Expression of a ß-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization.

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Combination of the mutations for improving activity of TEV protease in inclusion bodies.

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

Extraction and reimmobilization of used commercial lipase from industrial waste.

In industrial application, immobilized lipase are typically not reused and served as industrial waste after a certain process is completed. The capacity on the reusability of the spent lipase is not well studied. This current study embarks on reusing the remaining lipase from the spent immobilized enzyme. Active lipases were recovered using a simple reverse micellar extraction (RME). RME is the extraction process of targeted biomolecules using an organic solvent and a surfactant. This method was the first attempt reported on the recovery of the lipase from the used immobilized lipase. RME of the spent lipase was done using the nonionic Triton X-100 surfactant and toluene. Various parameters were optimized to maximize the lipase recovery from the used immobilized lipase. The optimum forward extraction condition was 0.075 M KCl, and backward conditions were at 0.15 M Triton X-100/toluene (pH 6, 2 M KCl) with recovery of 66%. The extracted lipase was immobilized via simple adsorption into the ethanol pretreated carrier. The optimum conditions of immobilization resulted in 96% of the extracted lipase was reimmobilized. The reimmobilized lipase was incubated for 20 h in pH 6 buffer at 50 °C of water bath shaker. The reimmobilized lipase still had 27% residual activity after 18 h of incubation, which higher thermal stability compared to the free lipase. In conclusion, the free lipase was successfully extracted from the spent immobilized lipase and reimmobilized into the new support. It exhibited high thermal stability, and the reusability of the spent lipase will promote continued use of industrial lipase and reduce the cost of the manufacturing process.

Enzymatic degradation of ginkgolic acids by laccase immobilized on core/shell Fe3O4/nylon composite nanoparticles using novel coaxial electrospraying process.

Enzyme immobilization can increase enzyme reusability to reduce cost of industrial production. Ginkgo biloba leaf extract is commonly used for medical purposes, but it contains ginkgolic acid, which has negative effects on human health. Here, we report a novel approach to solve the problem by degrading the ginkgolic acid with immobilized-laccase, where core/shell composite nanoparticles prepared by coaxial electrospraying might be first applied to enzyme immobilization. The core/shell Fe3O4/nylon 6,6 composite nanoparticles (FNCNs) were prepared using one-step coaxial electrospraying and can be simply recovered by magnetic force. The glutaraldehyde-treated FNCNs (FNGCNs) were used to immobilize laccase. As a result, thermal stability of the free laccase was significantly improved in the range of 60-90 °C after immobilization. The laccase-immobilized FNGCNs (L-FNGCNs) were applied to degrade the ginkgolic acids, and the rate constants (k) and times (τ50) were ~0.02 min-1 and lower than 39 min, respectively, showing good catalytic performance. Furthermore, the L-FNGCNs exhibited a relative activity higher than 0.5 after being stored for 21 days or reused for 5 cycles, showing good storage stability and reusability. Therefore, the FNGCNs carrier was a promising enzyme immobilization system and its further development and applications were of interest.

Recombinant ß-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv libraries.

Antibodies specific to ß-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein ß-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific binders were discovered and converted to full-size hIgG1 antibodies leading to highly stable binders with dissociation constants (Kd) in the range 10-40 nM. The antibodies were used further as ligands for affinity chromatography, where efficient and selective recovery of biologically active ß-Glucocerebrosidase from cultured media of Chinese hamster ovary cells was demonstrated. ß-Glucocerebrosidase was purified to nearly homogeneous state and had specific activity comparable to the commercially available preparations (40-44 U/mg protein). The obtained immunoaffinity sorbents have high capacity and can be easily regenerated.

Purification and immobilization of α-amylase in one step by gram-positive enhancer matrix (GEM) particles from the soluble protein and the inclusion body.

Immobilization of the enzyme benefits the catalytic industry a lot. The gram-positive enhancer matrix (GEM) particles could purify and immobilize the recombinant α-amylase in one step without changing the enzymatic character. The enzyme immobilized by GEM particles exhibited good reusability and storage stability. The denaturants dissolved some of the GEM particles and a part of the GEM particles could bear the denaturants. The GEM particles had strong binding ability to the recombination protein with the AcmA tag even when the denaturants existed. The inclusion body was dissolved by urea and then bound by the GEM particles. The GEM particles binding the recombination protein were separated by centrifugation and resuspended in the renaturation solution. GEM particles were recycled by repeating the boiling procedure used in preparing them. The recombination α-amylase without any tag was obtained by digestion and separated via centrifugation. Altogether, our findings suggest that GEM particles have the potential to function as both immobilization and purification materials to bind the soluble recombinant protein with the AcmA tag and the inclusion body dissolved in the denaturants.

"Fishing and Hunting"-Selective Immobilization of a Recombinant Phenylalanine Ammonia-Lyase from Fermentation Media.

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag.

Characterization of non-covalent immobilized Candida antartica lipase b over PS-b-P4VP as a model bio-reactive porous interface.

The design of interfaces that selectively react with molecules to transform them into compounds of industrial interest is an emerging area of research. An example of such reactions is the hydrolytic conversion of ester-based molecules to lipids and alcohols, which is of interest to the food, and pharmaceutical industries. In this study, a functional bio-interfaced layer was designed to hydrolyze 4-nitrophenyl acetate (pNPA) and Ricinus Communis (castor) oil rich in triglycerides using lipase b from Candida antarctica (CALB, EC 3.1.1.3). The attachment of CALB was performed via non-covalent immobilization over a polymer film of vertically aligned cylinders that resulted from the self-assembly of the di-block copolymer polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP). This polymer-lipase model will serve as the groundwork for the design of further bioactive layers for separation applications requiring similar hydrolytic processes. Results from the fabricated functional bio-interfaced material include cylinders with featured pore size of 19 nm, d spacing of 34 nm, and ca. 40 nm of thickness. The polymer-enzyme layers were physically characterized using AFM, XPS, and FTIR. The immobilized enzyme was able to retain 91% of the initial enzymatic activity when using 4-nitrophenyl acetate (pNPA) and 78% when exposed to triglycerides from castor oil.

Novel Synthesis Strategy for Biocatalyst: Fast Purification and Immobilization of His- and ELP-Tagged Enzyme from Fermentation Broth.

Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme; however, during the long-term fabrication process, the loss of enzyme activity is unavoidable, and the biomimetic mineralization mechanism is still poorly understood. Meanwhile, the purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, while the rapid, low-cost, and eco-friendly purification of biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Here, a binary tag composed of elastin-like polypeptide (ELP) and His-tag was presented for the first time to be fused with ß-glucosidase (Glu) to construct a recombinant Glu-linker-ELP-His (GLEH) with the aim of developing a fast synthesis strategy combining purification and immobilization processes for a biocatalyst with better stability and recyclability. The purification fold and activity recovery of GLEH reached 18.1 and 95.2%, respectively, once a single inverse transition cycling was conducted at 25 °C for 10 min. Then, efficient biomineralization of hybrid enzyme-Cu3(PO4)2 nanoflowers was realized in 15 min by the action of His-tag and ultrasonic-assisted reaction method. The activity recovery and relative activity reached the maximum at 90.3 and 111.0%, respectively. We demonstrate that the crystal growth process of a hybrid nanoflower involves obvious nucleation, self-assembly, and the Ostwald ripening process, and the enzyme GLEH acts as a "binder" to assemble Cu3(PO4)2 nanoflakes. The immobilized GLEH nanoflowers show outstanding operation stability and recyclability, and their catalytic efficiency is close to that of free Glu.

One-step immobilization and purification of genetic engineering CBD fusion EndoS on cellulose for antibodies Fc-glycan remodeling.

The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81-90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.

Enzymatic hydrolysis of inulin by an immobilized extremophilic inulinase from the halophile bacterium Alkalibacillus filiformis.

Bacterial inulinases are the key enzymes in the enzymatic hydrolysis of inulin and production of fructooligosaccharides (FOSs) and high fructose syrup (HFS). An extremophilic inulinase was purified from Alkalibacillus filiformis using 80% ethanol precipitation, ultrafiltration, and Q-Sepharose anion exchange chromatography. The purified inulinase was highly active in a wide range of pH, temperature, chemical reagents, and NaCl concentrations. The enzyme immobilization on cobalt ferrite magnetic nanoparticles (CoFe2O4 MNPs) was carried out by carrier binding method with covalent linkage and showed improved stability and reusability within a broad temperature and pH range, compared with the free enzyme. Using free and immobilized inulinases from A. filiformis, 122 g L-1 and 160 g L-1 fructose with 61% and 80% conversion, respectively, were obtained, with inulin as the substrate. The enzymatic properties, such as notable stability under extreme conditions, make the inulinase from A. filiformis a promising candidate for related biotechnological applications.

High-pressure study of magnetic nanoparticles with a polyelectrolyte brush as carrier particles for enzymes.

The recovery of enzymes from a reaction medium can be achieved in a convenient way by using magnetic nanoparticles (MNP) as carriers. Here, we present MNP with a polyelectrolyte brush composed of poly(ethylene imine) (PEI) to provide a benign environment for the immobilized enzyme molecules. Yeast alcohol dehydrogenase (ADH) has been tested for enzymatic activity when it is free in solution or adsorbed on the PEI brush-MNP. Furthermore, the effect of pressure on the enzymatic activity has been studied to reveal activation volumes, which are a sensitive probe of the transition state geometry. The results of this study indicate that the secondary structure of ADH is pressure-stable up to 9 kbar. The enzymatic activity of ADH can be analyzed using Michaelis-Menten kinetics free in solution and adsorbed on the PEI brush-MNP. Remarkably, no significant changes of the Michaelis constant and the activation volume are observed upon adsorption. Thus, it can be assumed that the turnover number of ADH is also the same in the free and adsorbed state. However, the maximum enzymatic rate is reduced when ADH is adsorbed, which must be explained by a lower effective enzyme concentration due to steric hindrance of the enzyme inside the PEI brush of the MNP. In this way, the pressure experiments carried out in this study enable a distinction between steric and kinetic effects on the enzymatic rate of adsorbed ADH.

One-step immobilization-purification of enzymes by carbohydrate-binding module family 56 tag fusion.

Immobilization of enzymes is an essential strategy with outstanding prospects in biocatalytic processes. Nontoxic, inexpensive immobilized enzyme approach is especially important for food enzymes. We here demonstrate that a carbohydrate-binding module family 56 domain (CBM56-Tag) mediates the immobilization of fusion enzymes with the curdlan (ß-1,3-glucan) particle support, thereby enabling the one-step immobilization-purification of target enzymes. CBM56-Tag exhibits an immunoglobulin-like ß-sandwich fold, which can be adsorbed by curdlan via hydrogen bond-mediated binding. The maximum adsorption capacity of a fusion chitosanase (CBM56-GsCsn46A) on curdlan is 50.72 mg/g. The immobilized enzyme could be directly used in the packed-bed reactor. This immobilization strategy utilizes a natural polysaccharide without any treatment, avoiding the negative environmental effects. Moreover, the one step immobilization-purification simplifies the purification step, which reduces the use of chemicals. Our study provides a nontoxic and inexpensive immobilization strategy for the biocatalytic reaction in food industry.

Continuous preparation and characterization of immunomodulatory peptides from type II collagen by a novel immobilized enzyme membrane reactor with improved performance.

In this study, a novel method of continuous coupling of immobilized enzymatic hydrolysis reactor and membrane separation (CIEH-MS) was used to isolate the immunomodulatory peptides from type II collagen (CII) in chick sternal cartilage. The immobilized neutral protease was successfully prepared with an activity of 400.5 U/g. The CIEH-MS system loaded with immobilized neutral protease had high operational stability with enzyme decay constant of 0.0077 and half-life of 89.61 hr. Using a CIEH-MS system, the immunomodulatory peptides were obtained with lymphocyte proliferation of 66.23%, peptide yield of 23.43%, degree of hydrolysis (DH) of 22.41%, and permeate flux of 6.17 L/m2 h. The peptide fractions were further purified and the P3-2-4 fraction (RGQLGPM) with 760.4 Da molecular weights exhibited the highest lymphocyte proliferation activity (85.54%) and binding ability to human leukocyte antigen-DRB1 (HLA-DRB1) molecules (133.2 ng/ml). The results demonstrated that CIEH-MS system is an effective way to obtain immunomodulatory peptides from CII. PRACTICAL APPLICATIONS: Chick sternal cartilage is one of the by-products of meat processing, and it is often discarded as waste or used for low-value purposes. CII is the most abundant collagen in chick sternal cartilage, and recently studies have demonstrated that CII peptides possess the ability to induce immunologic tolerance for the treatment of chronic disease. In order to find potential applications for this by-product, immunomodulatory peptides from CII hydrolysates in chick sternal cartilage were isolated using a novel CIEH-MS system. The result showed that CII peptides exhibited a high immunomodulatory activity, and had a potential application in functional foods and medical fields.

Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3.

AmyLa α-amylase gene from Laceyella sp. DS3 was heterologously expressed in E. coli BL21. E. coli BL21 maximally expressed AmyLa after 4 h of adding 0.02 mM IPTG at 37 °C. The recombinant AmyLa α-amylase was purified 2.19-fold through gel filtration and ion exchange chromatography. We immobilized the purified recombinant AmyLa α-amylase on four carriers; chitosan had the best efficiency. The recombinant free and the immobilized AmyLa α-amylase showed optimum activity in the pH ranges of 6.0-7.0 and 4.0-7.0, respectively and possessed an optimum temperature of 55 °C. The free enzyme had activation energy, Km, and Vmax of 291.5 kJ, 1.5 mg/ml, and 6.06 mg/min, respectively. The immobilized enzyme had activation energy, Km, and Vmax of 309.74 kJ, 6.67 mg/ml, and 50 mg/min, respectively. The immobilized enzyme was calcium-independent and insensitive (relative to the free enzyme) to metals. It could also be reused for seven cycles.

A novel peroxidase from Arabian balsam (Commiphora gileadensis) stems: Its purification, characterization and immobilization on a carboxymethylcellulose/Fe3O4 magnetic hybrid material.

A novel plant peroxidase was isolated from the stem of Arabian balsam (Commiphora gileadensis) and purified using ammonium sulfate, followed by ion exchange chromatography (DEAE-Sepharose) and gel filtration (Sephcryl S-200). The newly isolated peroxidase was characterized as having a specific activity of 9503.3 unit/mg of protein after 20.3-fold purification, which yielded a recovery of 18.5%. Based on the subunit size, the purified peroxidase was a 40 kDa monomeric structure and presented high thermostability, as it was entirely stable at 55 °C for 30 min and retained approximately 13.6% of its activity at 85 °C. The optimal pH exhibited a broad value range (pH 7.0- 7.5). The kinetic parameters for the purified peroxidase were obtained. To increase the enzyme durability, efficiency and reusability, the peroxidase was entrapped onto a carboxymethyl cellulose/Fe3O4 magnetic hybrid material. The immobilized enzyme was characterized by scanning electron microscopy (SEM) and FT-IR spectroscopy. It was tested at different pH values, storage times and temperatures, and its kinetic behavior was assessed. The immobilized enzyme maintained its activity upon storage at 4 and 25 °C for 8 weeks, and upon recycling for up to 15 uses. Arabian balsam peroxidase appears to be candidate for industrial applications.

Cellulose binding domain fusion enhanced soluble expression of fructosyl peptide oxidase and its simultaneous purification and immobilization.

Hemoglobin A1c (HbA1c) is a hemoglobin molecule in which the N-terminal valine residue of the ß subunit has been grafted with the glucose in blood. Its detection has important implications for the diagnosis of diabetes. Enzymatic colorimetric method using fructosyl peptide oxidase (FPO) is simple and rapid for HbA1c detection. A FPO mutant with enhanced activity was constructed and produced by E. coli; however, most of expressed mutant FPO was insoluble. Significantly enhanced expression solubility was achieved when cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of FPO mutant. Via the high affinity interaction between CBD and cellulose, the CBD fusion also facilitated the simultaneous purification and immobilization of FPO directly from E. coli cells lysate using bacterial cellulose (BC) nanofibrils as a matrix of very high specific area. A never-dried and water durable nanocellulose film with FPO activity could be easily obtained by collecting the FPO immobilized BC nanofibrils suspension on the surface of a microfiltration membrane. The activity of the ready-use FPO nanocellulose film was stable at least 7 days at room temperature for the detection of HbA1c level of 5.3-11% in blood samples.

Fabrication of nanobiocatalyst using encapsulated laccase onto chitosan-nanobiochar composite.

Laccase is one of the widely used enzymes for biotechnological processes. Immobilization of enzymes is a universally accepted approach to increase their reusability and stability. In this study, laccase enzyme from Trametes versicolor was encapsulated for the first time in a chitosan-nanobiochar matrix. The chitosan-tripolyphosphate gel formation technique was employed to produce homogeneous biocatalyst nanoparticles, with 35% effective binding efficiency and 3.5 Units/g apparent activity under the best configuration. The reusability of the encapsulated laccase was demonstrated towards the oxidation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) for several consecutive cycles, exhibiting 30% of the initial activity after 5 cycles. The encapsulated laccase showed a moderate increase in enzyme stability against pH and temperature variation compared to the free enzyme. Moreover, the storage stability of laccase at both 4 °C and 25 °C was increased after immobilization. Only 2% of laccase was leaked during a 5-day period from biocatalyst. Laccase in its free form showed no antibacterial activity against Gram positive and Gram-negative model microorganisms, while encapsulated laccase showed antibacterial activity towards Gram-positive ones. Thus, the encapsulation of the laccase is an efficient method to keep the enzyme active and stable for different applications.