Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K _m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t _50%) at 70° C was approximately 8 h. Received: 9 September 1996 / Accepted: 27 December 1996     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30?KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133?KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The K_m and V_max of purified enzyme when using pNPL hydrolysis were 4.0?mM and 73.53?nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27?°C. The enzyme was stable within a pH range of 6.5 to 7 at 27?°C for 1?h. The enzyme activity was enhanced up to 36% by KCl, BaCl₂, MgCl₂ and CaCl₂ while obviously inhibited (10–20%) by CoCl₂, ZnCl₂, MnCl₂ and CuCl₂. No inhibitory effects were observed with 1.0 and 5.0?mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0?mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0?mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Thermal stability of <Emphasis Type="Italic">α</Emphasis>-amylase in aqueous cosolvent systems

The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T _m )_app and activation energy (E _a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T _m )_app were 20°C, 14°C, 13°C and 9°C, respectively. The E _a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.     

Endo-β-Glucanase from Acetobacter xylinum: Purification and Characterization

A cellulose-producing acetic acid bacterium, Acetobacter xylinum KU-1, abundantly produces an extracellular endo-β-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography, Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel filtration. The purified enzyme showed the maximum activity at pH 5 and 50°C: it was stable up to 50°C at pH 5, activated by Co²⁺, and competitively inhibited by Hg²⁺; the apparent K _i was 7 μM. The molecular weight of the enzyme was determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the enzyme is monomeric. The enzyme hydrolyzed carboxymethylcellulose with an apparent K _m of 30 mg/ml and V _max of 1.2 μM/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did not act on cellobiose, cellotriose, or cellotetraose. Received: 3 October 1996 / Accepted: 5 November 1996     

Partial Purification and Characterization of Thermostable Acid Phosphatase from Thermoacidophilic Archaeon Sulfolobus acidocaldarius

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70°C, respectively. The apparent K _m value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75°C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein. Received: 8 July 1999 / Accepted: 9 August 1999     

Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144 kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K _m of 1.67 mg/ml. The enzyme was active at a broad range of pH (5–8) and temperatures (40–80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60 h with half-lives of 14 h, 6 h and 7 min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn²⁺, K⁺, cysteine, β-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12 h at 60°C and 6 h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri

Methanosarcina barkeri is a strictly anaerobic, cytochrome-containing, methane-forming archaeon. We report here that the microorganism contains a catalase, which was purified and characterized. The enzyme with an apparent molecular mass of 190 kDa was shown to be composed of four identical subunits of apparent molecular mass of 54 kDa. The heme-containing enzyme did not exhibit peroxidase activity, which indicates that it is a monofunctional catalase. This is substantiated by the primary structure, which is related to that of other monofunctional catalases rather than to that of bifunctional catalase-peroxidases. The enzyme showed an [S]_0.5V for H₂O₂ of 25 mM and an apparent V _max of 200,000 U/mg; it was inhibited by azide ([I]_0.5V = 1 μM) and cyanide ([I]_0.5V = 5 μM) and inactivated by 1,2,4-aminotriazole. The activity was almost independent of the pH (between pH 4 and 10) and the temperature (between 15 °C and 55 °C). Comparison of the primary structure of monofunctional catalases revealed that the enzyme from M. barkeri is most closely related to the monofunctional catalase of Dictyostelium discoideum. Received: 29 December 1998 / Accepted: 1 March 1999     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10?h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K_M values of 0.60?mM and 0.65?mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12?h with a half-life of 5.80?h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Production of a Thermostable Lipase by Humicola lanuginosa Grown on Sorbitol-Corn Steep Liquor Medium

For thermostable lipase production by Humicola lanuginosa No. 3, a simple optimized medium consisting of (%, w/v): sorbitol, 1.0; corn steep liquor, 1.0; NaCl, 0.5; CaCl₂–2H₂0, 0.01; Silicone Km-70 (antifoamer), 0.2; and whale oil or castor oil as a lipase inducer, 0.3, was used. The yield of the lipase was about 80 — 120U/ml after 25 hr aerobic cultivation at 45°C when the pH was maintained at 7 to 8. The acetone powder preparation of the enzyme was most active at pH 7.0 and 45°C. The enzyme retained 100% activity on incubation for 20 hr at 60°C. The enzyme was able to hydrolyze almost all forms of natural fats tested (14 kinds), coconut oil being the most rapidly hydrolyzed.     

Characterization of an inducible nitrilase from a thermophilic bacillus

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M _r, 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying k _cat/K _M values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced k _cat, while ortho-substitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65°C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50°C, 2.5 h at 60°C, 13 min at 70°C, and less than 3 min at 80°C. Received: March 1, 1999 / Accepted: August 3, 1999     

Production,purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21?U/mL, which was ～2.3-fold higher than that of the wild strain (2.04?U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32?°C, pH 7.0, 150?rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25?mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30?kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ～21-fold, and the mean overall yield was ～6%. The purified endoglucanase was active up to 80?°C and showed a half-life of 214?min at 60?°C in the absence of substrate at pH 8.0. The apparent K_m value for the purified endoglucanase was 0.70?mg/mL, while the V_max value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30?U of proteinase K/mg. The addition of Mg⁺² and Ca⁺² (5?mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5?mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4?°C and 75 days at ?20?°C signify the suitability of this enzyme for industrial applications as detergent additive.     

Immobilization/stabilization of acid urease on Eupergit® supports

The adsorption capacity and immobilization rate of two Eupergit® supports for acid urease was studied by varying the ionic strength and enzyme preparation concentration in the immobilizing solution at pH 7. Eupergit® C250 L yielded a series of derivatives with enzyme loadings (Y_P/B) ranging from 48 to 171 mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds). Use of drastic postimmobilization conditions at pH 9 for 3–9 days yielded a slight decrease (8–14%) in the initial activity of immobilized enzymes and a limited increase in the stabilization factor (1.1–1.5), as assessed by accelerated aging tests at 65°C. Further storage tests at 4°C in the wet state showed that the activity of several derivatives either stabilized or not was practically constant for as long as 547 days. Both free enzyme and immobilized acid urease derivatives exhibited a kinetic pattern of the Michaelis–Menten type. Using the Eadie–Hofstee diagram, the specific ammonia formation rate constant for free (k_cat) or immobilized (k′_cat) enzyme resulted to be little affected by immobilization (k_cat ≈ k′_cat ≈ 18.86 ± 0.34 IU/mg BSAE), whereas the apparent Michaelis constant for immobilized enzymes exhibited a statistically significant increase at P < 0.05 from the intrinsic value (2.55 ± 0.14 mM) for free enzyme to 5.38 ± 0.87 mM as Y_P/B increased to 171 mg BSAE/g ds. By estimating the observable Thiele modulus (?_obs), the activity of the biocatalyst with the greatest enzyme loading at the lowest urea concentrations tested (0.833 mM) was reduced by a factor of about 2 due to internal diffusional limitations. By operating in the pseudofirst‐order regime with immobilized derivatives at Y_P/B about 126 mg BSAE/g ds, their activity after grinding was no more limited by intraparticle diffusion and approached the value for free enzyme. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012