Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Summary Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml^–1) precipitated 1.2 g l^–1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.Offprint requests to: R. G. Burns     

Dye decolorisation by laccase entrapped in copper alginate

A novel immobilisation system was developed for dye decolorisation using laccase produced by Ganoderma sp. KU-Alk4. The enzyme showed high efficiency in dye decolorisation when entrapped in Cu–Al and Cu-alginate beads. The former gave the highest activity but the enzyme activity survived longer in the latter. An experimental design of two 3 × 3 Latin Square experiments was applied to evaluate the effects of three different alginate compositions (low, intermediate and high mannuronate), concentration of alginate, (1.5, 3.0 and 4.5% w/v) and concentration of cross-linking agent, CuSO₄ (0.075, 0.15 and 0.225 M) on the decolorisation of indigo carmine dye and residual laccase activity in beads. The most significant factor for residual activity was the concentration of the cross-linking agent (P < 0.05) followed by alginate composition (P < 0.1). Increasing the alginate concentration resulted in only small increase in the dye decolorisation. However, higher laccase activity remained in 3.0% w/v alginate beads. Maximal dye decolorisation was achieved when 3.6% w/v low mannuronate alginate and 0.15 M CuSO₄ was used. Optimal conditions were confirmed in an extended experimental run. Results are presented from 9 successive batch runs over 12 days, reaching 96% removal of the dye (216 mg/l).     

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Production of laccases in submerged process by <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis> PS-2001 in relation to carbon and organic nitrogen sources,antifoams and Tween 80

Some conditions in media composition for laccases production, such as different sources of carbon and organic nitrogen, antifoams and a surfactant, were studied in liquid cultures of Pleurotus sajor-caju strain PS-2001. Cultivation with fructose or glucose as carbon sources produced maximum enzyme activities of 37 and 36 U mL⁻¹, respectively. When sucrose was present in the medium, the best results were obtained using 5 g L⁻¹ of this carbohydrate, on the 11th day of the process, attaining laccase titres of 13 U mL⁻¹. In a medium without casein, practically no enzyme was produced during the experiments; among the sources of nitrogen studied, pure casein led to the highest titres of laccase activity. Different concentrations of pure casein and sucrose were also tested. As to the different concentrations of casein, the addition of 1.5 g L⁻¹ resulted in the highest titres of laccase activity. Negligible levels of manganese peroxidase activity were also detected in the culture medium. In low concentrations, polypropylene glycol or silicon-based antifoams and the surfactant Tween 80 have no significant influence on the formation of laccases by P. sajor-caju. However, enhanced concentration of polypropylene glycol negatively affected the production of laccases but favored the titres in total peroxidases, lignin peroxidase and veratryl alcohol oxidase.     

Production of laccase by a newly isolated deuteromycete fungus <Emphasis Type="Italic">Pestalotiopsis</Emphasis> sp. and its decolorization of azo dye

The effect of various carbon and nitrogen sources on the production of laccase by newly isolated deuteromycete Pestalotiopsis sp. was tested under liquid-state fermentation. Twenty grams per liter of glucose and 10 g l⁻¹ ammonium tartrate were found to be the optimized concentrations of carbon and nitrogen sources, respectively. The influence of different inducers and inhibitors on the laccase production was also examined. Adding the Cu up to optimum concentration of 2.0 mM in medium (include 20 g l⁻¹ glucose and 10 g l⁻¹ ammonium tartrate), the highest laccase activity of 32.7 ± 1.7 U ml^−l was achieved. Cu had to be supplemented after 2 days of growth for its maximal effect, an addition after 6 days of growth, during which laccase activity was dominantly formed, resulted in distinctly reduced laccase activity. In addition, Direct Fast Blue B2RL can be effectively decolorized by crude laccase, the decolorization percentage of which was 88.0 ± 3.2% at pH 4.0 within 12 h. The results suggest that Pestalotiopsis sp. is a high potential producer of the industrially important enzyme laccase.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-β-glucanase (endo-β-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a K_m value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent V_max of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced by hydrolytic efficiency to half.     

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30 °C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24 h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the “egg-box” model were observed using TEM.     

Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes

Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V _max and K _m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which creates an important environmental problem in the discharged textile dying solutions.     

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Bal?ova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.     

Laccase-mediated Remazol Brilliant Blue R decolorization in a fixed-bed bioreactor

A crude laccase mixture preparation from Pleurotus ostreatus cultures supplemented with copper and ferulic acid was used to decolorize the anthraquinonic dye Remazol Brilliant Blue R (RBBR). Performance of this enzymatic system was tested, and a maximum of 70% decolorization was achievable under optimal conditions. The crude preparation was immobilized by entrapment in copper alginate beads attaining 65% yield of laccase activity. Stability of the immobilized laccases was remarkably increased in comparison with that of the free enzyme preparation. Efficiency of the immobilized system was evaluated during stepwise dye additions in batch operations. Under the best conditions, 70% RBBR decolorization was achieved even after 20 cycles, although decolorization time exponentially increased after the 10th cycle. Different fixed-bed bioreactors were prepared and analyzed in continuous decolorization processes. The best performance was obtained by decreasing the amount of enzyme loaded and by improving laccase retention using chitosan-coated alginate beads.     

Evaluation of Chitosan/Alginate Beads Using Experimental Design: Formulation and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization

Bovine serum albumin-loaded beads were prepared by ionotropic gelation of alginate with calcium chloride and chitosan. The effect of sodium alginate concentration and chitosan concentration on the particle size and loading efficacy was studied. The diameter of the beads formed is dependent on the size of the needle used. The optimum condition for preparation alginate–chitosan beads was alginate concentration of 3% and chitosan concentration of 0.25% at pH 5. The resulting bead formulation had a loading efficacy of 98.5% and average size of 1,501 μm, and scanning electron microscopy images showed spherical and smooth particles. Chitosan concentration significantly influenced particle size and encapsulation efficiency of chitosan–alginate beads (p < 0.05). Decreasing the alginate concentration resulted in an increased release of albumin in acidic media. The rapid dissolution of chitosan–alginate matrices in the higher pH resulted in burst release of protein drug.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Decolorization of molasses spent wash by the white-rot fungus Flavodon flavus, isolated from a marine habitat

Flavodon flavus (Klotzsch) Ryvarden, a basidiomycete (NIOCC strain 312) isolated from decomposing leaves of a sea grass, decolorized pigments in molasses spent wash (MSW) by 80% after 8 days of incubation, when used at concentrations of 10% and 50%. Decolorizing activity was also present in media prepared with half-strength seawater (equivalent to 15 ppt salinity). Decolorizing activity was seen in low-nitrogen medium, nutrient-rich medium and in sugarcane bagasse medium. The percentage decolorization of MSW was highest when glucose or sucrose was used as the carbon source in the low-nitrogen medium. The production of lignin-modifying enzymes, manganese-dependent peroxidase (MNP) and laccase decreased in a medium containing MSW. MNP production and MSW decolorization were inversely correlated, suggesting no role for MNP in MSW decolorization. The decolorization of MSW was not effective when F. flavus was immobilized in calcium alginate beads. Decolorization was achieved best in oxygenated cultures. Besides color, total phenolics and chemical oxygen demand were reduced by 50% in MSW treated with F. flavus, suggesting its potential in the bioremediation of effluents.     

Immunoaffinity layering of enzymes

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae. Received: 16 November 1998 / Received revision: 22 March 1999 / Accepted: 26 March 1999     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997