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     

Effect of Immobilisation on the Production of α-Amylase by an Industrial Strain of Bacillus amyloliquefaciens

The effect of immobilisation of an industrial strain of Bacillus amyloliquefaciens in calcium alginate beads on production of α-amylase was investigated using lactose-based media in shake flasks and in a 0.3 dm³ glass fermenter. Although the microorganism was a good α-amylase producer in batch cultures of free cells, it was unable to produce the enzyme for extended periods either in repeated batch cultures, or in continuous cultivation. In each case, parallel tests with cells immobilised in calcium alginate beads gave still further reduced enzyme yields, and the free cells released into the broth from these beads probably contributed substantially to any amylase produced during these extended fermentations. After prolonged use, the core of the alginate beads accumulated hard insoluble material, with viable immobilised cells confined to a surface layer.     

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.     

Poly(allyl glycidyl ether-co-ethylene glycol dimethacrylate) copolymer beads as support for covalent immobilization of l-aminoacylase

Porous epoxy-activated copolymer beads were synthesized as support for the covalent immobilization of Aspergillus melleus l-aminoacylase. Here, a series of copolymer bead were synthesized using either glycidyl methacrylate (GMA) or allyl glycidyl ether (AGE) as monomer units and ethylene glycol dimethacrylate (EGDM) as cross-linking agent. The effect of monomer used and the effect of amount of cross-linking agent on covalent immobilization of aminoacylase were studied. Furthermore, the effect of porogen on immobilization of aminoacylase was also evaluated. AGE-co-EGDM copolymer beads gave higher binding of aminoacylase than GMA-co-EGDM copolymer beads. AGE-co-EGDM copolymer beads synthesized with lauryl alcohol as porogen and having 150% cross-linked density (i.e. AGE-(L)-150) gave maximum enzyme binding. Under optimum conditions, AGE-(L)-150 copolymer beads gave about 130 U/g of aminoacylase activity which corresponds to 72.24% of activity yield. Immobilized aminoacylase showed a broader pH, higher temperature and extended storage stability.     

Immobilization of malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103: properties and application in starch hydrolysis

BACKGROUND: A malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103 immobilized in calcium alginate beads was repeatedly used in batch processes of starch hydrolysis. The degree of starch degradation and operational stability of the immobilized system were optimized by varying the physical characteristics and composition of the beads. The products formed from hydrolysis of various starches by α‐amylase immobilized in different supports were analyzed. RESULTS: Immobilized beads prepared from 3% (w/v) alginate and 4% (w/v) CaCl₂ were suitable for up to 10 repeated uses, losing only 25% of their efficiency. On addition of 1% silica gel to alginate prior to gelation, the operational stability of the immobilized enzyme was enhanced to 20 cycles of operation, retaining > 90% of the initial efficiency. Distribution of malto‐oligosaccharides in the starch hydrolyzate depended on the type of starch, reaction time and mode of immobilization. Soluble starch and potato starch formed a wide range of malto‐oligosaccharides (G1–G5). Starches from wheat, rice and corn formed a narrow range of smaller oligosaccharides (G1–G3) as the major products. CONCLUSION: The immobilized beads of α‐amylase from KCC103 prepared from alginate plus silica gel showed high efficiency and operational stability for hydrolysis of starch. This immobilized system is useful for production of malto‐oligosaccharides applied in the food and pharmaceutical industries. Since this KCC103 amylase can be produced at low cost utilizing agro‐residues in a short time and immobilized enzyme can be recycled, the overall cost of malto‐oligosaccharide production would be economical for industrial application. Copyright © 2008 Society of Chemical Industry     

Poly(hydroxyethyl methacrylate‐co‐glycidyl methacrylate) reactive membrane utilised for cholesterol oxidase immobilisation

Poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) p(HEMA–GMA) membrane was prepared by UV‐initiated photopolymerisation of 2‐hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) in the presence of an initiator, azobisisobutyronitrile (AIBN). Cholesterol oxidase was immobilised directly on the membrane by forming covalent bonds between its amino groups and the epoxide groups of the membrane. An average of 53 µg of enzyme was immobilised per cm² of membrane, and the bound enzyme retained about 67% of its initial activity. Immobilisation improved the pH stability of the enzyme as well as its temperature stability. The optimum temperature was 5 °C higher than that of the free enzyme and was significantly broader. The thermal inactivation rate constants for free and immobilised preparations at 70 °C were calculated as k_{i (free)} 1.06 × 10^?1 min^?1 and k_{i (imm)} 2.68 × 10^?2 min^?1, respectively. The immobilised enzyme activity was found to be quite stable in the repeated experiments. © 2002 Society of Chemical Industry     

Optimization of L-Asparaginase Immobilization onto Calcium Alginate Beads

In this study, anti-leukemic enzyme L-asparaginase (E.C.3.5.1.1) from Escherichia coli ATCC 11303 was modified by the microencapsulation technique onto calcium alginate beads. Using response surface methodology (RSM), a three-level full factorial design, the values of concentration of sodium alginate, concentration of calcium chloride, and enzyme loading were investigated to obtain the highest residual L-asparaginase (L-ASNase) activity % (immobilized enzyme activity/free enzyme activity). The effects of the studied factors on immobilization were evaluated The predicted values by the model were close to the experimental values, indicating suitability of the model. The results presented that an increase in sodium alginate concentration increased the percent of residual activity of L-ASNase at any given calcium chloride concentration and the moderate amount of enzyme loading increased the percent residual activity. The optimal immobilization conditions were as follows: sodium alginate 1.98% (w/v), calcium chloride concentration 3.70% (w/v), and enzyme load 46.91% (v/v). The highest residual L-ASNase activity % obtained was 34.49%.     

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     

Chitosaneous hydrogel beads for immobilizing neutral protease for application in the preparation of low molecular weight chitosan and chito‐oligomers

Enzyme hydrolysis with immobilized neutral protease was carried out to produce low molecular weight chitosan (LMWC) and chito‐oligomers. Neutral protease was immobilized on (CS), carboxymethyl chitosan (CMCS), and N‐succinyl chitosan (NSCS) hydrogel beads. The properties of free and immobilized neutral proteases on chitosaneous hydrogel beads were investigated and compared. Immobilization enhanced enzyme stability against changes in pH and temperature. When the three different enzyme supports were compared, the neutral protease immobilized on CS hydrogel beads had the highest thermal stability and storage stability, and the enzyme immobilized on NSCS hydrogel beads had the highest activity compared to those immobilized on the other supports, despite its lower protein loading. Immobilized neutral protease on all the three supports had a higher K_m (Michaelis‐Menten constant) than free enzyme. The V_max (maximum reaction velocity) value of neutral protease immobilized on CS hydrogel beads was lower than the free enzyme, whereas the V_max values of enzyme immobilized on CMCS and NSCS hydrogel beads were higher than that of the free enzyme. Immobilized neutral protease on CS, CMCS, and NSCS hydrogel beads retained 70.4, 78.2, and 82.5% of its initial activity after 10 batch hydrolytic cycles. The activation energy decreased for the immobilization of neutral protease on chitosaneous hydrogel beads. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3743–3750, 2006     

Immobilization of lipase with alginate hydrogel beads and the lipase‐catalyzed kinetic resolution of α‐phenyl ethanol

The immobilization of enzymes is one of the key issues in both the field of enzymatic research and industrialization. In this article, we report a facile method for immobilizing Candida antarctica lipase B in an alginate carrier. In the presence of calcium cations, an enzyme–alginate suspension was crosslinked to form beads with a porous structure at room temperature, and the enzymes were well dispersed in the beads. The chiral resolution of α‐phenyl ethanol in the organic phase was tested by the enzyme–alginate beads. The effects of the reaction parameters, such as the enzyme concentration, temperature, and molar ratio of the substrate to the solvent, on the resolution behavior are discussed. Reuse cycle experiments for the chiral resolution of α‐phenyl ethanol demonstrated that the activity of the enzyme–alginate beads was maintained without marked deactivation up to five repeated cycles. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40178.     

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 µg g⁻¹ bead and the retained activity of the immobilized enzyme was found to be 44%. The K_m values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the V_max values were found to be 1890 and 760 U mg⁻¹ for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 °C and for the immobilized enzyme was 45 °C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. © 2000 Society of Chemical Industry     

Dual‐responsive semi‐interpenetrating network beads based on calcium alginate/poly(N‐isopropylacrylamide)/poly(sodium acrylate) for sustained drug release

To improve the mechanical strength of natural hydrogels and to obtain a sustained drug‐delivery device, temperature‐/pH‐sensitive hydrogel beads composed of calcium alginate (Ca‐alginate) and poly(N‐isopropylacrylamide) (PNIPAAm) were prepared in the presence of poly(sodium acrylate) (PAANa) with ultrahigh molecular weight (M_η ≥ 1.0 × 10⁷) as a strengthening agent. The influence of PAANa content on the properties, including the beads stability, swelling, and drug‐release behaviors, of the hydrogels was evaluated. Scanning electron microscopy and oscillation experiments were used to analyze the structure and mechanical stability of the hydrogel beads, respectively. The results show that stability of the obtained Ca‐alginate/PNIPAAm hydrogel beads strengthened by PAANa the alginate/poly(N‐isopropyl acrylamide) hydrogel bead (SANBs) was significantly improved compared to that of the beads without PAANa (NANBs) at pH 7.4. The swelling behavior and drug‐release capability of the SANBs were markedly dependent on the PAANa content and on the environmental temperature and pH. The bead sample with a higher percentage of PAANa exhibited a lower swelling rate and slower drug release. The drug release profiles from SANBs were further studied in simulated intestinal fluid, and the results demonstrated here suggest that SANBs could serve as a potential candidate for controlled drug delivery in vivo. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Properties of native and immobilised preparations of β-D-glucosidase from Aspergillus niger

The enzyme β-D-glucosidase from Aspergillus niger has been immobilised through its carbohydrate moiety on concanavalin A-Sepharose and on cyanogen bromide-activated Sepharose after aminoalkylation of the carbohydrate side chains of the enzyme. For comparison, the enzyme was also immobilised on microcrystalline cellulose through its protein moiety. High retention of activity and a decrease in K_m and V_max. were observed when β-D-glucosidase was immobilised by these methods. An increase in the thermal stability of the immobilised β-D-glucosidase preparations over the soluble enzyme was achieved if it was treated with glutaraldehyde before its adsorption on concanavalin A-Sepharose or if the enzyme immobilised on cyanogen bromide-activated Sepharose was subsequently treated with glutaraldehyde. Treatment of β-D-glucosidase immobilised on microcrystalline cellulose with glutaraldehyde hardly increased its thermal stability over the soluble enzyme.     

Removal efficiency of heavy oil by free and immobilised microorganisms on laboratory‐scale

This study explored free and immobilised microorganisms to degrade heavy oil. Two oil‐degrading bacterial strains (W‐1 and W‐2) were isolated from heavy oil wastewater samples collected from Shengli Oil Field in China. W‐1 and W‐2, identified as Rhosococcus sp. and Bacillus cereus sp., respectively, were tested for their growth behaviour and optimal growth conditions in the laboratory. The obtained results showed that the optimal growth conditions for W‐1 and W‐2 were identified as pH of 8, temperature of 40°C, and salinity of 2% and 4%, respectively. The environmental conditions affecting oil‐degrading efficiency by W‐1 and W‐2 were optimised in the media containing 0.3% heavy oil. The results showed that the optimal degradation and optimal growth conditions were similar, and the oil degradation rates of W‐1 and W‐2 were about 34.6% and 45.3%, respectively after 5 days. W‐1 and W‐2 capable of degrading oil was immobilised in calcium alginate gel beads containing active carbon and used for degradation of heavy oil. The heavy oil biodegradability of immobilised bacteria improved dramatically, compared with that of the free ones. The heavy oil biodegradation rates of immobilised W‐2 were found to be maximal at the same optimal growth conditions of pH, temperature, and salinity as the free ones. The best biodegradation rate of immobilised W‐2 reached above 78%, which is 33% than that of the free W‐2. © 2011 Canadian Society for Chemical Engineering     

乙酸乙烯酯接枝改性海藻酸钙凝胶微球

为了降低海藻酸钙凝胶微球的溶胀度,以乙酸乙烯酯（VAc)对海藻酸钠进行自由基接枝共聚,进而制备具有较低溶胀度的聚乙酸乙烯酯改性海藻酸钙(Ca-SA-PVAc)凝胶微球。红外光谱表明,改性之后海藻酸钙的分子上生成新的化学键;热重分析表明,改性微球受热失水行为发生变化,热稳定性提高;扫描电镜表明,改性微球结构孔隙结构发达;接枝反应条件如反应温度、VAc的浓度、引发剂用量、海藻酸钠浓度、钙离子浓度及反应时间等对改性凝胶微球在生理盐水中的抗溶胀性具有不同程度的影响。通过改变反应条件以控制接枝反应参数,可以获得溶胀行为可控的改性海藻酸钙凝胶微球。     

Studies on biodegradation of phenol using response surface methodology

Biodegradation of phenol was studied using Pseudomonas pictorum (NCIM 2077) immobilized on alginate and activated carbon – alginate beads. Control experiments were also performed using free cells and non‐inoculated activated carbon – alginate beads. The entrapped alginate and activated carbon – alginate beads suffer from a concentration gradient for oxygen in the interior of the beads and hence free cells showed better degradation at lower concentrations of phenol. The results on entrapped alginate beads were modeled using response surface methodology which determines the dependency of the maximum percentage of phenol degraded as a function of the independent variables, namely initial phenol concentration, initial pH, temperature, and diameter of the immobilized beads. The predicted values are in close agreement with the experimental values with the coefficient of correlation equal to 0.9999 and 0.9993 for both P pictorum – alginate beads and activated carbon – P pictorum – alginate beads respectively. © 2002 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.     

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     

Immobilised cellulolytic and hemicellulolytic enzymes from Macrophomina phaseolina

Cellulolytic enzymes from Macrophomina phaseolina were immobilised in acrylamide polymer. The immobilised enzyme preparation showed activity towards filter paper and cotton. However, the degree of hydrolysis of highly organised cellulose, particularly cotton, appears to be low in comparison with that of soluble substrate. The kinetic studies of immobilised enzymes indicated the presence of diffusional limitations by the increase in V_max as the particle size decreased. The operational studies suggested that the immobilised enzymes retained the original activities up to 25–29 times in the reuse cycle.     

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.