Thermostable,salt-tolerant amylase fromBacillus sp. 64

A thermostable, salt-tolerant amylase was produced byBacillus sp. 64, with maximum amylase production (8.0 U/ml culture filtrate) after 24-h growth. Partially purified amylase was stable at 60°C for 30 min and 80% of the original activity was retained when incubated in 5m NaCl over 24 h. Starch or dextrin was the best carbon source and peptone the best nitrogen source for the production of the enzyme. Amylase was secreted over a wide pH range (5 to 11) with the maximum activity between pH 7 and 8. Ca²⁺ and Mg²⁺ stimulated growth and enzyme production.NCL Communication No. 5209.     

Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale

Fish scale, the chief waste material of fish processing industries was processed and tested for production of extracellular protease by mutant Aspergillus niger AB₁₀₀. Protease production by A. niger AB₁₀₀ was greatly enhanced in presence of processed fish scale powder. Where as among the three complex nutrients tested, soya bean meal shows maximum stimulatory effect over protease production (2,776 μmol/ml/min) when used in combination with glucose (5% w/v) and urea (2.5% w/v). The protease was optimally active at pH 7.0, retaining more than 60% of its activity in the pH range of 5–9. The enzyme was found to be most active at 50°C and stable at 30°C for 1 h. Purification of enzyme by CM-Cellulose and SDS-PAGE resulted in about 26-fold increase in the specific activity of the enzyme with a molecular weight of 30.9 kDa. HPLC study shows the purity of the enzyme as 75.92%. By the activating effect of divalent cations (Fe²⁺, Zn²⁺, Mn²⁺, Ca²⁺and Mg²⁺) and inhibiting effect of chelating agent (EDTA) and Hg²⁺, the enzyme was found to be a metalloprotease.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Production of milk clotting protease by a local isolate of Mucor circinelloides under SSF using agro-industrial wastes

Agro-industrial residues, a cheap source of energy have high potential in the area of fermentation for the production of enzymes. Twenty agro-industrial residues were evaluated to check the possibility of potential utilization of substrates in SSF for milk clotting enzyme protease production by Mucor circinelloides. In this study, dhal husk holds the greatest promise for cost effective production of the milk clotting enzyme. The dhal husk supported maximum milk clotting protease production, and yield was improved with the supplementation of sucrose and yeast extract as carbon and nitrogen source, respectively. Among all the physico-chemical parameters tested, the best results were obtained in a medium having moisture content of 20% at pH 7.0, when inoculated with 30% of spore suspension and incubated at 30°C for 5 days. The activity was increased further on addition of Ca²⁺, Cu²⁺, and Mg²⁺ ions. The purified milk-clotting protease obtained from M. circinelloides was successfully applied and compared with commercial rennet in the manufacture of a cheddar cheese.     

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.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

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.     

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.     

Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

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

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

In vitro fibrinolytic activity of an enzyme purified from Bacillus amyloliquefaciens strain KJ10 isolated from soybean paste

A fibrinolytic protease secreting producing Bacillus amyloliquefaciens strain KJ10 was initially screened from the fermented soybean. Maximum productivity was obtained in the culture medium after 40 h incubation, 34 °C incubation temperature at pH 8.0. Fibrinolytic protease production was enhanced in the culture medium with 1% sucrose (3712 ± 52 U/mL), 1% (w/v) yeast extract (3940 ± 28 U/mL) and 0.1% MgSO₄ (3687 ± 38 U/mL). Enzyme was purified up to 22.9-fold with 26%recovery after Q-Sepharose HP column chromatography. After three steps purification, enzyme activity was 1606U/mg and SDS-PAGE analysis revealed 29 kDa protein and enzyme band was detected by zymograpy. Enzyme was highly active at pH 8.0, at wide temperature ranges (40 °C ? 55 °C) and was activated by Mn²⁺ (102 ± 3.1%) and Mg²⁺ (101.4 ± 2.9%) ions. The purified fibrinolytic enzyme was highly specific against N-Suc-Ala-Ala-Pro-Phe-pNA (189 mmol/min/mL) and clot lytic activity reached 28 ± 1.8% within 60 minin vitro. The purified fibrinolytic enzyme showed least erythrocytic lysis activity confirmed safety to prevent various health risks, including hemolytic anemia. Based on this study, administration of fibrinolytic enzyme from B. amyloliquefaciens strain KJ10 is safe for clinical applications.     

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii

Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 °C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

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.     

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.     

Optimization of Fermentation Conditions for Phytase Production by Two Strains of Bacillus licheniformis (LF1 and LH1) Isolated from the Intestine of Rohu, Labeo rohita (Hamilton)

The culture conditions for extracellular production of phytase by two strains of Bacillus licheniformis (LF1 and LH1) isolated from the proximal and distal intestine of rohu (Labeo rohita) were optimized to obtain maximum level of phytase. Both the strains were cultured TSA broth for 24 h at 37 ± 2 °C, when average viable count of 9.75 × 10⁷ cells ml^?1 culture broth was obtained. This was used as the inoculum for the production medium. Sesame (Sesamum indicum) oilseed meal was used as the source of phytic acid (substrate). The effects of moisture, pH, temperature, fermentation period, inoculum size, different nitrogen sources, vitamins and surfactants on phytase production by these two strains were evaluated. Phytase yield was highest (1.87 U in LF1 and 1.57 U in LH1) in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 5.5–6.5. Minimum phytase production was observed at 50 °C, while maximum production was obtained at 40 °C. To standardize the fermentation period for phytase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points for the strain LF1 and up to 2.0 % for the strains LH1. Ammonium sulphate as the nitrogen source was most effective in LF1, while beef extract proved useful to maximize enzyme production by LH1.     

Addendum to Issue 1 - ENZITEC 2012 Cheese whey and passion fruit rind flour as substrates for protease production by Bacillus sp. SMIA-2 strain isolated from Brazilian soil

Abstract

Application of wastes from the food processing industry as carbon sources in enzyme production processes reduces the cost of production, and also helps in solving problems of their disposal. In this work, we demonstrated that sweet cheese whey, in combination with passion fruit rind flour, can be successfully used for the production of protease by Bacillus sp. SMIA-2, opening perspectives for the use of these agricultural byproducts as novel and cost-effective culture media for the production of protease. The maximum production of the enzyme was observed in a sweet cheese whey-based culture medium preparation (0.5%, w/v) containing 0.25% (w/v) passion fruit rind flour and supplemented with different metal salts at an initial pH of 7.5–8.0, incubated at 50°C for 48 h. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 70°C. These characteristics presented by the protease produced by Bacillus sp. SMIA-2 could be very useful when thinking about biotechnological applications.     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl₂.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.