Extracellular Alkaline Protease Research Articles

Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei

A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

Immobilization ofBacillus amyloliquefaciens SP1 and its alkaline protease in various matrices for effective hydrolysis of casein.

An extracellular alkaline protease producing B. amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth-promoting activities. B. amyloliquefaciens SP1 protease was immobilized using various concentrations of calcium alginate, agar and polyacrylamide to determine the optimum concentration for formation of the beads. Enzyme activity before immobilization (at 60 °C, pH 8.0 for 5 min) was 3580 µg/ml/min. The results of immobilization with various matrices revealed that 3 % calcium alginate (2829.92 µg/ml/min), 2 % agar (2600 µg/ml/min) and 10 % polyacrylamide (5698.99 µg/ml/min) were optimum concentrations for stable bead formation. Immobilized enzyme reusability results indicated that calcium alginate, agar and polyacrylamide beads retained 25.63, 22.05 and 34.04 % activity in their fifth repeated cycle, respectively. In cell immobilization technique, the free movement of microorganisms is restricted in the process, and a semi-continuous system of fermentation can be used. In the present work, this technique has been used for alkaline protease production using different matrices. Polyacrylamide (10 %) was found with the highest total alkaline protease titer, i.e., 24,847 µg/ml/min semi-continuously for 18 days as compared to agar (total enzyme titer: 5800 in 10 days) and calcium alginate (total enzyme titer: 13,010 in 15 days). This present study reported that polyacrylamide (10 %) among different matrices has maximum potential of immobilization of B. amyloliquefaciens SP1 and its detergent stable alkaline protease with effective application in bloodstain removal.

Production of Thermostable Alkaline Protease from <i>Streptomyces</i> sp-A54

Bacterial extracellular alkaline proteases have been found to have broad spectrum industrial applications because of their stability characteristics among the bacteria. The Actinomyces are of enormous importance as they can be recovered easier than other bacteria after fermentation. Thus, the study was aimed at sourcing for potential protease producers among the Actinomycetes species and determining the cultural conditions for their optimal protease yields. Among 67% of the recovered soil isolates that demonstrated potentials for protease production on skimmed milk agar, only Norcardia sp-A2 2 , Micromonospora sp-A2 3 , Streptosporangium A-5 2 , and Streptomyces sp-A5 4 produced protease with higher activity(18.5-20.5mm) than the others (8.5-16.5mm). Preliminary protease assay in submerged shake-flask fermentation confirmed Streptomyces sp-A5 4 as the highest producer. Further studies on protease production by Streptomyces sp-A5 4 showed that maximum protease yield was achieved within 72h at 0.4% inoculum concentration. All the test organic carbon and nitrogen substrates supported protease yield (> 1.5/2.0 u/ml) but soybean and sweet potato meal gave the highest support (> 2.5/2.53 u/ml). However, their protease yields were significantly (p< 0.05) less than that of refined glucose and peptone when substituted as the carbon and nitrogen sources. The produced crude enzyme demonstrated optimal activity at pH 8.0 and temperature of 50 0 C. Therefore, the ability of Streptomyces sp-A5 4 to produce thermostable protease at a relatively low concentration indicated its potential as a good source for industrial applications. Keywords - Streptomyces -spA5 4 ,Protease-Fermentation, Optimization, Cultural Condition.

Production and optimization of extracellular alkaline protease from halotolerant chromate resistant Bacillus circulans isolated from Tannery solid waste

Production and optimization of extracellular alkaline protease from halotolerant chromate resistant Bacillus circulans isolated from Tannery solid waste

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.

Enzymatic dehairing of goat skins using alkaline protease from Bacillus sp. SB12

The present paper reports the production, purification and biochemical characterization of an extracellular alkaline protease from Bacillus sp. SB12. The enzyme has been used as an alternative to conventional chemicals treatment for dehairing of goat skins. The protease was optimally active at 37 °C and pH 9. Starch at 2% (w/v) was used as the best carbon source and the addition of yeast extract and peptone at 1% each supported the maximum level of protease production in the presence of 5 mM Ca2+. Protease purification was performed with ammonium sulphate precipitation at 70% saturated fraction followed by dialysis and gel filtration chromatography using Sephadex G-100. The purified enzyme was homogeneous on non-denaturing PAGE and appeared as a single band with an apparent molecular weight of 41 kDa. This enzyme was moderately thermostable and has a wide pH stability range extending from pH 7 to 11. It showed high tolerance toward surfactants agents and organic solvents while it was completely inhibited by PMSF indicating the serine protease type. Purified protease was used to remove hair from goat skin proving its potential application in leather processing industry. The results revealed that the protease has enhanced the quality and physico-chemical properties of the skins while reducing the pollution.

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes

Alkaline proteases are hydrolytic enzymes that cleave peptide bonds in proteins and peptides in alkaline conditions, which occupy a pivotal importance with respect to their industrial applications. This study aimed to isolate new alkaline protease producing alkaliphilic bacteria from Egyptian soda lakes and optimize the fermentation process to enhance the enzyme production. The extensive screening process of the samples collected from Egyptian soda lakes resulted in isolation of a potent alkaline protease producing alkaliphilic strain AK-R. The isolate was identified as Bacillus agaradhaerens strain AK-R based on 16S rRNA gene analysis (99%). Wheat bran and gelatin supported maximum alkaline protease production as carbon and nitrogen sources, respectively. Strain AK-R is halo-tolerant thermo-tolerant alkaliphilic bacterium in nature, as it can grow over a wide range of NaCl concentrations (up to 25%) and up to 55 °C, with maximal growth and enzyme production at 2.5-5%, and pH 11 at 35 °C. Among the tested cations, only Mg 2+ and Ca 2+ ions significantly enhanced the enzyme production by about 1.2, and 1.3 fold compared to control, respectively. Alkaline protease secretion was coherent with the growth pattern, reaching maximal yield after about 32 h (mid stationary phase). In conclusion a new halo-tolerant thermo-tolerant alkaliphilic alkaline protease producing Bacillus agaradhaerens strain AK-R was isolated from Egyptian soda lakes. Optimization of the nutritional and cultivation conditions resulted in increase of enzyme yield by 20 fold. Strain AK-R and its extracellular alkaline protease with salt, pH and temperature, tolerance signify their potential application in laundry and pharmaceuticals industries.

Production and Partial Characterization of an Extracellular Thermophile Alkaline Protease from a Selected Strain of Bacillus sp Isolated from Abattoir Soil in the North Region of Cameroon

The present study aims at screening and isolates thermostable alkaline protease producing bacteria from soil sample in the northern part of Cameroon, and characterizes their enzyme. Samples of soil were collected from four (04) North Cameroonian towns (Ngaoundere, Garoua, Figuil and Maroua). Six (06) isolates showed important proteolytic activity on skim milk agar. The strain N2.3.5 from Ngaoundere showed the largest hydrolysis halo (24 mm) and was selected for protease production. The partial identification of strain N2.3.5 through morphological and biochemical characterization enabled us to classify it as belonging to the genus Bacillus sp. The growth of the strain N2.3.5 and its enzyme production profile showed that the growth of the bacteria was a diauxic type, and that the protease production was not directly associated with the microbial growth. In a medium composed of soya flour, corn flour, yeast extract and at alkaline pH, following the monitoring of each of these parameters by a complete factorial experimental design, the pH value of 10 was found to be significant for an optimal growth. Meanwhile, the corn flour, the soya flour and the yeast extract did not have a consequent influence on the protease production by the bacteria, under the chosen experimental conditions. The extracellular protease produced by Bacillus sp N235 was partially purified using a precipitation with ammonium sulphate, a dialysis and a gel filtration on sephadex G-75. A specific activity of 1516.7 U/mg and a purification yield of 37.63% were obtained. The partially purified enzyme was able to maintain its activity after been heat-treated at 80°C for 30 min. The maximal activity was exhibited at temperature of 80°C and to a pH 12. The enzyme activity and stability was enhanced in presence of the Ca2+.

One Factor at A Time based optimization of protease from poultry associated Bacillus licheniformis

The results obtained from the present investigation indicate enhanced production of alkaline protease from Bacillus licheniformis strain 018 under optimized medium components and culture conditions by using traditional OFAT method. The most significant findings of this study were the ability of the isolate to produce maximum level of extracellular alkaline protease after 24 h of incubation, and the stability of enzyme at high temperature and pH. The best process parameters were found to be casein as the sole carbon source and yeast extract as nitrogen source. The isolate of the present context could be a good alternative of the commercial strains in tanneries, pharmaceutical and food processing industries as biotechnological and physiological aspects due to the thermo-alkaline nature of protease obtained. Further studies are in progress in order to purify the alkaline protease from the isolate and the scaling up of the enzyme for commercial applications.

Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: purification, characterization, effects of surfactants and organic solvents

In this study, the extracellular thermostable alkaline protease out of A10 strain was purified 1.38-fold with 9.44% efficiency through the ammonium sulfate precipitation-dialysis and DE52 anion exchange chromatography methods. The molecular weight of the enzyme in question along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be approximately 40.55 kDa, whereas the optimum pH and temperature ratings were identified as 9.0 and 70 °C, respectively. It was seen that the enzyme had remained stable between pH 7.5–10.5 range, protecting more than 90% of its activity in the wake of 1 h incubation at 60–70 °C. It was also observed that the enzyme enhanced its activity in the presence of Mg2+, Mn2+, K+, while Fe2+, Ni2+, Zn2+, Ag+ and Co2+ decreased the activity. Ca2+, however, did not cause any change in the activity. The enzyme was seen to have been totally inhibited by phenylmethylsulfonyl fluoride, therefore, proved to be a serine alkaline protease.

Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30.

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of- flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80°C in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50 °C and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni(2+), Mn(2+), and Cu(2+) to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax values were estimated to be 35.7 mg/ml, and 5 × 10(4) U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere.

A thermostable extracellular alkaline protease producing Bacillus amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth promoting activities. Strain SP1 was purified to 6.48-fold using four-step purification protocol and characterized in detail for its robustness and ecofriendly application in leather and detergent industries. Structural analysis revealed that the protease was monomeric and had a molecular weight of 43 kDa. It exhibited optimum activity at 60°C in alkaline environment (pH 8.0) and stable in the presence of surfactants and oxidizing agents. Enzyme was thermostable at 50°C and retained more than 70% activity after 30 min incubation. It has shown stain removal property and dehairing of goat skin without chemical assistance and hydrolyzing fibrous proteins. This protease showed Km of 0.125 mg ml(-1) and V(max) of 12820 μg ml(-1) indicating its excellent affinity and catalytic role. Thermal inactivation of the pure enzyme followed first-order kinetics. The half life of the pure enzyme at 50, 60, and 65°C was 77, 19.80, and 13.33 min, respectively. The activation energy was 37.19 KJ mol(-1). The results suggest that the B. amyloliquefaciens SP1 has a potential application in different industries.

Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis

Caldicoprobacter guelmensis isolated from the hydrothermal hot spring of Guelma (Algeria) produced high amounts of extracellular thermostable serine alkaline protease (called SAPCG) (23,000U/mL). The latter was purified by ammonium sulphate precipitation, UNO Q-6 FPLC and Zorbex PSM 300 HPLC, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 55,824.19Da. The 19 N-terminal residue sequence of SAPCG showed high homology with those of microbial proteases. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine protease family. It showed optimum protease activity at pH 10 and 70°C with casein as a substrate. The thermoactivity and thermostability of SAPCG were enhanced in the presence of 2mM Ca2+. Its half-life times at 80 and 90°C were 180 and 60min, respectively. Interestingly, the SAPCG protease exhibited significant compatibility with iSiS and Persil, and wash performance analysis revealed that it could remove blood-stains effectively. Overall, SAPCG displayed a number of attractive properties that make it a promising candidate for future applications as an additive in detergent formulations.

Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudoalteromonas sp. 129-1.

An extracellular alkaline protease produced by marine bacteria strain Pseudoalteromonas sp. 129-1 was purified by ammonium sulphate precipitation, anion exchange chromatography, and gel filtration. The purity of the protease was confirmed by SDS-PAGE and molecular mass was estimated to be 35 kDa. The protease maintained considerable activity and stability at a wide temperature range of 10-60 °C and pH range of 6-11, and optimum activity was detected at temperature of 50 °C and pH of 8. Metallo-protease inhibitor, EDTA, had no inhibitory effect on protease activity even at concentration up to 15 mM, whereas 15 mM PMSF, a common serine protease inhibitor, greatly inactivated the protease. The high stability of the protease in the presence of surfactants (SDS, Tween 80, and Triton X-100), oxidizing agent H(2)O(2), and commercial detergents was observed. Moreover, the protease was tolerant to most of the tested organic solvents, and saline tolerant up to 30%. Interestingly, biofilm of Pseudomonas aeruginosa PAO1 was greatly reduced by 0.01 mg ml(-1) of the protease, and nearly completely abolished with the concentration of 1 mg ml(-1). Collectively, the protease showed valuable feathers as an additive in laundry detergent and non-toxic anti-biofilm agent.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

High-throughput transformation method for Yarrowia lipolytica mutant library screening.

As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided.

Characterisation of the role of an alkaline protease from Vibrio midae SY9 in enhancing the growth rate of cultured abalone fed a probiotic-supplemented feed

Abstract South Africa has a rapidly developing abalone aquaculture industry based largely on the cultivation of Haliotis midae . The prophylactic use of probiotic microorganisms has been shown to enhance the health, growth and nutritional status of a variety of cultured aquaculture species. Furthermore, research has shown that H. midae fed a high protein artificial diet supplemented with the proteolytic marine bacterium Vibrio midae SY9 have enhanced digestive protease activity levels, protein digestion and absorption, and growth. The predominant V. midae SY9 extracellular alkaline protease, VmproA, has been previously shown to be present in the crop/stomach and intestinal regions of H. midae fed a diet supplemented with V. midae SY9. Therefore, VmproA may be responsible for the probiotic properties of the bacterium in farmed abalone. In this study, the role of VmproA on abalone growth was investigated using the mutant strain V. midae SY9Mut2, which is unable to produce VmproA. Over the course of a 180 day laboratory-based growth trial the growth rate of small (28 mm) abalone improved by 6.5 and 14.5% following dietary supplementation with V. midae SY9 and V. midae SY9Mut2, respectively, in comparison to abalone fed a basal diet. In situ protease activity within the H. midae digestive tract was significantly increased in animals fed the V. midae SY9 supplemented diet compared to abalone fed either the basal feed or the V. midae SY9Mut2 supplemented diet. Thus, VmproA does not appear to be the factor responsible for increased growth of probiotic-supplemented abalone. Statement of relevance The manuscript addresses the mechanism of how probiotics function in a farmed abalone species. This is important since probiotic supplementation of feed is proving successful in increasing the growth of these molluscs, and yet very little is known regarding the underlying relationship between the host animal and the probiont.

Isolation, identification, partial purification, optimization and characterization of alkaline protease from Neisseria flavescence

Microbial alkaline proteases are among the important hydrolytic enzyme and have been used extensively since the advent of enzymology. Bacterial extracellular alkaline proteases are of great importance due to its wide spectrum applications in detergent industries, bioremediation, food industries, and leather processing and bio-film degradation. From the various niches eighteen isolates were screened for alkaline protease production, out of which four isolates showed efficient alkaline protease production. Out of four bacterial species one of the isolate i.e. N. flavescence showed significant enzyme activity. Optimization of the pH and temperature conditions for enzyme production was determined and was found to be 7.0 and 600C, respectively. Optimization of carbon, nitrogen sources and metal ions for enzyme production were determined and was found to be 4.64U/ml for sucrose, 0.91U/ ml for gelatin and 0.21U/ml for zinc chloride for the isolates. The yield of alkaline protease was inhibited by copper sulphate. Enzyme activity was assayed using tyrosine-casein method. The purified enzyme preparations of having enzyme activity 0.26U/ ml was also excellent in destaining of ink colour. The molecular weight of different b and s of alkaline protease from N. flavescence ranged from 25kD to 83kD.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0–20%), showing maximal growth and enzyme production at 0–5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

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.