Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Bioreduction of platinum salts into nanoparticles: a mechanistic perspective

A mechanism for the bioreduction of H₂PtCl₆ and PtCl₂ into platinum nanoparticles by a hydrogenase enzyme from Fusarium oxysporum is proposed. Octahedral H₂PtCl₆ is too large to fit into the active region of the enzyme and, under conditions optimum for nanoparticle formation (pH 9, 65°C), undergoes a two-electron reduction to PtCl₂ on the molecular surface of the enzyme. This smaller molecule is transported through hydrophobic channels within the enzyme to the active region where, under conditions optimal for hydrogenase activity (pH 7.5, 38°C) it undergoes a second two-electron reduction to Pt(0). H₂PtCl₆ was unreactive at pH 7.5, 38°C; PtCl₂ was unreactive at pH 9, 65°C.     

Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> MA-4680

A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP⁺ as electron acceptor. Apparent K _m values were 4.98 μM for NADP⁺ and 6,620 μM for NAD⁺, respectively, using Mn²⁺ as divalent cation. The enzyme performed a 33,000-fold greater specificity (k _cat/K _m) for NADP⁺ than NAD⁺. Moreover, SaIDH activity was entirely dependent on the presence of Mn²⁺ or Mg²⁺, but was strongly inhibited by Ca²⁺ and Zn²⁺. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP⁺.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml⁻¹ in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45°C over 1 h and was stable between pH 5.8 and 12 at 4°C for 24 h and up to 40°C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l⁻¹) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Isolation and purification of an enzyme hydrolyzing ochratoxin A from <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Ochratoxin A is a mycotoxin produced by several Aspergillus and some Penicillium species which may be present in food and feed products. It can be enzymatically hydrolyzed into ochratoxin α and l-β-phenylalanine, thereby decreasing its toxicity. The ochratoxin A degradation capacity of Aspergillus niger is well known and here we report the isolation and purification of a novel enzyme from A. niger that hydrolyzes this mycotoxin. A wheat germ medium supplemented with ochratoxin A was used to produce the enzyme, which was purified from culture filtrate by acetone precipitation and anion exchange chromatography. An overall purification of 2.5-fold with a recovery of 68% and a final specific activity of 36 U/mg was obtained. The enzyme is a metalloenzyme as it was inhibited at 10 mM EDTA, whereas PMSF had no effect. The ochratoxin A hydrolytic enzyme presented a V _max of 0.44 μM/min and a K _m of 0.5 mM when the reaction was carried out at pH 7.5 and 37°C.     

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated<Emphasis Type="Italic">Bacillus subtilis</Emphasis> PE-11

The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca²⁺, Mg⁺², and Mn⁺². Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl₂ and 1M glycine. The addition of 10mM CaCl₂ and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl₂ and 1M glycine.     

Purification and characterization of fibrinolytic alkaline protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. BLB

Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH₄)₂SO₄ and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Enhanced Production and Partial Characterization of Thermostable α-galactosidase by Thermotolerant Absidia sp.WL511 in Solid-state Fermentation using Response Surface Methodology

Summary Response surface methodology was applied to optimize medium components for maximum production of a thermostable α-galactosidase by thermotolerant Absidia sp. WL511. First, the Plackett-Burman screening design was used to evaluate the effects of variables on enzyme production. Among these variables, MgSO₄ and soybean meal were identified as having the significant effects (with confidence level (90%). Subsequently, the concentrations of MgSO₄ and soybean meal were further optimized using central composite designs. The optimal parameters were determined by response surface and numerical analyses as 0.0503% (g/g) MgSO₄ and 0.406% (g/g) soybean meal and allowed α-galactosidase production to be increased from 4.4 IU g⁻¹ to 117.8 IU g⁻¹. The subsequent verification experiments confirmed the validity of the model. The optimum pH of enzymatic activity was 7.5 and the enzyme was stable at pH values ranging from 5.0 to 9.0. The optimum temperature was 73 °С. The enzyme was fairly stable at temperatures up to 60 °С and had 87% of its full activity at 65 °С after 2 h of incubation.     

Characterization of a xylanase from a thermophilic strain of <Emphasis Type="Italic">Anoxybacillus pushchinoensis</Emphasis> A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and V_max and K _m were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Comparison of angiotensin converting enzyme-like activity in the Antarctic teleosts <Emphasis Type="Italic">Trematomus bernacchii</Emphasis> and <Emphasis Type="Italic">Chionodraco hamatus</Emphasis>

Biochemical parameters of the angiotensin converting enzyme-like activity (ACELA) in the gills of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii were characterized. Enzymatic activity was revealed following hydrolysis of a specific substrate of angiotensin-converting enzyme N-[3-(2-furyl)acryloyl]l-phenylalanyl-glycyl-glycine (FAPGG) and metabolites were separated by reverse phase HPLC analysis. The results showed similar Km values for the substrate FAPGG at 5°C for the two species with an increase of Km value for T. bernacchii at 25°C. The optimum pH value was 8.5 at 25°C and optimum chloride concentrations were about 300 mM. In T. bernacchii the optimum temperature for maximum enzyme activity was 50°C, while maximum activity in C. hamatus occurred at 35°C. Lisinopril was more efficient in inhibiting ACELA in C. hamatus with an I ₅₀ value of 16.83 ± 5.11 nM, compared to an I ₅₀ value of 30.66 ± 5.19 nM in T. bernacchii. In conclusion, it appears that some biochemical parameters of ACELA in C. hamatus differ from those in T. bernacchii, probably due to different ways that the enzyme adapts to the constantly cold temperatures of the animal’s environment.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.