Production,characterization, and immobilization of partially purified surfactant–detergent and alkali-thermostable protease from newly isolated Aeromonas caviae

Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0–11.0) and moderate salinity (0–5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128?U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl₂?·?2H₂O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42?hr of incubation at 37°C. It was active over broad range of pH (7.0–12.0), temperature (15–100°C), and salinity (0–9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0–12.0 and salinities of 0–5% NaCl for 48?hr. Cu²⁺, Mn²⁺, Co²⁺, and Ca²⁺ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant–detergent-stable partially purified extracellular protease from A. caviae.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

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.     

Purification and Characterisation of an F16L Mutant of a Thermostable Lipase

A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40–60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8–9. Metal ions such as Ca²⁺, Mn²⁺, Na⁺, and K⁺ enhanced the lipase activity, but Mg²⁺, Zn²⁺, and Fe²⁺ inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.     

A novel low-temperature resistant alkaline lipase from a soda lake fungus strain Fusarium solani N4-2 for detergent formulation

The effects of temperature, pH, and various components in a detergent on the activity and stability of a lipase produced by a soda lake fungus strain Fusarium solani N4-2 were studied in a preliminary evaluation for use in detergent formulation. The molecular mass of the lipase was 31.6 kDa by SDS-PAGE after purification using acetone fractionation and Q-sepharose ion exchange technique. In aqueous solutions, the lipase showed maximal activity at pH 9.0 in a glycine–NaOH buffer at 30 °C. At 0 °C and 10 °C, 52.3% and 82.6% of its maximum activities were detained, respectively. Among the metal ions tested, additions of Cu²⁺, Ca²⁺, Mg²⁺, Al³⁺ and Mn²⁺ ions were found to enhance the enzyme activity while Zn²⁺, Ba²⁺ and Hg²⁺ ions showed inhibitory effects. Compared with the commercial lipase Lipolase^®, the enzyme showed better stability towards selected surfactants, commercial detergents, oxidizing agents and proteases. The remarkable resistance capability of the lipase makes it a potential additive for better detergent formulation.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

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 characterisation of alkaline cellulase produced by a novel isolate,<Emphasis Type="Italic"> Bacillus sphaericus</Emphasis> JS1

A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4--glucanase, E.C. 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5. CMCase was purified 192-fold by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23%. The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa. Using SDS-PAGE a single band is found at 42 kDa. This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases. The activity of the enzyme was significantly inhibited by bivalent cations (Fe³⁺ and Hg²⁺, 1.0 mM each) and activated by Co²⁺, K⁺ and Na⁺. The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose. Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.     

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.     

Thermostable alkaline protease from newly isolated <Emphasis Type="Italic">Vibrio</Emphasis> sp.: extraction,purification and characterisation

An extracellular alkaline protease-producing Vibrio sp. was isolated from mangrove sediments of Vellar estuary. A 9.36-fold purification was achieved by a three-step purification procedure and the molecular weight of the enzyme was determined as 33 kDa by SDS-PAGE. The enzyme was active in a broad range of pH (6.0–11.0) and temperature (30–70°C), the optimum being at pH 9.0 and temperature 55°C. The enzyme was stable at alkaline pH range of 9–11 and up to a temperature of 60°C, after incubation for 1 h. Metals like Co²⁺, Hg²⁺, Ni²⁺ and Cu²⁺ inhibited the enzyme activity, whereas Fe²⁺, Ca²⁺ and Mn²⁺ were found to enhance the activity. The protease was found to be highly stable in the presence of oxidizing agents like H₂O₂, detergents such as SDS and Triton-X-100 and also some of the commonly used commercial detergents. The organic solvents like xylene, isopropanol, hexane and benzene were found to enhance as well as stabilize the enzyme activity. The extracellular production of the enzyme, the pH and thermal stability, and the stability in presence of oxidants, surfactants, commercial detergents and organic solvents, altogether suggest that it can be used as a laundry additive.     

Production,purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20–50 °C) and pH values (6–11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na⁺, Ba²⁺, Ca²⁺, and Mn²⁺). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s⁻¹mM⁻¹, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.     

Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere

An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL'S Microsep centrifugal device (10 kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49 kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca²⁺, Na⁺, Cu²⁺, Mg²⁺, Fe²⁺, or Hg²⁺ at 10 mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10 mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10 mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1 mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Influence of cultivation conditions on the production of a thermostable extracellular lipase from Amycolatopsis mediterranei DSM 43304

Among several lipase-producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture conditions and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28°C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. Under optimal culture conditions, the lipase activity was enhanced 12-fold with a twofold increase in lipase specific activity. The lipase showed maximum activity at 60°C and pH 8.0. The enzyme was stable between pH 5.0 and 9.0 and temperatures up to 60°C. Lipase activity was significantly enhanced by Fe³⁺ and strongly inhibited by Hg²⁺. Li⁺, Mg²⁺ and PMSF significantly reduced lipase activity, whereas other metal ions and effectors had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24 h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions.     

Purification and characterization of alkaline soda-bleach stable protease from Bacillus sp. APP-07 isolated from Laundromat soil

The detergent-compatible alkaline protease was produced from the bacterial strain Bacillus sp. APP-07 isolated from Laundromat soil of Solapur, Maharashtra, India. The culture was grown in 1000?ml capacity baffled flask with a working volume of 100?ml and incubated at 55?°C for 33?h on a rotary shaker. After incubation, alkaline protease was partially purified by the sequential method of acetone precipitation followed by nominal molecular weight limit (NMWL) cut-off ultrafiltration using 50?K and 10?K filters. Finally, Sephadex G-100 gel filtration chromatographic purification was performed to obtain 3.12 fold purified alkaline protease enzyme with a 66.67% final yield. The purified enzyme showed 31907.269 units (U) of enzyme activity containing 8741.718?U/mg of specific enzyme activity. The molecular weight of the enzyme was confirmed about 33.0?kDa (kDa) by the SDS-PAGE analysis. The purified enzyme was stable at higher pH and temperature range, with an optimum pH 10.5 and temperature 55?°C. The enzyme showed excellent stability and compatibility in various detergents, surfactants, bleach, and oxidizing agents. The enzyme activity enhanced in the presence of Ca²⁺, Cu²⁺, and surfactants, whereas; the phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate (DFP) completely inhibit the enzymatic activity, which pointed out that the enzyme affiliated to serine-centered metalloproteases family.In conclusion, the remarkable tolerance and stability of the enzyme explored the promising candidature for the several potential applications in the laundry detergents. The sustainability of the enzyme might serve several possible applications in the laundry detergents, leather industries, and other harsh industrial processes.     

A novel thermostable and halostable carboxymethylcellulase from marine bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>AU01

A novel thermostable, halostable carboxymethylcellulase (CMCase) from a marine bacterium Bacillus licheniformisAU01 was purified 10.4-fold with 18% yield with a specific activity of 88.43 U/mg and the molecular weight was estimated as 37 kDa. The enzyme was optimally active at pH 9–10 and temperature 50–60°C and it was most stable up to pH 12 and 20–30% of NaCl concentration. The enzyme activity was reduced when treated with Hg²⁺, Fe²⁺ and EDTA and stimulated by Co²⁺, Mn²⁺, Mg²⁺ and Ca²⁺. Various cationic, anionic detergents and commercial detergents were not much affected CMCase activity.     

Purification and properties of the lipases from Rhodotorula pilimanae Hedrick and Burke

Summary The Rhodotorula pilimanae CBS 5804 strain secretes into the culture medium two lipases: their pH optima are 4 and 7. The two lipases were purified by precipitation with acetone followed by chromatography on SP-Sephadex C50 and Sephadex G200. The purification factors achieved in comparison with the supernatant culture were x74 for lipase I and x90 for lipase II. The molecular weights were estimated at 172,800 and 21,400 for lipase I and lipase II, respectively. Their activities are optimal between 45°C and 55°C. The activation energies were 5.9 kcal·mole^-1 for lipase I and 12.4 kcal·mole^-1 for lipase II. The inactivation energies were about 21.9 and 17.7 kcal·mole^-1 for lipase I and lipase II, respectively. The enzymes are slightly inhibited by Cu²⁺, Co²⁺, Hg²⁺, Mn²⁺, N-acetylacetone, acetic acid and sodium lauryl sulphate. EDTA did not affect their enzymatic activity. These two lipases are secreted in the culture media in the absence of inducer; their biosynthesis is not inhibited by glucose. These lipases hydrolyse primarily the 1-(or 3-)position of all triglycerides tested.     

Purification and Properties of a Chromobacterium Lipase with a High Molecular Weight

A lipase with a high molecular weight was purified from Chromobacterium viscosum by chromatography using the Amberlite CG–50 and Sephadex G–75. The purified lipase (Lipase A) was found to be homogeneous by disc electrophoresis.

Lipase A had an optimum pH around 7 for lipolysis of olive oil and the enzyme was stable at the range of pH 4 to 9 and below 50°C. Zn²⁺, Cu²⁺, Fe³⁺ and high concentrations of l-cysteine, iodoacetic acid and NBS had remarkable inhibitory effects. Bile salts were activator. Lipase A was more active on water insoluble esters than water soluble esters. The isoelectric point of the enzyme was pH 4.7.     

Biochemical properties of a serine protease from Aspergillus flavus and application in dehairing

The proteases are enzymes produced by several filamentous fungi with important biotechnological applications. In this work, a protease from Aspergillus flavus was characterized. The culture filtrate of A. flavus was purified to homogeneity by Sephacryl S-200 column chromatography followed by CM–cellulose. The molecular weight of the purified enzyme was estimated to be approximately 32?kDa by SDS–PAGE. The enzyme hydrolysed BTpNA (N-α-benzoyl-dl-tyrosyl-p-nitroanilide), azo-casein and casein as substrates. Optimal temperature and pH were 55?°C and 6.5, respectively. The enzyme was stimulated by Mg²⁺, Ca²⁺, Zn²⁺ and inhibited by Hg²⁺ and Ag²⁺ and Cu²⁺. The protease showed increased activity with detergents, such as Tween 80 and Triton X, and was stable to the reducing agents, such as β-mercaptoethanol. The protease activity was strongly inhibited in the presence of phenylmethylsulfonyl fluoride, indicating it is a serine protease. The enzyme entrapped in calcium alginate beads retained its activity for longer time and could be reused up to 10 times. The thermostability was increased after the immobilization and the enzyme retained 100% of activity at 45?°C after 60?min of incubation, and 90% of residual activity at 50?°C after 30?min. In contrast, the free enzyme only retained 10% of its residual activity after 60?min at 50?°C. The enzymatic preparation was demonstrated to be efficient in the capability of dehairing without destruction of the hide. The remarkable properties such as temperature, pH and immobilization stability found with this enzyme assure that it could be a potential candidate for industrial applications.     

Cloning,expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg²⁺, Mn²⁺, Li⁺, Ca²⁺, Ni²⁺, CHAPS, DTT, and EDTA and inhibited by Hg²⁺, Ag⁺, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.