Lipase immobilization on glutaraldehyde activated graphene oxide/chitosan/cellulose acetate electrospun nanofibrous membranes and its application on the synthesis of benzyl acetate.

In this research, lipase Km12 was immobilized on the glutaraldehyde-activated graphene oxide/chitosan/cellulose acetate nanofibers (GO/Chit/CA NFs) prepared by the electrospinning method. This immobilized lipase exhibited a higher activity value than the free lipase in the acidic pH region. This enzyme showed a 10 °C shift in the maximum temperature activity. Results displayed that the Vmax value of NFs-lipase was 0.64 µmol/min, while it was gained 0.405 µmol/min for the free lipase. The activity of NFs-lipase was reserved 100% after 10 min maintaining at 60 °C, in which the free lipase only kept 75% of its original activity. Moreover, a 20% enhancement in the lipase activity was observed for NFs-lipase after 180 min of incubation at 60 °C, compared to the free enzyme. Reusability studies exhibited that the immobilized lipase well-kept 80% of its original activity after 10 cycles of reusing. Results displayed that 14% of the protein was leaked from NFs-lipase at the same condition. Transesterification results indicated that the free lipase exhibited 65% and 85% conversation level of benzyl acetate after 12 and 24 h of incubation. Besides, the immobilized lipase showed 80% and 95% conversation level at the same condition. These results indicated the high performance of free and immobilized lipase in the production of benzyl acetate for applications in the perfume and cosmetic industries.

One-pot synthesis and biochemical characterization of a magnetic collagenase nanoflower and evaluation of its biotechnological applications.

Recently, hierarchical magnetic enzyme nanoflowers have been found extensive attention for efficient enzyme immobilization due to high surface area, low mass transfer limitations, active site accessibility, promotion of the enzymatic performance, and facile reusing. Herein, we report the purification of the Bacillus collagenase and then synthesis of magnetic cross-linked collagenase-metal hybrid nanoflowers (mcCNFs). The catalytic efficiency (kcat/Km) value of the immobilized collagenase was 2.2 times more than that of the free collagenase. The collagenase activity of mcCNFs enhanced about 2.9 and 4.6 at 85 and 90 °C, respectively, compared to free collagenase. Thermal stability of mcCNFs increased about 31% and 24% after 3 h of incubation at 50 and 60 °C, respectively. After 10 cycles of reusing, the mCNFs collagenase showed 83% of its initial activity. Results showed that the mcCNFs revealed 1.4 times more activity than the free collagenase in 0.16% protein waste. Furthermore, the hydrolysis value of chicken pie protein wastes by the immobilized enzyme obtained 4 times more than the free collagenase after 240 min incubation at 40 °C. Finally, our results showed that the construction of mcCNFs is an efficient method to increase the enzymatic performance and has excessive potential for the hydrolysis of protein wastes in the food industry.

Lipase immobilization on a novel class of Zr-MOF/electrospun nanofibrous polymers: Biochemical characterization and efficient biodiesel production.

In this study, due to the favorable properties of MOF compounds and fibrous materials, new nanostructures of Zr-MOF/PVP nanofibrous composites were synthesized by electrospinning procedure. The related features of these samples were characterized by relevant analyzes, including SEM, BET surface area analysis, XRD, and FTIR spectroscopy. The final product showed significant properties such as small particle size distribution, large surface area, and high crystallinity. This strategy for producing these nanostructures could lead to new compounds as novel alternative materials for biological applications. Lipase MG10 was successfully immobilized on the mentioned nanofibrous composites and biochemically characterized. The lipase activity of free and immobilized lipases was considered by measuring the absorbance of pNPP (500 µM in 40 mM Tris/HCl buffer, pH 7.8, and 0.01% Triton X100) at 37 °C for 30 min. Different concentrations of glutaraldehyde, different crosslinking times, different times of immobilization, different enzyme loading, and different pH values have been optimized. Results showed that the optimized immobilization condition was achieved in 2.5% glutaraldehyde, after 2 h of crosslinking time, after 6 h immobilization time, using 180 mg protein/g support at pH 9.0. The immobilized enzyme was also totally stable after 180 min incubation at 60 °C. The free enzyme showed the maximum activity at pH 9.0, but the optimal pH of the immobilized lipase was shifted about 1.5 pH units to the alkaline area. The immobilized lipase showed about 2.7 folds (78%) higher stability than the free enzyme at 50 °C. Some divalent metal ions, including Cu2+ (22%), Co2+ (37%), Mg2+ (12%), Hg2+ (11%), and Mn2+ (17%) enhanced the enzyme activity of immobilized enzyme. The maximum biodiesel production (27%) from R. communis oil was obtained after 18 h of incubation by lipase MG10. The immobilized lipase displayed high potency in biodiesel production, about 83% after 12 h of incubation. These results indicated the high potency of Zr-MOF/PVP nanofibrous composites for efficient lipase immobilization.

Cross-linked Enzyme Aggregates of Fibrinolytic Protease BC1 Immobilized on Magnetic Chitosan Nanoparticles (CLEAs-Fib-mChi): Synthesis, Purification, and Characterization.

Bacterial fibrinolytic proteases achieved more attention in the prevention and treatment of cardiovascular diseases, so purification, characterization, and activity enhancement are of prime importance. In this study, a fibrinolytic serine metalloprotease was purified from the culture supernatant from Bacillus sp. BC1. It was purified to homogeneity by a two-step procedure with a 24-fold increase in specific activity and a 33.1% yield. It showed 28 kDa molecular weight, while its optimal pH and temperature were obtained 8 and 50-60 °C. The cross-link enzyme aggregates of this fibrinolytic BC1 successfully immobilized on magnetic chitosan nanoparticles. A 52% activity enhancement was obtained by immobilized enzyme at pH 6.0, compared to free protease. Km values of the free and immobilized proteases were obtained about 0.638 and 0.61 mg/ml, respectively. The free and immobilized enzymes did not show any activity concerning transferrin, γ-globulins, and hemoglobin, as blood plasma proteins. The in vitro blood clot lysis test of the free and immobilized proteases showed a maximum of 42 and 50% clot lysis, which was comparatively higher than that revealed by streptokinase and heparin at the same condition. These results indicated that the free and immobilized proteases have the potential to be effective fibrinolytic agents.

One-pot synthesis and biochemical characterization of protease metal organic framework (protease@MOF) and its application on the hydrolysis of fish protein-waste.

A protease from Bacillus sp. CHA410 was purified and immobilized by a one-step MOF-embedded approach. The immobilized protease characterized using transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). The optimal pH activity of protease CHA410 and protease@MOF was obtained at pH 8.0 and 9.0, respectively. Thermostability results after 160 min incubation showed that a 25 and 41 % enhancement in the relative activity of protease@MOF observed at 60 and 70 °C, respectively, compared to free protease. Km of the free and immobilized protease@MOF in the presence of casein was 0.685 and 0.033 mg/mL, respectively. Also, Km of the free and immobilized protease@MOF in the presence of fibrin was 0.292 and 0.145 mg/mL, respectively. The Fibrinolytic activity/Caseinolytic activity ratio (F/C ratio) of the free and immobilized proteases was 0.36 and 0.43, respectively. Protease activity of both forms of the enzyme was increased in the presence of some divalent cations, including Ca2+, Mn2+, Mg2+, and Zn2+ ions, while it intensely diminished by phenylmethylsulfonyl fluoride (PMSF), proposed as serine-protease. A 10 and 82 % enhancement in protease activity of free and immobilized proteases was achieved in the presence of butanol, respectively. Storage stability results showed that the immobilized enzyme retained about 70 % of its original activity at the end of this period, while the free enzyme only showed 22 % of its initial activity. The hydrolysis degree of immobilized protease CHA410 in the hydrolysis of fish protein waste was obtained about 46 % after 2 h of incubation at 50 °C. In comparison, it was gained about 20 % for protease CHA410 at a similar situation. Finally, results indicated that the free and immobilized protease could be used in the food industry for the hydrolysis of fish protein waste.

Fabrication of an efficient and sensitive colorimetric biosensor based on Uricase/ Th-MOF for uric acid sensing in biological samples.

Recently, nanomaterial-based artificial enzymes have expected abundant consideration because of cheap, accessibility, and respectable stability. In this study, we report a Th-MOF artificial peroxidase, which can oxidize 3,3,5,5-tetramethylbenzidine by hydrogen peroxide to yield a blue product. The catalytic behavior of Th-MOF tracked Michaelis-Menten equation and the affinity of this nanozyme to the substrate was higher than horseradish peroxidase as a natural enzyme. The absorbance value of oxidized TMB is linearly associated with the hydrogen peroxide concentration. Since, hydrogen peroxide is the oxidative end product of uric acid (UA) by uricase, an efficient and sensitive approach for uric acid determination was also established. Results showed that Km value of Th-MOF with TMB as a substrate is much lower than that of other mentioned catalysts. The linear regression equations for uric acid substrate was stated as A = 0.0039C + 0.0519 with a correlation coefficient of 0.9955. The linear range for uric acid was from 4.0 to 70 µM, and the LOD was measured as 1.15 µM. Furthermore the absorbance of assay reaction was approximately constant in the following four cycles, demonstrating that Th-MOF catalyst has outstanding stability. Results showed that the public interfering substances had no obvious absorbance values and it was less than 0.1. Results indicated that the recoveries for UA in serum fluids were between 93.10% and 99.04%. The relative standard deviation (RSD, n = 3) at each concentration value was less than 4.3%. UA determination in serum fluids has been confirmed through a comparison between the recommended technique and tedious clinical approaches. Good recovery and accuracy of UA measurement indicated that this established colorimetric sensing system is appropriate for UA revealing in actual experimental samples.

Construction of CLEAs-lipase on magnetic graphene oxide nanocomposite: An efficient nanobiocatalyst for biodiesel production.

In the present work, cross linked enzyme aggregates of Lipase (CLEAs-lip) was synthesized and immobilized on the magnetic amino functionalized graphene oxide (maGO-CLEAs-lip) nanocomposites. The immobilized lipase showed a broad range of temperature activity about 40-60 °C, as compared to free enzyme. In the case of maGO-CLEAs-lip nanocomposite, the observed lower Km value state 2.25 folds affinity for the p-nitrophenyl palmitate. Enzyme activity of maGO-CLEAs-lip nanocomposite was the highest up to 5 cycles. Storage stability results displayed maGO-CLEAs-lip retained about 75% of its original activity after 30 days of incubation. Remarkably, maGO-CLEAs-lip formed the highest biodiesel construction (78%) from R. communisoil after 24 h of incubation. The biodiesel yield of this nanocomposite was 3.0 folds higher than free enzyme, making it talented as an excellent nanobiocatalyst for efficient production of biodiesel.

Ultrasound assisted reverse micelle efficient synthesis of new Ta-MOF@ Fe3O4 core/shell nanostructures as a novel candidate for lipase immobilization.

In the present study, Ta-MOF@Fe3O4 core/shell nanostructures were synthesized in optimal conditions using the rapid, efficient, and novel ultrasound assisted reverse micelle method. FTIR, TGA/DTG, XRD, TEM, EDS and N2 adsorption/desorption isotherms were conducted in order to obtain samples with desirable properties. Results showed that the synthesized products had the thermal stability of 200 °C, particle-size distribution of 38 nm and surface area of 740 m2/g. Also, the VSM test showed that these compounds have desirable magnetic properties which provide the opportunity for recovery. Based on these obtained properties, final products were used as a novel candidate for enzyme immobilization. Results of SEM images revealed that the Bacillus licheniformis Km12 lipase is efficiently loaded on the Ta-MOF@Fe3O4 core/shell substrate. The stability test indicated the high stability of the enzyme loaded into these nanostructures. The synthesis method and the results obtained from enzyme immobilization developed in this study can be a new strategy for various applications of these novel compounds in diverse biological fields.

Improve Salinivibrio zinc-metalloprotease function in less polar organic solvents by increasing surface hydrophobicity.

Organic solvents tend to strip water from protein and thereby disrupt non-covalent forces and decrease enzyme activity and stability. In the present study, we have replaced the surface charge residues in Salinivibrio zinc-metalloprotease (SVP) with hydrophobic ones (E12V, D22I, D24A and D310I) in order to study the effects of surface hydrophobicity with hydrophobic strength of organic solvents. Compared to SVP, D24A exhibited an increase in kcat and catalytic efficiency and a reduction in thermal inactivation rate in aqueous solvent. Structural studies indicated that the replacement of surface charge residues with hydrophobic residues would not induce conformational changes. C50 value (the value of solvent concentration where 50% of enzyme activity remains), ki (irreversible thermoinactivation rate), and kinetic parameters of E12V, D22I, and D24A were higher in isopropanol and n-propanol. D24A is found to be the most efficient mutant for its remarkable decrease in ki value in the presence of isopropanol and n-propanol and a reduction in ki value in the presence of dimethylformamide (DMF) and methanol. C50 value in this variant was increased about 1.2% in DMF, 2% in methanol and isopropanol and 2.5% in n-propanol. Results revealed that, there was a correlation between surface hydrophobicity of SVP and hydrophobic strength of organic solvents.

Biochemical characterization of a novel cold-active, halophilic and organic solvent-tolerant lipase from B. licheniformis KM12 with potential application for biodiesel production.

Nowadays, finding a talented lipase with high potency in biodiesel production has been attracted to researchers. In this study, an extracellular salt tolerant, cold-active and organic solvent-stable lipase producing bacterium was isolated from oil-contaminated environments located in Kerman province (Iran) and identified as Bacillus licheniformis KM12. Lipase was purified by 36.0% recovery and molecular weight was estimated about 33 kDa by SDS-PAGE. The optimal pH and temperature for lipase activity were found to be 8.0 and 35 °C, respectively. Lipase activity was remarkably increased about 37% after 20 min incubation at 20 °C. Km value of KM12 lipase was 0.53 mM for p-nitrophenyl palmitate (p-NPP) and maximum activity was detected for pNP-decanoate and glyceryl-decanoate substrates. KM12 lipase displayed significant stability in different organic solvents after 7 and 21 days of incubation, especially in polar organic solvents. Biodiesel production with 78% yields was achieved with the one-step addition of methanol at around 18 h by using Myrtus oil (as a non-edible oils feedstocks). These unique properties of KM12 lipase make it talented as a potential biocatalyst for biodiesel production.

Production and characterization of an acido-thermophilic, organic solvent stable cellulase from Bacillus sonorensis HSC7 by conversion of lignocellulosic wastes.

The acidophilic and thermophilic cellulase would facilitate the conversion of lignocellulosic biomass to biofuel. In this study, Bacillus sonorensis HSC7 isolated as the best thermophilic cellulose degrading bacterium from Gorooh hot spring. 16S rRNA gene sequencing showed that, this strain closely related to the B. sonorensis. CMCase production was considered under varying environmental parameters. Results showed that, sucrose and (NH4)2SO4 were obtained as the best carbon and nitrogen sources for CMCase production. B. sonorensis HSC7 produced CMCase during the growth in optimized medium supplemented with agricultural wastes as sole carbon sources. The enzyme was active with optimum temperature of 70 °C and the optimum CMCase activity and stability observed at pH 4.0 and 5.0, respectively. These are characteristics indicating that, this enzyme could be an acidophilic and thermophilic CMCase. Furthermore, the CMCase activity improved by methanol (166%), chloroform (152%), while it was inhibited by DMF (61%). The CMCase activity was enhanced in the presence of Mg+2 (110%), Cu+2 (116%), Triton X-100 (118%) and it retained 57% of its activity at 30% NaCl. The compatibility of HSC7 CMCase varied for each laundry detergent, with higher stability being observed in the presence of Taj® and darya®. This enzyme, that is able to work under extreme conditions, has potential applications in various industries.

L-asparaginase production in the pseudomonas pseudoalcaligenes strain JHS-71 isolated from Jooshan Hot-spring.

L-asparaginase has lots of medical and industrial applications. Ever since L-asparaginase anti-tumor activity was first demonstrated, its production using microbial systems has attracted considerable attention owing to their cost-effective and eco-friendly nature. The aim of this study is to obtain L-asparaginase producing bacteria and determining the enzyme activity. Samples were picked up from Jooshan hot springs located in the Sirch, Kerman. The L-asparaginase producing bacteria were screened on the agar medium supplied with L-asparagine and phenol red indicator dye (pH-7.0). L-asparaginase activity was detected on the basis of pink color around the colony. Enzyme production was also performed based on ammonia detection by Nessler method. Among 24 strains, there were 7 strains which could produce L-asparaginase. Sequencing of 16S rRNA showed that, the best isolates producing L-asparaginase belongs to the Pseudomonas genus. Enzyme activity after 24 and 48 h of incubation showed that Pseudomonas pseudoalcaligenes strain JHS-71 was the best strain that produced L-Asparaginase about 240 (U/ml) after 48h of incubation. Results showed that, L-Asparaginase activity enhanced about 27% in the presence of Co+2. L-asparaginase JHS-71 retained more than 50% of its initial activity in the presence of Cu+2, Mn+2, Zn+2, Mg+2 and Fe+2. Because of various applications of L-asparaginase in biotechnology, P. pseudoalcaligenes strain JHS-71 can be used as a suitable candidate in these fields.

Purification and biochemical properties of a thermostable, haloalkaline cellulase from Bacillus licheniformis AMF-07 and its application for hydrolysis of different cellulosic substrates to bioethanol production.

A thermophilic strain AMF-07, hydrolyzing carboxymethylcellulose (CMC) was isolated from Kerman hot spring and was identified as Bacillus licheniformis based on 16S rRNA sequence homology. The carboxymethylcellulase (CMCase) enzyme produced by the B. licheniformis was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography. The purified enzyme gave a single band on SDS- PAGE with a molecular weight of 37 kDa. The CMCase enzyme was highly active and stable over broad ranges of temperature (40-80ºC), pH (6.0-10.0) and NaCl concentration (10-25%) with an optimum at 70ºC, pH 9.0 and 20% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high activity in the presence of cyclohexane (134%) and chloroform (120%). Saccharification of rice bran and wheat bran by the CMCase enzyme resulted in respective yields of 24 and 32 g L-1 reducing sugars. The enzymatic hydrolysates of rice bran were then used as the substrate for ethanol production by Saccharomyces cerevisiae. Fermentation of cellulosic hydrolysate using S. cerevisiae, reached maximum ethanol production about 0.125 g g-1 dry substrate (pretreated wheat bran). Thus, the purified cellulase from B. licheniformis AMF-07 utilizing lignocellulosic biomass could be greatly useful to develop industrial processes.

Production and Characterization of a Nitrilase from Pseudomonas aeruginosa RZ44 and its Potential for Nitrile Biotransformation.

BACKGROUND: The conversion of nitriles into amides or carboxylic acids by nitrilase has taken its application into consideration, as the scope of its applications has recently been extended. OBJECTIVES: In this study, P. aeruginosa RZ44 was isolated from sewage in the Kerman which has Nitrile-degradation activity. In order to improve the nitrilase production, several optimization were done on environmental condition. Nitrilase activity was characterized against different pHs, temperatures, ions, and substrates. MATERIALS AND METHODS: Enzyme activity was evaluated by determining the production of ammonia following to the modification of the phenol/hypochlorite method. Different factors that affect production of the enzyme by P. aeruginosa RZ44 were optimized and evaluated in the culture mediums. RESULTS: The results showed that degradation of the acetonitrile by P. aeruginosa RZ44 increased the pH of the growth medium from the initial pH 7.0 to 9.37. Optimizing the medium for P. aeruginosa RZ44, it was found that glucose and starch (5 g.L-1) have strongly supported nitrilase production, compared to the control. As well, urea (5 g.L-1) and yeast extract (15 g.L-1) have favored an increased biomass and nitrilase production, as the nitrogen sources. These results show that nitrilase production increases in the pH range 5.0 to 7.0 and then start decreasing. Addition of the Mg2+, Fe2+ and Na+ has supported the biomass and nitrilase production. Co2+, Mn2+ and Cu2+ were confirmed to inhibit cell growth and enzyme production. Enzyme characterization results show that, P. aeruginosa RZ44 nitrilase exhibits comparatively high activity and stability at pH 7.0 and 40°C. Nitrilase was completely inhibited by CoCl2 and CaCl2, whereas, the inhibition in the presence of MnSO4 and CuSO4 was about 60%. Time course analysis of the nitrile conversion by the resting P. aeruginosa RZ44 cells showed that nitrile substrates (i.e. acetonitrile) was hydrolyzed within 8 h. CONCLUSIONS: these results indicate that P. aeruginosa RZ44 has the potential to be applied in the biotransformation of nitrile compounds.

Purification and characterization of a thermo- and organic solvent-tolerant alkaline protease from Bacillus sp. JER02.

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. Km and Vmax of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H2O2 (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.

Strategies for optimizing DNA hybridization on surfaces.

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.

Enhanced activity and stability in the presence of organic solvents by increased active site polarity and stabilization of a surface loop in a metalloprotease.

Salinivibrio zinc-metalloprotease (SVP) is an enzyme which was isolated from Salinivibrio proteolyticus, a moderately halophilic species from a hypersaline lake in Iran. A195E and G203D mutants were constructed to increase polarity near the active site in order to preserve the hydration layer against organic solvents [dimethylformamide (DMF), methanol, isopropanol and n-propanol]. A268P was constructed to stabilize a surface loop far from the active site and A195E/A268P was constructed to investigate the combined effects of these two mutations. Results showed that relative C(50) values of A195E increased to approximately 26 and 11% in DMF and methanol whereas an increase of approximately 32 and 41% was observed in the presence of isopropanol and n-propanol. The irreversible thermoinactivation rate (k(i)) for A195E was estimated to be 60 and 130 (x10(-3) min(-1)) in the presence of DMF and n-propanol, respectively, while k(i) for SVP was 90 and 190 (x10(-3) min(-1)). G203D exhibited similar k(i) as A195E in the presence of methanol and isopropanol, but the calculated k(i) in the presence of DMF and n-propanol was 70 and 160 (x10(-3) min(-1)), respectively. A268P and A268P/A195E variants marginally increased the thermoresistance of the enzyme in this condition.

Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization.

Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K(M) and k(cat) were calculated 535 microM and 127 s(-1) for ABTS, 53 microM and 3 s(-1) for 2, 6-DMP and 5 microM and 20 s(-1) for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80 degrees C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70 degrees C and 2.4-fold after 10 min preincubation at 80 degrees C. Preincubation of the enzyme in 70 degrees C for 30 min raised the activity four-fold with ABTS as the substrate. Also, L-dopa was used as a substrate. The enzyme was able to oxidize L-dopa with the K(M) and k(cat) of 1,493 microM and 194 s(-1), respectively.