Purification Of Alkaline Protease Research Articles

Dissolution of clotted blood from slaughtered cattle by alkaline protease produced by Streptomyces violaceoruber isolated from saline soil in Saudi Arabia

Saudi Arabia suffers from the spread of diseases linked to clotted blood leaking from slaughtered livestock. Anticoagulant proteases produced by Streptomyces are used to safely dissolve clotted blood. Halophilic Streptomyces isolates were extracted from saline soil in Saudi Arabia and tested to dissolve clotted blood by protease. The most protease-producing isolate was identified using classical and genetic methods. The protease was precipitated with saturated ammonium sulfate from a batch of blood clots inoculated with the most protease-producing isolates. Streptomyces isolates (410) were recovered from thirty saline soil samples collected from Dammam (170 isolates, 41.4 %), Al-Khobar (126 isolates, 30.8 %) and Al-Ahsa (114 isolates, 27.8 %). Proteolytic activity was observed in 305 isolates including the most protease-producing isolate S295, which showed the highest activity (33.8 mm), and was identified as S. violaceoruber (99 % similarity). Among all isolates, 241 showed partial hemolysis (alpha) and 64 showed complete hemolysis (beta), including isolate S295 that presented the highest hemolytic activity (0.58 %). The alkaline protease was precipitated with saturated ammonium sulfate (60 %), and it was found that its activity was 5.2 U/ml, total activity 24,960 U/ml, specific activity 463 U/mg, purification fold 15.2 and yield 78 %. The purified alkaline protease was electrophoresed as a single band at 45 kDa.

Immobilization of alkaline protease produced by Streptomyces rochei strain NAM-19 in solid state fermentation based on medium optimization using central composite design.

This study evaluated Streptomyces rochei strain NAM-19 solid-state fermentation of agricultural wastes to produce alkaline protease. Alkaline protease production increased with flaxseed, rice bran, and cheese whey fermentation reaching 147U/mL at 48h. Statistical optimization of alkaline protease production was performed using the central composite design (CDD). Results of CDD and the optimization plot showed that 4.59g/L flaxseed, 4.31g/L rice bran, 4.17mL cheese whey, and a vegetative inoculum size of 7.0% increased alkaline protease production by 27.2% reaching 186U/mL. Using the 20-70% ammonium sulfate fractionation method, the optimally produced enzyme was partially purified to fivefold. The partially purified alkaline protease was then covalently immobilized on a biopolymer carrier, glutaraldehyde-polyethylene-imine-κ-carrageenan (GA-PEI-Carr), with 90% immobilization efficiency. Characterizations revealed that immobilization improved thermostability, reusability, optimum temperature, and sensitivity towards metal ions of the free enzyme. The optimal temperature for free and immobilized enzymes was 40 and 50°C, respectively. Both enzymes had the same optimum pH of 10. Immobilization increased Km from 19.73 to 26.52mM and Vmax from 56.7 to 62.5mmolmin-1L-1. The immobilized enzyme retained 35% of its initial activity at 70°C, while the free enzyme retained only 5%. The immobilized enzyme kept 80% of its initial activity at the 20th cycle. After 7weeks of storage, the free enzyme lost all its initial activity, whereas the immobilized enzyme retained 50%. The free and immobilized enzymes were able to hydrolyze gelatin, and azo-casein demonstrating different relative activity, 85, 80, 90 and 95%, respectively, compared to casein (100%).

Biocatalytic Profiling of Free and Immobilized Partially Purified Alkaline Protease from an Autochthonous Bacillus aryabhattai Ab15-ES

Partially purified alkaline protease produced by an indigenous bacterial strain, Bacillus aryabhattai Ab15-ES, was insolubilized in alginate beads using an entrapment technique. Maximum entrapped enzyme activities of 68.76% and 71.06% were recorded at optimum conditions of 2% (w/v) sodium alginate and 0.3 M calcium chloride. Biochemical profiling of free and immobilized proteases was investigated by determining their activity and stability as well as kinetic properties. Both enzyme preparations exhibited maximum activity at the optimum pH and temperature of 8.0 and 50 °C, respectively. However, in comparison to the free enzyme, the immobilized protease showed improved pH stability at 8.0–9.0 and thermal stability at 40–50 °C. In addition, the entrapped protease exhibited a higher Vmax and increased affinity to the substrate (1.65-fold) than the soluble enzyme. The immobilized protease was found to be more stable than the free enzyme, retaining 80.88% and 38.37% of its initial activity when stored at 4 °C and 25 °C, respectively, for 30 d. After repeated use seven times, the protease entrapped in alginate beads maintained 32.93% of its original activity. These findings suggest the efficacy and sustainability of the developed immobilized catalytic system for various biotechnological applications.

Characterization and Purification of Alkaline Protease from Novel Bacillus subtilus MS1 for Detergent Additives

Characterization and Purification of Alkaline Protease from Novel Bacillus subtilus MS1 for Detergent Additives

Biochemical properties and repression studies of an alkaline serine protease from a haloalkaliphilic actinomycete, Nocardiopsis dassonvillei subsp. albirubida OK-14

A marine haloalkaliphilic actinomycete identified as Nocar diopsis dassonvillei subsp. albirubida (strain OK-14) was screened for its ability to produce extracellular alkaline protease under various physical conditions pertinent to different applications. The strain utilized amino acids as the sole source of nitrogen, displaying repression and de-repression phenomenon above the threshold levels. The activity of the partially purified alkaline protease was 1421.7 U/ml/min. The enzyme was optimally active at pH 10 and 60 °C. The activity was assessed under various concentrations of NaCl, organic solvents and metal ions. The partially purified alkaline protease was then examined for its suitability in blood de-staining, gelatine degradation and keratinase activity, implicating its relevance in detergent industries, gelatine extraction from X-ray films and degradation of bio-wastes. • Repression and de-repression of protease by different amino acids. • Protease stability in solvents, metal ions and various other agents. • Highly efficient in gelatin deproteinization and silver recovery. • Compatible as detergent supplement.

Expression, purification, and molecular characterization of a full-length thermostable alkaline protease gene from Bacillus subtilis DMA-09

The present study aim was to isolate bacterial strains, producing protease, from soil samples of different industrial wastes and dumping areas of slaughter houses. For this, 25 bacterial isolates forming clear zones in skimmed milk agar plates as a result of casein hydrolysis were selected. One bacterial isolate showing the highest protease activity (400 U/ml) was identified as Bacillus subtilis through 16S rRNA gene sequencing. Full-length alkaline protease gene was amplified, sequenced, cloned in expression vector pT7-7, and transformed in E. coli BL21C. Over-expression of protease gene was determined in isopropyl β-d-1-thiogalactopyranoside (0.5 mM) at 37 °C for 3 h. Expressed alkaline protease was purified and 10 folds increased enzyme activity was determined as compared to the wild type B. subtilis DMA-09. The purified protease was detected on SDS-PAGE as a single band of 31 kDa and was stable at 90 °C and 85% enzyme activity was retained at 100 °C. The optimum enzyme activity was obtained at pH 10 and was enhanced in the presence of 5 mM each Mg2+ and Ca2+ while phenylmethylsulfonyl fluoride (PMSF) inhibited its activity at 0.5 mM. The expressed purified alkaline protease may find potential applications in food and biotechnological industries.

Antioxidant and Enzyme Inhibitory Activities of Metapenaeus monoceros By-Product Hydrolysates Elaborated by Purified Alkaline Proteases

This study investigated the antioxidant potential and enzymatic inhibitory power of shrimp by-product protein hydrolysates (hyd) prepared using two purified serine alkaline proteases namely; alkaline protease from Anoxybacillus kamchatkensis strain M1V (SAPA) and alkaline protease from Aeribacillus pallidus strain VP3 (SPVP). The characterization of their molecular weights showed the presence of peptides less than 15 kDa. SAPA-hyd displayed the highest reducing power activity. Likewise, SAPA-hyd was found to be active against angiotensin I-converting enzyme (ACE), tyrosinase (Tyr), and amylase (Amy) enzymes with IC50 values of 10.01, 6.13, and 4.11 µg/mL, respectively. SPVP-hyd revealed the highest radical scavenging power, β-carotene protection, and ferrous chelating activity with EC50 values of 11.79, 3.69, and 0.61 µg/mL, respectively. After fraction nation, all fractions displayed a high inhibitory potential against ACE. Only fractions F2 and F3 from SAPA-hyd showed the highest anti-tyrosinase activity with IC50 values of 25.10 and 12.5 ng/mL, respectively. These results suggest that a simple, clean, economic, and controllable bioprocess for the utilization of shrimp by-product was investigated. This study gives an unprecedented report on the anti-Tyr activity of the protein hydrolysate from fish bio-waste. Additionally, two peptide hydrolysates with multifunctional biological activities were elaborated from Metapenaeus monoceros by two bacterial purified proteases.

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non-covalent immobilization to poly(vinylimidazole)/clay hydrogel.

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K m, V m, and k cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG≠ (activation free energy) and ΔG≠ E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG≠ ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS≠ value implied that the immobilized form of the enzyme was more ordered than its free form.

Potential application of Bacillus pseudofirmus SVB1 extract in effluent treatment

Abstract: The wastewater generated from food industries like dairy and meat processing industries are rich in organic compounds and nutrients having high concentrations of BOD, COD and suspended solids. However, most of the conventional techniques used for treatment are not so eco-friendly. The eco-friendly approaches mostly employ biological aid but are usually ineffective in treating high organic load under extreme conditions. This study uses alkaline protease isolated from Bacillus pseudofirmus SVB1 to treat effluents from dairy industry and domestic sludge. The results obtained from this study indicate that both crude and partially purified alkaline protease performed significantly well in the treatment of effluents from dairy and domestic sewage. They were also able to effectively lyse both Gram positive and Gram negative bacteria. Both crude and partially purified enzyme procured from SVB1 performed efficiently when compared to the already available commercial enzymes used in this study. The efficiency of the extract reduces the overall cost of treatment, hence can potentially be used for treating effluents, enhancing the economy of the entire process.

Characterization of partially purified alkaline protease secreted by halophilic bacterium Citricoccus sp. isolated from agricultural soil of northern India

Abstract The present study reports the isolation of protease producing bacteria Bact2 from agricultural soil of Regional Centre of Soil Salinity Research Institute, Lucknow, India. It was identified as Citricoccus sp. (KC522120.1) by 16S rRNA gene sequencing. The optimum pH and temperature for maximum enzyme production was observed to be at 10 and 40 °C respectively. The enzyme protease was partially purified and recovery of enzyme was 77.16%. A 1.66 fold increase in enzyme activity was also observed in comparison to original. After purification, the partially purified enzyme (PPE) was characterized. The optimum pH and temperature of partially purified enzyme (PPE) activity was observed to be at 10 and 40 °C respectively. The activity of PPE was enhanced in presence of Ca2+ and Mg2+ while it was inhibited by Cu++ and Na+. The PPE was completely inhibited by Phenyl methyl sulphonyl fluoride (PMSF) indicating that it belongs to serine protease group. Finally, enzyme was found to be compatible with detergents and effective in removing stains of tea and ink indicating it's application in detergent industry. Besides, to our knowledge till date production of alkaline protease from Citricoccus sp. has been reported first time in the present manuscript.

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.

Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634

An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0–11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and Vmax = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.

English

Proteases constitute most important enzymes owing to their wide variety of functions and have immense applications in various industries viz., medical, pharmaceutical, biotechnology, leather, detergent, and food industries. Despite of their wide spread occurrence in various sources, microorganisms present remarkable potential for proteolytic enzymes production due to their extensive biochemical diversity and susceptibility to genetic manipulation. The present study was aimed at isolating alkaline protease producing fungal members from dairy form effluents, designing the process variables for maximizing the protease production and determining the fibrinolytic potential of the partially purified alkaline protease. To achieve the specified objectives, the dairy form effluent was processed for the isolation of proteolytic fungi using suitable microbiological medium. All the fungal isolates were screened for their protease producing ability and the isolate showing highest alkaline protease production was selected for further studies. Optimization of different fermentative variables like carbon, nitrogen sources, pH, temperature and incubation period were carried out to enhance enzyme production. Ammonium sulphate fractionation was employed to partially purify the enzyme following which its fibrinolytic potential was determined. Based on morphological and microscopic studies, the selected fungal isolate was identified as Aspergillus niger. Optimization studies using OVAT (one variable at a time) method revealed an enhanced protease production in the presence of fructose as additional carbon source and ammonium sulphate as nitrogen source. The optimum incubation period, temperature and pH for enzyme production by the selected fungal isolate was found to be 92 h, 50°C and 10, respectively. The partially purified alkaline protease was efficient in the removal of blood stains emphasizing its fibrinolytic ability. An alkaline protease producing Fungal sp. was screened and isolated from dairy form effluent and it was found to be efficient in the removal of blood stains proving its fibrinolytic potential. Enzymes produced from microorganisms that can survive under extremes of pH could be particularly useful for commercial applications under high alkaline conditions. Key words: Alkaline protease, dairy form effluent, optimization, Aspergillus niger, fibrinolytic potential.

Production, purification and characterization of halophilic organic solvent tolerant protease from marine crustacean shell wastes and its efficacy on deproteinization.

The quantum of marine fish wastes produced by fish processing industries has necessitated to search new methods for its disposal. Hence, this study is focused on production and purification of halophilic organic solvent tolerant protease (HOSP) from marine Alcaligenes faecalis APCMST-MKW6 using marine shell wastes as substrate. The candidate bacterium was isolated from the marine sediment of Manakudi coast and identified as A. faecalis APCMST-MKW6. The purified protease showed 16.39-fold purity, 70.34 U/mg specific activity with 21.67 % yield. The molecular weight of the purified alkaline protease was 49 kDa. This purified protease registered maximum activity at pH 9 and it was stable between pH 8–9 after 1.30 h of incubation. The optimum temperature registered was 60 °C and it was stable between 50 and 60 °C even after 1.30 h of incubation. This enzyme also showed maximum activity at 20 % NaCl concentration. Further, manganese chloride, magnesium chloride, calcium chloride and barium chloride influenced this enzyme activity remarkably and it was also found to be enhanced by many of the tested surfactants and solvents. The candidate bacterium effectively deproteinized the shrimp shell waste compared to the other tested crustaceans shell wastes and also attained maximum antioxidant activity.

Optimized production, characterization and application of alkaline proteases from taxonomically assessed microbial isolates from Lonar soda lake, India

Lonar soda lake of India was explored for isolation of the industrially important alkaline protease producers. The isolates from Lonar lake were identified based on their morphological characters, microscopic features, enzymatic profile, biochemical pattern and 16S rRNA partial gene sequences as Brachybacterium sp. LAP214, Bacillus cohnii LAP217, Bacillus pseudofirmus LAP220, Brevibacterium casei LAP223 and Halomonas venusta LAP515 and deposited in Microbial Culture Collection, NCCS, Pune (India) for public access. Production of the alkaline proteases from these isolates was optimized by adopting a one-variable-at-a-time (OVAT) approach in a submerged fermentation system in modified fermentation medium (MFM). More than 3 fold enhancement in the alkaline protease production was recorded from all these isolates after optimization of the physicochemical parameters. Molecular weights of partially purified alkaline proteases from LAP515, LAP214 and LAP223 were recorded as 20.1, 14.3 and 43 kDa respectively. Molecular weights of partially purified alkaline proteases from LAP220 and LAP217 were recorded as 29 kDa. Partially purified alkaline proteases from LAP515, LAP214, LAP220, LAP217 and LAP223 exhibited Vmax values 1000, 1290.323, 526.316, 1250 and 2000 U/mg respectively. Catalytic activities of alkaline proteases from all these isolates were partially and completely inhibited in presence of a serine protease inhibitor PMSF, at the concentrations of 1 and 10 mM respectively. The metal cations Mg2+, Ba2+, and Ca2+ enhanced the catalytic activities of these proteases. Remarkable stability of these alkaline proteases was observed in presence of selected solvents and commercial detergents. These proteases therefore can be used in various biotechnological applications.

Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis

An extracellular alkaline protease produced by the alkali-tolerant Cellulomonas bogoriensis was purified by a combination of ammonium sulfate precipitation and cation exchange chromatography. The purity of the protease was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was confirmed to be 18.3 kDa. The enzyme showed optimum activity at 60 °C and pH 11. The stability of the protease was maintained at a wide temperature range of 4–60 °C and pH range of 3–12. Irreversible inhibition of the enzyme activity by phenylmethylsulfonyl fluoride and tosyl-l-phenylalanine chloromethyl ketone demonstrated that the purified enzyme is a chymotrypsin of the serine protease family. The Km and Vmax of the protease activity on casein were 19.2 mg/mL and 25000 μg/min/mg, respectively. The broad substrate specificity and remarkable stability in the presence of organic solvents, salt, and commercial detergents, as well as its excellent stain removal and dehairing capability, make the purified alkaline protease a promising candidate for industrial applications.

Purification and Characterization of Halophilic Organic Solvent Tolerant Protease from Marine Bacillus sp. APCMST-RS7 and Its Antioxidant Potentials

Halophilic organic solvent tolerant proteolytic bacterium was isolated from the marine sediment of Rajakkamankalam estuary and identified as Bacillus sp. APCMST-RS7. The alkaline protease synthesized by Bacillus sp. APCMST-RS7 was purified by using ammonium sulphate precipitation, dialysis and DEAE-Sepharose Fast Flow column. The purified protease showed 6.60-fold purity, 26.02 U/mg specific activity with 24.30 % yield. The molecular weight of the purified alkaline protease was 32 kDa. This purified protease registered maximum activity at pH 8 and it was stable between pH 8–9 after 1.30 h of incubation. The optimum temperature registered was 50 °C and it was stable between 30 and 40 °C even after 1.30 h of incubation. This enzyme also showed maximum activity at 1.5 M NaCl concentration. The Km and Vmax values registered were, 0.0002 g/l and 1428.57 U/ml, respectively. Further, magnesium chloride, barium chloride and copper sulphate influenced this enzyme activity remarkably and it was also found to be enhanced by many of the tested surfactants and solvents. Here, the serine protease inhibitor totally inhibited the enzyme activity; hence it is of serine protease family. The candidate bacterium effectively deproteinized the shrimp shell waste with maximum protease activity. The shrimp shell hydrolysate also exhibited maximum antioxidant activity.

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

ABSTRACT In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey) as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35 o C for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40 o C, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The K m , K cat , V max and K cat /K m values of purified protease were 7.0 mg/mL, 3.8 x10 2 S -1 , 54.30 µmol/min and 54.28 s -1 mg -1 .mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

Wealth from waste: Optimized alkaline protease production from agro-industrial residues by Bacillus alcalophilus LW8 and its biotechnological applications

An efficient alkaline protease producer was isolated from the water of the hyperalkaline-saline Lonar soda lake and identified as Bacillus alcalophilus LW8 by culture-dependent techniques by its morphological, microscopic, biochemical, physiological and molecular characteristics. The 16S rRNA gene sequence was submitted to the GenBank nucleotide repository under accession number KC689353. Alkaline protease production was optimized by adopting a one-variable-at-a-time approach in a submerged fermentation system in modified fermentation medium (MFM). The optimized components of MFM were (w/w) casein (1%), sugarcane molasses (1%), NaCl (1%), ammonium sulphate (0.5%), KH2PO4 (0.05%), K2HPO4 (0.05%) and Na2CO3 (1%). Optimized culture conditions were used for alkaline protease production. The final yield of partially purified alkaline protease after dialysis was 53.35%. The molecular mass of the dialysed alkaline protease was 27kDa. On a Lineweaver–Burk plot, the calculated Km and Vmax values were 24mg/mL and 1000U/mg, respectively. The enzyme was remarkably stable in the pH range 7.0–12.0, with optimum activity at pH 10.0. LW8 alkaline protease was completely inhibited by phenylmethylsulphonyl fluoride at 10mmol/L, indicating that the enzyme belongs to the serine protease class. The metal ions Ca2+, Ba2+, Mg2+, Zn2+, Fe3+, Cu2+ and Mn2+ increased the catalytic activity of partially purified alkaline protease. The protease effectively decomposed the gelatinous coating on an X-ray film, hydrolysed blood clot, a blood-stain from a piece of cotton fabric and hairs from a piece of goat skin.

Purification and characterization of detergent-compatible protease from Aspergillus terreus gr.

The possibility of using Aspergillus terreus protease in detergent formulations was investigated. Sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis indicated that the purified alkaline protease (148.9 U/mg) is a monomeric enzyme with a molecular mass of 16 ± 1 kDa. This was confirmed by liquid chromatography–mass spectrometry. The active enzyme degraded the co-polymerized gelatin. The protease demonstrated excellent stability at pH range 8.0–12.0 with optimum at pH 11.0. It was almost 100 % stable at 50 °C for 24 h, enhanced by Ca2+ and Mg2+, but inhibited by Hg2+, and strongly inhibited by phenylmethyl sulfonyl fluoride. It showed maximum activity against casein followed by gelatin; its Vmax was 12.8 U/ml with its corresponding KM of 5.4 mg/ml. The proteolytic activity was activated by Tween-80, Triton-100 and SDS, and remained unaltered in the presence of H2O2 and NaClO. The enzyme exhibited higher storage stability at 4, 28 and −20 °C. It was stable and compatible to the desired level in the local detergents. The addition of the protease to the Super wheel improved its blood stain removal. The isolated protease can thus be a choice option in detergent industry.