Immobilization Parameters Research Articles

Effective production of keratinase by gellan gum-immobilised Alcaligenes sp. AQ05-001 using heavy metal-free and polluted feather wastes as substrates.

The ability of gellan gum-immobilised cells of the heavy metal-tolerant bacterium Alcaligenes sp. AQ05-001 to utilise both heavy metal-free and heavy metal-polluted feathers (HMPFs) as substrates to produce keratinase enzyme was studied. Optimisation of the media pH, incubation temperature and immobilisation parameters (bead size, bead number, gellan gum concentration) was determined for the best possible production of keratinase using the one-factor-at-a-time technique. The results showed that the immobilised cells could tolerate a broader range of heavy metal concentrations and produced higher keratinase activity at a gellan gum concentration of 0.8% (w/v), a bead size of 3mm, bead number of 250, pH of 8 and temperature of 30°C. The entrapped bacterium was used repeatedly for ten cycles to produce keratinase using feathers polluted with 25ppm of Co, Cu and Ag as substrates without the need for desorption. However, its inability to tolerate/utilise feathers polluted with Hg, Pb, and Zn above 5ppm, and Ag and Cd above 10ppm resulted in a considerable decrease in keratinase production. Furthermore, the immobilised cells could retain approximately 95% of their keratinase production capacity when 5ppm of Co, Cu, and Ag, and 10ppm of As and Cd were used to pollute feathers. When the feathers containing a mixture of Ag, Co, and Cu at 25ppm each and Hg, Ni, Pb, and Zn at 5ppm each were used as substrates, the immobilised cells maintained their operational stability and biological activity (keratinase production) at the end of 3rd and 4th cycles, respectively. The study indicates that HMPF can be effectively utilised as a substrate by the immobilised-cell system of Alcaligenes sp. AQ05-001 for the semi-continuous production of keratinase enzyme.

Imobilizacija Aspergillus Niger celulaze na Lifetech TM nosače i primena u reakciji hidrolize lignocelulozne frakcije suncokretove sačme

Cellulases are enzymes which catalyse cellulose hydrolysis and are widely used in various industry branches. Lately, their application in treatment of different agroindustrial waste materials which could serve for fuel production is being extensively explored. In order to increase their stability and cost-effectiveness of their usage, application of their immobilized forms are preferred over free enzymes. Hereby, we tested eight different LifetechTM immobilization supports differing in polarity, porosity and functional groups as carriers for Asspergillus Niger cellulase immobilization. Most promising carrier was methacrylate based, with primary amino groups, C6 "space arm" and pores with diameter of 60-120 nm - LifetechTM ECR8409F. For this support, most important immobilization parameters were investigated and after 3 h at pH 6 with initial protein concentration of 23.3 mg/g support immobilized cellulase with 406 IU/g (with carboxymethyl cellulose as a substrate) was obtained. This preparation was successfully applied in the hydrolysis of lignocellulosic fraction of sunflower seed meal, which is widely available byproduct of sunflower seed meal fractionation for protein-rich fractions production. Initial reaction rates and yields of reducing sugars were unchanged comparing to free enzyme, indicating that there were no significant diffusion limitations for substrate to approach active sites of A. Niger cellulase molecules immobilized onto LifetechTM ECR8409F support.

Immobilized cells of Rhodococcus strain UCC 0004 as source of green biocatalyst for decolourization and biodegradation of methyl orange

Abstract The bulk presence of methyl orange is contributed by anthropogenic sources which poses substantial threat to health and the environment. Locally isolated Rhodococcus strains were used as alternatives to develop greener and cost effective methyl orange removal technology. Rhodococcus strain UCC 0004 in particular demonstrated complete removal of 0.5 g/L methyl orange after 2 days of incubation and this strain exhibited 513% higher methyl orange removal efficiency under static mode in comparison to agitated mode. Adopting the technology of immobilization enabled the decolourization of higher concentration of methyl orange which resulted in 66% greater methyl orange removal activity as opposed to the non-immobilized cells. Optimization of immobilization parameters at 0.4% (w/v) alginate concentration, 0.3 M CaCl2, 3 mm bead diameter and 50 beads led to improved methyl orange-decolourizing activity and reduced the time taken to achieve complete decolourization by 67%. The immobilized beads could be reused up to nine batches without significant loss of methyl orange-decolourizing ability which is advantageous economically. The immobilized cells demonstrated higher substrate affinity (Km = 0.3771 g/L) and accelerated rate of disappearance of methyl orange (Vmax = 0.05903 g/L/h) compared to free cells. No germination inhibition was observed on Triticum aestivum and Vigna radiata indicating complete removal of dye toxicity. The decolourization of methyl orange occurred through biodegradation as supported by complete disappearance of the major peak from UV–vis spectrum. Therefore, this strain has a huge potential to be developed as biological tool for efficient removal of wastewater containing methyl orange.

Extraction of nanosilica from oil palm leaves and its application as support for lipase immobilization

The study reports the preparation of a composite consisting of magnetite coated with nanosilica extracted from oil palm leaves (OPL) ash as nanosupports for immobilization of Candida rugosa lipase (CRL) and its application for the synthesis of butyl butyrate. Results of immobilization parameters showed that ∼ 80% of CRL (84.5 mg) initially offered was immobilized onto the surface of the nanosupports to yield a maximum protein loading and specific activity of 67.5 ± 0.72 mg/g and 320.8 ± 0.42 U/g of support, respectively. Surface topography, morphology as well as information on surface composition obtained by Raman spectroscopy, atomic force microscopy, field emission scanning electron microscopy and transmission electron microscopy showed that CRL was successfully immobilized onto the nanosupports, affirming its biocompatibility. Under optimal conditions (3.5 mg/mL protein loading, at 45 ℃, 3 h and molar ratio 2:1 (1-butanol:n-butyric acid) the CRL/Gl-A-SiO2-MNPs gave a maximum yield of 94 ± 0.24% butyl butyrate as compared to 84 ± 0.32% in the lyophilized CRL. CRL/Gl-A-SiO2-MNPs showed an extended operational stability, retaining 50% of its initial activity after 17 consecutive esterification cycles. The results indicated that OPL derived nanosilica coated on magnetite can potentially be employed as carrier for lipase immobilization in replacement of the non-renewable conventionalsilica sources.

Facile immobilization of Pseudomonas fluorescens lipase on polyaniline nanofibers (PANFs-PFL): A route to develop robust nanobiocatalyst

Herein, we demonstrate the immobilization of Pseudomonas fluorescens lipase (PFL) on polyaniline nanofibers (PANFs) via physical adsorption. Polyaniline nanofibers (PANFs) were synthesized by the oxidative polymerization of aniline. The developed robust nanobiocatalyst (PANFs-PFL) exhibited eight times higher activity than free PFL. In addition, immobilization of lipase on PANFs imparted operational stability of the nanobioconjugate. The various reaction parameters for immobilization (viz. reaction time, pH, stirring rate and enzyme-support ratio) were optimized using statistical design in terms of lipase activity and loading. Furthermore, facile separation, enhanced reusability (upto 6 cycles) and thermostability (upto 75 °C) were additional advantages of the nanobioconjugate. The catalytic prowess of nanobioconjugate was examined in the kinetic resolution of (RS)-N-(4-(3-chloro-2-hydroxypropoxy)phenyl)acetamide and (RS)-1-(1-naphthyl) ethanol in comparison to free PFL. PANFs-PFL demonstrated 49.9% and 48.1% conversion for (RS)-N-(4-(3-chloro-2-hydroxypropoxy)phenyl)acetamide and (RS)-1-(1-naphthyl) ethanol, respectively which signified its importance as a nanobiocatalyst.

Magnetic biocatalysts of pectinase and cellulase: Synthesis and characterization of two preparations for application in grape juice clarification

In the present study, we prepared two different magnetic biocatalysts of pectinase and cellulase: carrier-free magnetic CLEAs (CLEA-MP*) and immobilization on glutaraldehyde-activated magnetite (Enz-Glu-MP*). The biocatalysts were compared to their magnetic properties, immobilization parameters, stability and grape juice clarification. Enz-Glu-MP* presented higher magnetic properties than CLEA-MP*, whereas this presented higher surface area and pore volume. The KM of the enzyme immobilized on Enz-Glu-MP* was 25.65mM, lower in comparison to the CLEA-MP* (33.83mM). On the other hand, CLEA-MP* was the most active and stable biocatalyst, presenting higher recovered activity (33.4% of cellulase), higher thermal stability (2.39 stabilization factor) and improved reusability (8cycles). The integration of magnetic technology with enzymatic immobilization emerges as a possibility to increase the recover and reuse of biocatalysts for application in juice technology.

Oxime-functionalized cryogel disks for catalase immobilization

Catalase is a protective enzyme against oxidative stress and converts hydrogen peroxide into water and molecular oxygen. In the current study, catalase immobilization was applied onto the oxime-functionalized cryogel disks. Cryogel disks were produced by free radical polymerization. After cutting as circular disks, oxime ligand (4-biphenylchloroglyoxime, BPCGO) was attached and oxime-functionalized cryogel disks were obtained. After optimization of several immobilization parameters such as pH, initial catalase concentration, temperature and ionic strength, maximum catalase load was detected as 261.7 ± 11.2mg/g for cryogel disk at pH5.0. Activity studies indicated that immobilization enhanced the enzyme activity in basic pH region, the temperature range of 15–35°C and at ionic strengths between 0.2 and 1.0M NaCl. Km was detected as 9.9 and 11.0mM and Vmax was 357.1 and 769.2μmol min−1 for free and immobilized catalase, respectively. kcat and Km/kcat values showed that immobilization enhanced the catalytic efficiency. Storage stability experiments demonstrated that immobilization increased the usability period. Furthermore, catalase desorption was achieved by 1.0M NaSCN at pH8.0 successfully and catalase adsorption capacity of oxime-functionalized cryogel disk was decreased by 9.9% at the end of 5 adsorption-desorption cycle.

Development of a reusable and sustainable biocatalyst by immobilization of soybean peroxidase onto magnetic adsorbent

In this work we synthesized an activated carbon/magnetite composite by a simple co-precipitation method. The activated carbon (AC) was synthesized from the solid waste obtained in the extraction process of the peroxidase enzyme and the magnetic composite was used as support for the immobilization of soybean peroxidase (SP). After the determination of the optimal immobilization parameters, a 100% yield was achieved under the following conditions: support:enzyme proportion of 1.0:0.05 g, equilibration time of 7 h, pH 3.0 (citrate buffer phosphate 0.1 mol L−1) and temperature of 50 °C. The determination of pH to the point of zero charge was also done to assist in the understanding of the immobilization process at different pH values. Several characterization techniques were used, such as thermogravimetric analysis, elemental analysis composition, X-ray powder diffraction, Fourier transform infrared spectroscopy and Scanning electron microscopy. The biocatalyst presented excellent operational stability and was reused for 11 consecutive cycles. The magnetic properties inserted in the AC contributed to the removal of the biocatalyst from the reaction medium without interfering in the adsorptive characteristics of the AC. Thus, the activated carbon/magnetite composite can be applied to different research fields with high performance.

Catalytic characteristics and application of l-asparaginase immobilized on aluminum oxide pellets

l-asparaginase from Escherichia coli (l-ASNase) was covalently immobilized on aluminum oxide pellets (AlOPs) using a cross-linking agent, glutaraldehyde. Maximum immobilization yield (85.0%) was obtained after optimizing immobilization parameters using response surface methodology (RSM). Both free and immobilized l-ASNase (AlOP–ASNase) were optimally active at 37°C and pH7.5. However, the bioconjugate exhibited enhanced activity and stability at different pH and temperatures. It had higher affinity (low Km) and was comparatively more stable in presence of some solvents (ethyl acetate, acetone, acetonitrile), metal ions (Ag+, Zn2+) and β-mercaptoethanol. AlOP–ASNase was reused in a glass column reactor for l-asparagine hydrolysis upto nine successive cycles without any loss in activity. The AlOP–ASNase was effective in lowering l-asparagine level in blanched potato chips indicating its potential use in mitigating acrylamide formation in starchy foods. This cost-effective enzyme preparation had shelf-life of more than 30days and can be effectively used in starch based food industries.

Using Response Surface Methodology for Amperometric Glucose Biosensor Construction

In this study, construction of amperometric glucose biosensor was carried out by immobilizing of
 glucose oxidase (GOD) on platinum electrode with 0.09 cm2 surface area which coated with
 polypyrrole (PPy) by cyclic voltammetry technique. Because measured current values in the
 presence of glucose would be affected from the electrode preparing and working conditions,
 experimental parameters should be optimized by response surface methodology (RSM). To this,
 State Ease Design Expert 8.0.7.1. (Serial Number:0021-6578) programe was used. PPy synthesis
 conditions of pyrrole (Py) monomer concentration and scan rate were optimized according to
 current response in presence of glucose. Optimal Py monomer concentration and scan rate for PPy
 synthesis were determined as 10 mM and 50 mV/s, respectively. Immobilization parameters such
 as concentrations of chitosan, GOD and glutaraldehyde (GAL) also were optimized by RSM as 1.0
 %, 4 mg/ml and 0.0625 %, respectively. The digital photos of electrodes at each stage were
 obtained. All electrodes well characterized in absence and in the presence of glucose by cyclic
 voltammetry and impedance techniques and it was observed that electrodes were sensitive to
 glucose molecule. Finally the effect of working pH and applied potential on the current response
 was investigated by RSM. The highest current response was obsreved when pH of glucose solution
 and applied potential were 6.0 and 0.8, respectively.

UiO-66-NH2@PMAA: A Hybrid Polymer–MOFs Architecture for Pectinase Immobilization

A hybrid polymer-MOFs architecture UiO-66-NH2@PMAA was synthesized by tethering polymethacrylic acid (PMAA) onto the surface of the metal–organic frameworks UiO-66-NH2, and it was further employed for pectinase immobilization by electrostatic interactions. The hybrid architecture was prepared through a combination of atom transfer radical polymerization (ATRP), click chemistry and post-synthetic modification (PSM). The optimal immobilization parameters were at 25 °C and pH 4.0 with 10 U/mL of pectinase for 2 h, under which pectinase showed the highest enzymatic activity (1.215 U/mg) and protein loading (448.5 mg/g), respectively. Compared with free pectinase, the immobilized was found to exhibit higher pH, thermo and storage stability. Additionally, the immobilized pectinase had remarkable reusability, the residual activity reached as high as 81% after 8 cycles continuously. Meanwhile, the desorption of immobilized pectinase can be achieved by changing pH values of the medium. Obviously, the UiO-66-NH2@PM...

Immobilization of Cellulase on a Functional Inorganic–Organic Hybrid Support: Stability and Kinetic Study

Cellulase from Aspergillus niger was immobilized on a synthesized TiO2–lignin hybrid support. The enzyme was effectively deposited on the inorganic–organic hybrid matrix, mainly via physical interactions. The optimal initial immobilization parameters, selected for the highest relative activity, were pH 5.0, 6 h process duration, and an enzyme solution concentration of 5 mg/mL. Moreover, the effects of pH, temperature, and number of consecutive catalytic cycles and the storage stability of free and immobilized cellulase were evaluated and compared. Thermal and chemical stability were significantly improved, while after 3 h at a temperature of 50 °C and pH 6.0, the immobilized cellulase retained over 80% of its initial activity. In addition, the half-life of the immobilized cellulase (307 min) was five times that of the free enzyme (63 min). After ten repeated catalytic cycles, the immobilized biocatalyst retained over 90% of its initial catalytic properties. This study presents a protocol for the production of highly stable and reusable biocatalytic systems for practical application in the hydrolysis of cellulose.

Covalent Immobilization of Laccase onto Nanofibrous Membrane for Degradation of Pharmaceutical Residues in Water

Enzymatic degradation with ligninolytic enzyme, e.g., laccase, is a potential green solution for removal of pharmaceutical compounds that are released into the environment through wastewater effluent. However, the deficiencies of using the enzyme in its free forms, such as reusability and stability, should be addressed before industrial applications. In this study, laccase was immobilized onto tailor-made polyacrylonitrile–biochar composite nanofibrous membrane through covalent bonding, and the parameters of immobilization were optimized. The obtained biocatalyst was utilized for removal of chlortetracycline (CTC), carbamazepine (CBZ), and diclofenac (DCF) at an environmentally relevant concentration in batch mode. These pharmaceutical compounds represented three main categories of pharmaceutical compounds, i.e., antibiotics, antidepressant, and anti-inflammatory. The results showed that the immobilized laccase has improved storage, temperature, and pH stability compared to free laccase. Also, it maintain...

Investigating and optimizing the immobilization of levansucrase for increased transfructosylation activity and thermal stability

Abstract Levansucrase (LS) represents a key enzyme in glycoside synthesis of novel prebiotics and β-2,6-levan. The study of the effects of immobilization parameters of LS, produced from Bacillus amyloliquefaciens , onto glyoxyl agarose-iminodiacetic acid/Cu (glyoxyl agarose-IDA/Cu) by response surface methodology revealed the significance of their interactive effects. Retention of activity was altered by interactive effects from buffer molarity/time and buffer pH/buffer molarity. The optimized immobilization conditions were identified to be a protein loading of 9.09 mg protein/g support, a buffer concentration of 608 mM at pH 6.8 and an incubation time of 49 h. Normally a reducing agent is applied to the immobilized enzyme in order to promote the formation of covalent bonds. This step was replaced with the addition of the ionic polymer polyethylenimine (PEI), which provided a better compromise between retained activity and thermal stability of the immobilized LS. Indeed, LS immobilized onto glyoxyl agarose-IDA/Cu/PEI had a retention of activity of 70.91% with a protein yield of 44.73% and an activity yield of 54.69%, while exhibiting a half-life 4.7 times higher than that of the free LS at 50 °C.

Development of nanobiocatalysts through the immobilization of Pseudomonas fluorescens lipase for applications in efficient kinetic resolution of racemic compounds

The present work reports covalent immobilization of Pseudomonas fluorescens lipase (PFL) on functionalized multiwalled carbon nanotubes (MWCNTs) as a nanobiocatalyst (NBC). This nanobiocatalyst facilitates efficient kinetic resolution of (RS)-1-phenylethanol into (S)-1-phenylethanol [C=49.7%, eep=99.5%, ees=98.1% and E value=191.4]. The immobilized preparation (MWCNTs-PFL) showed ten-fold increase in activity, thermal stability upto 80 °C and recyclability (8 cycles). MWCNTs-PFL nanobioconjugate demonstrated better stability and enhanced activity compared to covalently immobilized PFL on other matrices (silver nanoparticles, gold nanoparticles and chitosan beads) used for the study. A statistical design [response surface methodology (RSM)] employed for the optimization of enzyme immobilization parameters made this study statistically more significant. Overall, the newly developed nanobiocatalyst has applications towards the kinetic resolution of racemic compounds.

Highly effective antibacterial activity by the synergistic effect of three dimensional ordered mesoporous carbon-lysozyme composite

Aiming at developing a safe and efficient alternative to traditional drinking water disinfection, this work successfully synthesized a novel antibacterial material with high surface area, ultra large pore size and tunable loading of immobilized lysozyme. The immobilized enzymes exhibit high antibacterial efficacy without forming carcinogenic disinfection byproducts. Critical immobilization parameters were optimized to keep the activity of the immobilized enzyme at a high level. The immobilization of lysozymes on 3DOm COOH could be confirmed by the characterizations of transmission electron microscopy, X-ray diffraction and Zeta-Potential. In addition, the structural stability of lysozymes on 3DOm COOH were studied by Fourier transform infrared spectroscopy. The antibacterial performance of 3DOm COOH-Lyz were specifically investigated based on the disinfection efficacy of Staphylococcus aureus in water. The results revealed that the immobilization capacity and relative activity of immobilized lysozyme were 814mg/g carrier and 80%, respectively, under the optimal immobilization conditions. And the antibacterial material with the initial mass ratio of lysozyme and 3DOm COOH as 3:1 exhibited maximum bacteria removal efficiency (98.1%) at pH 5. Moreover, the reusability test indicated that 3DOm COOH-Lyz has certain operational stability, and remains 82% bacterial removal efficiency even in the fifth cycle, which provides a promising application for safe and efficient drinking water disinfection in small-scale and emergency water treatment.

Optimum Immobilization of <i>Candida Antartica</i> B Lipase on Natural Silica by RSM

The immobilization of the lipase of Candida antartica B (LCAB) by adsorption on a natural silica support carried out to develop the adsorbent local supports. Immobilization conditions and characterization of the immobilized enzyme were investigated. Response surface methodology (RSM) and 3-level–3-factor fractional factorial design were employed to evaluate the effects of immobilization parameters, such as immobilization time (5-25 hour), immobilization temperature (25-45°C), and enzyme/support ratio (0.1-0.5, w/w), on yield of lipase immobilization on the support. The optimum immobilization conditions were as follows: immobilization time 18 hours, immobilization temperature 20°C, and enzyme / support ratio 0.5 (w/w); with a yield immobilization of 56,13 mg / g. The immobilization lipase shows hydrolytic and synthesis satisfactory activity.

Comparison of the performance of entrapped and covalently immobilized lipase in the synthesis of pear flavor

Abstract Introduction Although aroma esters are produced mostly by a chemical method that requires the use of aggressive chemical catalysts, enzymatic esterification has been very favored during previous two decades. The main aim of this study was to investigate the commercially important pear flavor, hexyl acetate (HAc), by the catalysis of immobilized lipase samples and to optimize the immobilization and operational conditions in detail. Methods Lipases from Candida rugosa (CRL) and porcine pancreas (PPL) were immobilized using biodegradable polysaccharides, by entrapment in calcium alginate/chitosan composite gel (CRLCa-Alg/Chi and PPLCa-Alg/Chi) and by covalent binding onto the chitosan (CRLChi and PPLChi) carrier. Results Among the several organic media, the highest esterification activites were observed in heptane. HAc yield decreased when substrate concentrations were higher than 50 or 75 mM. Yield increased with increase in reaction time up to the 5th hour in the batch type reactor while it increased during the 8 h reaction time for fixed bed reactors with lower yields. Discussion and conclusions As well as synthesis conditions, immobilization parameters also affected HAc productivity. The amount of water in the microenvironment of lipase is so important not only for gaining its active conformation but only for the reaction direction.

Screening Complex Biological Samples with Peptide Microarrays: The Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings.

The generation of robust analytical data using microarray platforms strictly relies on optimal ligand-target interaction at the sensor surface, which, in turn, is inherently bound to the correct immobilization scheme of the interrogated bioprobes. In the present work, we performed a rigorous comparative analysis of the impact of peptide ligands immobilization strategy in the screening of Burkholderia cepacia complex (BCC) infections in patients affected by cystic fibrosis (CF). We generated arrays of previously validated Burkholderia derived peptide probes that were selectively oriented on polymeric coatings by means of different click-type reactions including thiol maleimide, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and strain-promoted azide-alkyne cycloaddition (SPAAC). We compared immobilization efficiency among the different chemoselective reactions, and we evaluated diagnostic performances at a statistically significant level, also in contrast to random immobilization strategies. Our findings clearly support the favorable role of correct bioprobe orientation in discriminating seronegative from infected individuals and, in the last analysis, in generating more-reliable and more-reproducible data. Spacing biomolecules from the sensor surface by means of small hydrophilic linkers also positively affects the analytical performance and leads to increased statistical significance of data. Overall, all of the click immobilization strategies that were considered displayed a good efficiency. Interestingly, SPAAC-mediated conjugation using DBCO cyclooctyne for some peptides resulted in sequence-dependent autofluorescence in the Cy5 emission range wavelength, which could be circumvented by using a different fluorescence detection channel. On the basis of our results, we critically discuss the immobilization parameters that need to be carefully considered for peptide ligand immobilization purposes.

Biotransformation of l-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae

The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62μg/ml under optimized conditions (1.5% sodium alginate, 2ml inoculum, and 2mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62μg/ml) than did wild-type I-2 (21μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.