Purification and characterization of Bacillus cereus protease suitable for detergent industry

An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease. Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly.     

Purification and partial characterization ofRhizomucor miehei lipase for ester synthesis

A commercialRhizomucor miehei lipase was purified by ammonium sulfate precipitation. Phenyl Sepharose 6 Fast Row hydrophobic interaction chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The recovery of lipase activity was 32% with a 42-fold purification. The molecular size of the purified enzyme was 31,600 Dalton and the pI 3.8. The enzyme was stable for at least 24 h within a pH range of 7.0-10.0, and 96.8% of the enzyme activity remained when kept at 30‡C for 24 h. Further, about 10–30% of the lipase activity was inhibited by K⁺, Li⁺, Ni⁺, Co²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Cu²⁺, Ba²⁺, Ca²⁺, and Fe²⁺ ions and by SDS, but EDTA had no effect. Under the experimental conditions, the optimum temperature for the hydrolysis of olive oil was 50‡C (pH 8.0), and for the synthesis of 1-butyl oleate, 37‡C. It was concluded that hydrolytic activity of lipase alone is not a sufficient criterion for its synthetic potential. The optimal molar ratio of oleic acid and 1-butanol was 2:1 for 1-butyl oleate synthesis. The 1-butyl oleate yield was unaffected by purification of the enzyme after 12 h.     

Purification and characterization of two xylanases from alkalophilic and thermophilic Bacillus licheniformis 77-2

The alkalophilic bacteria Bacillus licheniformis 77-2 produces significant quantities of thermostable cellulase-free xylanases. The crude xylanase was purified to apparent homogeneity by gel filtration (G-75) and ionic exchange chromatography (carboxymethyl sephadex, Q sepharose, and Mono Q), resulting in the isolation of two xylanases. The molecular masses of the enzymes were estimated to be 17 kDa (X-I) and 40 kDa (X-II), as determined by SDS-PAGE. The K _m and V _max values were 1.8 mg/mL and 7.05 U/mg protein (X-I), and 1.05 mg/mL and 9.1 U/mg protein (X-II). The xylanases demonstrated optimum activity at pH 7.0 and 8.0–10.0 for xylanase X-I and X-II, respectively, and, retained more than 75% of hydrolytic activity up to pH 11.0. The purified enzymes were most active at 70 and 75°C for X-I and X-II, respectively, and, retained more than 90% of hydrolytic activity after 1 h of heating at 50°C and 60°C for X-I and X-II, respectively. The predominant products of xylan hydrolysates indicated that these enzymes were endoxylanases.     

Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium

Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was reali-zed by applying successively ammonium sulfate precipitation, dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration, reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was found that the weight of enzyme was 45 kDa by using SDS-PAGE.     

Purification and characterization of an alkaline protease prot 1 frombotrytis cinerea

Alkaline thiol protease named Prot 1 was isolated from a culture filtrate ofBotrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0–10.0 and 60°C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50°C over 120 min; it maintained 50% of activity after 60 min of heating at 60°C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25°C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.     

Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain

N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8-9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40-60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555 U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46 ± 1 kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190 kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40 °C for a period of 45 min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5-9.5 at 40 °C, specifically at pH 9.0 for a 45 min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn²⁺, Cu²⁺, Sn²⁺, Co²⁺, Zn²⁺ and Hg²⁺, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01 M of dithiothreitol (DTT) or 0.06 M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively.     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Purification and characterization of lamb pregastric lipase

Lamb pregastric lipase was purified from a commercial source using delipidation, solubilization with KSCN, acid-precipitation, pepsin-digestion, affinity chromatography with agarose-Cibacron Blue F3GA, gel filtration, and elution from a native 10% (w/v) polyacrylamide gel. The enzyme had a single subunit of 68,000 Da with maximum esterase activity when measured at pH 6.0 and 30 degrees C. The enzyme preferentially hydrolyzed short- and medium-chain (C4, C6, and C8) synthetic esters and short-chain (C4 and C6) monoacid triglycerides. The NH2-terminal sequence demonstrated high homology with gastric and lingual lipases.     

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1+orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.     

Production of the Enzyme Cyclodextringlycosyltransferase from Bacillus firmus Alkalophilic

Different culture media have been testedfor the production of the enzyme CGTase (cyclodextringlycosyltransferase) from Bacillus firmus (strain #37). The concentration of different carbon and nitrogen sources have been varied and the enzyme activity, cell concentration, reducing sugars, total reducing sugars, soluble protein and pH have been followed during cultivation. Results indicate that higher concentrations of yeast extract and polypeptone lead to increased synthesis of CGTase, whereas when starch is substituted by glucose there is a drastic inhibition of CGTase production.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Purification and characterization of theD-hydantoinase from bacillus circulans

AD-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity fromBacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0–10.0 and a temperature of 75‡C. The enzyme is the most stable in a pH range of 8.5–9.5 and up to 60‡C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Purification and characterization of a xylanase from the thermophilic ascomyceteThielavia terrestris 255b

Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II). The latter enzyme could be purified to > 99% homogeneity using anion-exchange chromatography and gel filtration. Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6–4.0 and 60–65°C, respectively. The ratio of the enzyme’s activity against xylan and carboxymethylcellulose was 500–1000 to 1, indicating a possible application of this enzyme in biobleaching processes. The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi.     

Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7

A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH₄)H₂PO₄ 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively. Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application.     

Purification and characterization of a laccase from the white-rot fungus Trametes multicolor

The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.     

Reductive alkylation of lipase

Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.     

Purification and properties of three cellobiases from Aspergillus niger A20

Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gelelectrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55–60°C. The pattern of their aminoacid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K_m values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co²⁺ and Mg²⁺ ions stimulated cell obiase A. The purified enzymes hydrolyzed cellobiose and aryl-β-d-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylate reaction, and the major product formed from cellobiose was tetramer of glucose.     

Purification, characterization, kinetic properties, and thermal behavior of extracellular polygalacturonase produced by filamentous fungus Tetracoccosporium sp

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30°C in an orbital shaker at 160 rpm for 48h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40°C, and the K _m and V _max values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 μmol/min, respectively. Ag⁺, Co²⁺, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al³⁺, Ba²⁺, Ca²⁺, Fe²⁺, Fe³⁺, Ni²⁺, Mg²⁺, Mn²⁺, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t _1/2 of 63 min at 60°C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min⁻¹, and 35.18 mL/(min·mg), respectively. The activation energy (ΔE ^#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters ΔG ^#, ΔH ^#, and ΔS ^# were also calculated, revealing a potential application for the industry.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Kinetic studies of lipase from Candida rugosa

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K _d=0.98 h⁻¹), whereas for the immobilized lipase it was 1.10 h (K _d=0.63 h⁻¹). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The K _m was 0.15 mM and the V _max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.