent that the diffusion limited the rate of reaction in the immobilised system. The bioreactor operated at high conversions at relatively low inlet concentrations of substrates. As the substrate concentration was raised the percentage conversion fell even though the amount of product produced overall rose substantially.     

Properties of cathepsin B immobilized in calcium alginate beads

Cathepsin B (EC 3.4.22.1), purified from goat brain, was immobilized in calcium alginate beads in the presence of bovine serum albumin. The immobilized enzyme retained ∼63% of the original activity and could be used for seven successive batch reactions with retention of 22–30% of the initial activity. Immobilized cathepsin B hydrolysed α-N-benzoyl-D ,L -arginine-β-naphthylamide (BANA) maximally at pH 5·5, exhibiting a shift of 0·5 pH unit from that of the soluble enzyme (pH optima 6·0). It showed enhanced stability in acidic as well as alkaline environments in comparison to the free enzyme. The optimal temperature and thermal stability were not altered significantly after immobilization. The K_m value for the immobilized enzyme was two-fold higher than for the soluble enzyme.     

Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

Production of lactic acid from beet molasses by calcium alginate immobilized Lactobacillus delbrueckii IFO 3202

Lactic acid was produced from pretreated beet molasses by the homofermentative organism Lactobacillus delbrueckii subsp delbrueckii IFO 3202 entrapped in calcium alginate gel using batch, repeated batch and continuous fermentation systems. In batch fermentation studies successful results were obtained with 2.0–2.4 mm diameter beads prepared from 2% sodium alginate solution. The highest effective yield (82.0%) and conversion yield (90.0%) were obtained from substrate concentrations of 52.1 and 78.2 g dm⁻³ respectively. The gel beads produced lactic acid for 14 consecutive batch fermentations without marked activity loss and deformation. In the continuous fermentation, the highest lactic acid (4.22%) was obtained at a dilution rate of 0.1 h⁻¹ while the highest productivity (13.92 g dm⁻³ h⁻¹) was obtained at a dilution rate of 0.4 h⁻¹. © 1999 Society of Chemical Industry     

The effects of oxygen on β-lactam biosynthesis by alginate entrapped streptomyces clavuligerus

The physiology of Streptomyces clavuligerus has been studied by investigating oxygen uptake and β-lactam (clavulanic acid) production by free and alginate entrapped cells. The specific oxygen uptake rate of cells entrapped in strontium or calcium alginate was only about 35% of the rate in free cells. This reduction in oxygen uptake was due to reduced internal diffusion of oxygen; in addition calcium, barium and strontium ions could also cause oxygen uptake in free cells to be inhibited. Measurements of the deformation of individual gel beads showed that maximum hardness was reached after 15 min exposure to 0.14 mol dm^?3 Ca²⁺. Exposure of cells to Ca²⁺ for this time caused a 15% reduction in the rate of oxygen uptake. Levels of clavulanic acid produced by free cells were higher than those produced by immobilized cells; production was dependent on the degree of aeration of the cells.     

Optimization of pullulan production using Ca‐alginate‐immobilized Aureobasidium pullulans by response surface methodology

BACKGROUND: The production of pullulan from synthetic medium by Aureobasidium pullulans P56 immobilized in Ca‐alginate beads was investigated using batch and repeated batch fermentation systems. RESULTS: The highest pullulan concentration (19.52 ± 0.37 g dm^?3) was obtained with 2.0‐2.4 mm beads prepared from 2% sodium alginate solution. Pullulan production was mainly accomplished by immobilized fungal cells since leaked cells in the fermentation medium comprised 17.4% of the total fungal population at the end of fermentation. The pullulan proportion was 84.5% of the total polysaccharide in the fermentation medium. Response surface methodology was used to investigate the effects of three fermentation parameters (initial pH, agitation speed and incubation time) on the concentration of pullulan. Results of the statistical analysis showed that the fit of the model was good in all cases. The maximum pullulan concentration of 21.07 ± 0.48 g dm^?3 was obtained at the optimum concentrations of process variables (pH 7.31, agitation speed 191.5 rpm, incubation time 101.2 h). The gel beads produced pullulan under the optimized conditions for six consecutive batch fermentations without marked activity loss and deformation. CONCLUSION: The results of this study suggest that the immobilization of A. pullulans cells in Ca‐alginate gel beads is suitable for batch and repeated batch production of pullulan. Copyright © 2007 Society of Chemical Industry     

Modelling of phenol biodegradation by Candida tropicalis immobilised in alginate gel beads

Phenol‐degrading yeast Candida tropicalis were immobilised in alginate gel beads and photographed by scanning electron microscopy. Batch phenol biodegradation experiments were done in shaking flasks under varying conditions such as initial phenol concentrations and bead loadings. A mathematical model was proposed to simulate the batch phenol biodegradation process in the immobilised system, which took into account the internal and external mass transfer resistances of phenol and oxygen and the double‐substrate phenol–oxygen intrinsic kinetics. The validation of this model was done by the comparison between the model simulations and the experimental measurements of phenol concentration profiles in the main liquid phase. Moreover, the time and radius courses of phenol, oxygen, and cell concentration profiles within the alginate gel beads were reasonably predicted by the proposed model.     

Production of Hexanoic Acid by Free and Immobilised Cells of Megasphaera elsdenii: Influence of in-situ Product Removal Using Ion Exchange Resin

The objective of this work was to improve the production of hexanoic acid by the anaerobic rumen bacterium, Megasphaera elsdenii, using product removal and immobilised cell approaches. Hexanoic acid, the major product of glucose metabolism by M. elsdenii strain ATCC25940, was produced at concentrations of 2–3 g dm⁻³ in stirred batch cultures. With pH controlled manually at 7, maximum concentrations of hexanoic acid increased to 6–8 g dm⁻³ with yields (g product per g glucose used) of approximately 30%. When an anion exchange resin, Amberlite IRA 400, was added during early stages of culture to minimise product inhibition, growth was not impaired and cell lysis, which was commonly seen during the stationary phase in control fermentations, was prevented. The presence of resin in pH-controlled, stirred batch fermentations increased the rate of glucose consumption and doubled hexanoic acid productivity: the equivalent of 11 g dm⁻³ of hexanoic acid was made with an estimated yield of up to 39%. Cells were immobilised successfully in κ-carrageenan and, when cell densities in inocula were sufficiently high, rates of glucose consumption and product formation were similar to free cells. Including resin in cultures of immobilised cells had effects similar to those above. Using a fed-batch mode with immobilised cells cultured in the presence of resin further increased final concentrations of hexanoic acid (up to 19 g dm⁻³) but yields were lower (20–30%) and productivity did not increase. These results show that production of volatile fatty acids can be improved significantly by product stripping onto an anion exchange resin. © 1997 SCI.     

Characterization of the structure and diffusion behavior of calcium alginate gel beads

A simple and novel method using gel shrinkage to indirectly characterize the structure of calcium alginate gel (CAG) beads during the calcium alginate gelation process was presented in this study. The effect of preparation process parameters (gelling cations, bead diameter, and alginate M _w and concentration) on the structure of the CAG bead formation process was thoroughly investigated. It was found that (a) the concentration of the Na⁺ and Ca²⁺ ion in gel bath was found to be the determining factor in the gel structure formation process by regulating the dissociation of alginate and the complexation of the calcium; (b) Na⁺ acts as a competitor with calcium and a screen in the electrostatic repulsion; (c) the effect of beads size below 700 μm on the structure of CAG beads can be neglected; and (d) the sodium alginate concentration has no significant effect on the gel formation process. Furthermore, the diffusion of bovine serum albumin (BSA) was controlled by the density of CAG bead. Consequently, a faster diffusion rate of BSA within the looser structure of beads can be observed. These results are keys to understanding the behavior and performance of beads in their utilization medium. © 2020 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48923.     

Immobilisation of α-amylase from Aspergillus niger onto polyaniline

α-Amylase from Aspergillus niger culture medium was immobilised on glutaraldehyde-modified polyaniline (PANIG-AMY) in a yield of 42% activity retention. Compared with the free enzyme, PANIG-AMY was less sensitive to inhibition by Zn²⁺, Cu²⁺ and Fe²⁺. The catalytic efficiencies of hydrolysis of starch (potato, cassava, wheat, maize and rice/maize) were similar for free and PANIG-AMY. Oligosaccharides were formed following the hydrolysis of potato starch by PANIG-AMY whereas the free enzyme produced oligosaccharides and glucose. PANIG-AMY retained 50% of its activity after repeated assay and storage at 5 °C and pH 7.0. The thermal stability of PANIG-AMY was increased by the presence of CaCl₂. The repeated batch-wise hydrolysis of potato starch using 5.0 mg of PANIG-AMY (0.8 U α-amylase) produced 1.7 μmol of reducing sugar per cycle, yielding a total of 25.5 μmol of reducing sugar after 15 cycles. The same yield would require 12.5 U of free α-amylase.     

Comparative studies on bioconversion of benzaldehyde to L‐phenylacetylcarbinol (L‐PAC) using calcium alginate‐ and barium alginate‐immobilized cells of Torulaspora delbrueckii

Comparison of the production of L ‐phenylacetylcarbinol (L ‐PAC) from benzaldehyde by cells of Torulaspora delbrueckii immobilized in calcium alginate and barium alginate showed that cells immobilized in calcium alginate beads were usable for up to six cycles compared with nine cycles for barium alginate‐immobilized cells. Barium alginate‐immobilized cells yielded a total of 2.17 g of L ‐PAC in nine cycles whereas in the case of calcium alginate‐immobilized cells, a total of 1.17 g of L ‐PAC was formed when 400 mg benzaldehyde and 400 mm³ of acetaldehyde were used in each cycle as substrate and co‐substrate respectively. Copyright © 2003 Society of Chemical Industry     

Intraparticle cell growth and cell leakage in cultures of Nicotiana tabacum cells immobilized in calcium alginate gel beads

Immobilized Nicotiona tabacum cells in calcium alginate gel beads were prepared under various conditions and then were cultivated. The effects of different conditions of preparation, in relation to concentration of calcium ions (Ca²⁺), on intraparticle cell growth and cell leakage from beads were investigated experimentally. As the amount of Ca²⁺ incorporated into the beads increased, the numbers of cells leaked from the beads into the medium decreased. However, cell growth was inhibited by high Ca²⁺ concentrations in the beads. Optimal conditions existed, which prevented cell leakage without inhibiting intraparticle cell growth. The effect of adding CaCl₂ to the culture medium was also studied. The Ca²⁺, used for the alginate crosslinking, gradually leached from the beads with increasing cultivation time, such that the beads gradually became brittle and fragile. The addition of CaCl₂ was effective in preventing Ca²⁺ loss from the beads and cell leakage. © 2000 Society of Chemical Industry     

Stabilisation in bacillus stearothermophilus of a recombinant plasmid carrying the homologous α-amylase gene

The phenotypic stability of the recombinant plasmid pAT9 (11.5 MD), which contained the cloned α-amylase gene from B. stearothermophilus, was studied in batch and continuous culture. Irrespective of the type of culture (batch or continuous; or using glucose or maltose as carbon source), deletion plasmids of the phenotype Km^rAmy^? the same size (7.3 MD) emerged with time. DNA sequencing analysis and Southern hybridization of a region adjacent to both ends of the pAT9 HindIII fragment containing the α-amylase gene showed that almost the entire part of the HindIII fragment was lost. Homology between fragments contiguous to the pAT9 HindIII terminals allowed exclusion of a fragment that did not encode the α-amylase gene and a new recombinant plasmid, pATHP9 (Km^rAmy⁺; 7.5 MD), was constructed which had enhanced phenotypic stability in B. stearothermophilus.     

Lipolytic Enzyme Production by Immobilized Rhizopus oryzae

In this paper, a suitable technique as well as an adequate material for the immobilization of the fungus Rhizopus oryzae were investigated. This organism has been shown to have potential in the field of food aroma due to the production of extracellular lipolytic enzymes. However, an efficient production system at bioreactor scale for its application to the flavor compounds production is still needed. Among the supports studied, alginate beads were the best carrier materials, leading to the highest lipolytic activities of up to 400 U/L after 3 days of cultivation. Repeated batch cultures were carried out to improve cell concentration and lipolytic activity. The gel beads produced lipolytic enzyme under optimized conditions for consecutive batch fermentations without marked activity loss and deformation attained a maximum level of 715 U/L after three batches. The production of lipolytic enzyme by immobilized Rhizopus oryzae in a 2‐litre airlift bioreactor with the optimized conditions was evaluated. Lipolytic activities of 487 U/L were attained, operating in successive batches without operational problems and the bioparticles (the fungus grows in alginate beads) maintained their shape throughout fermentation.