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An alkaline protease from halotolerantBacillus licheniformis BA17, isolated from Van Lake in Turkey, was purified 5.4 fold with 58% yield. The molecular weight was 19.7 kDa and the optimum
temperature and pH were 60°C and 10, respectively. The half-life of the pure enzyme was 38 h, 93 min, 14 min and 6 min at
40, 50, 60 and 70°C, respectively. BA17...
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Citations
... The purified protease exhibited a single band on the SDS-PAGE, suggesting its homogeneity and it showed molecular weight of about 25 kDa (Figure 2c). In comparison with other bacillus proteases, purified SH21 had a lower molecular weight than Bacillus stearothermophilus (28 kDa) [19], Bacillus amyloliquefaciens (43 kDa) [20], but higher compared with those produced by Bacillus subtillis (17.1 kDa) [21], and Bacillus licheniformis (19.7 kDa) [22]. For the purified sample, the zymogram activity specified a caseinolytic zone with a projected molecular weight of 25 kDa, which was seen co-migrating with a protein. ...
... The purified protease exhibited a single band on the SDS-PAGE, suggesting its homogeneity and it showed molecular weight of about 25 kDa (Figure 2c). In comparison with other bacillus proteases, purified SH21 had a lower molecular weight than Bacillus stearothermophilus (28 kDa) [19], Bacillus amyloliquefaciens (43 kDa) [20], but higher compared with those produced by Bacillus subtillis (17.1 kDa) [21], and Bacillus licheniformis (19.7 kDa) [22]. For the purified sample, the zymogram activity specified a caseinolytic zone with a projected molecular weight of 25 kDa, which was seen co-migrating with a protein. ...
Proteases are important enzymes that are engaged in a variety of essential physiological functions and have a significant possible use in industrial applications. In this work, we reported the purification and biochemical characterization of a detergent stable, antimicrobial, and antibiofilm potential protease (SH21) produced by Bacillus siamensis CSB55 isolated from Korean fermented vegetable kimchi. SH21 was purified to obtain homogeneity via ammonium sulfate precipitation (40–80%), Sepharose CL-6B, and Sephadex G-75 column. By analyzing the SDS-PAGE and zymogram, it was determined that the molecular weight was around 25 kDa. The enzyme activity was almost completely inhibited in the presence of PMSF and DFP, which indicated that it was a member of the serine protease family. SH21 showed excellent activity with a broad range of pH and temperature, with its maximum pH of 9.0 and temperature of 55 °C. The enzyme had estimated Km and Vmax values of 0.197 mg/mL and 1.22 × 103 U/mg, respectively. In addition, it preserved good activity in the presence of different organic solvents, surfactants, and other reagents. This enzyme showed good antimicrobial activity that was evaluated by MIC against several pathogenic bacteria. Furthermore, it exhibited strong antibiofilm activity as determined by MBIC and MBEC assay and degraded the biofilms, which were analyzed by confocal microscopic study. These properties established that SH21 is a potent alkaline protease that can be used in industrial and therapeutic applications.
... The most potent inhibitor at 5 mM concentration was PMSF (the well-known serine-protease inhibitor), followed by DNTB both of which produced residual activities of 0.09% and 29.16% respectively. Ö ztürk et al. [40] reported complete loss of activity when their alkaline protease from Bacillus sp. strain BA17 was exposed to 1-5 mM of PMSF as observed in this study. ...
A strain of Stenotrophomonas acidaminiphila, isolated from fermenting bean-processing wastewater, produced alkaline protease in pretreated cassava waste-stream, but with low yield. Strain improvement by alternate combinatorial random mutagenesis and bioprocess optimization using comparative statistical and neural network methods enhanced yield by 17.8-fold in mutant kGy-04-UV-25. Kinetics of production by selected mutant modeled by logistic and modified Gompertz functions revealed higher specific growth rate in mutant than in the parent strain, likewise volumetric and specific productivities. Purification by PEG/Na⁺ citrate aqueous two-phase system recovered 73.87% yield and 52.55-fold of protease. Its activity was stable at 5-35% NaCl, 45-75°C, and was significantly enhanced by 1-15 mM sodium dodecyl sulfate (SDS). The protease was inhibited by low concentrations of phenyl-methyl-sulfonyl fluoride but was activated by 1-5 mM Mn²⁺ suggesting a manganese-dependent serine-protease. The 45.7 kDa thermo-halo-stable alkaline protease demonstrated keratinolytic and blood-stain removal potentials showing prospects in textile and detergent industries, respectively.
... The enzymatic activity at each step was determined using 2 mM Suc-AAPF-pNA at 30 °C in 50 mM Tris-HCl (pH=7). Suc-AAPF-pNA as a synthetic peptide peptidyl-pNA was used to determine the enzymatic activity because the extracellular serine protease and alkaline protease showed the highest specificity towards Suc-AAPF-pNA (31,32). KB-SP was purified 8.60±0.09-fold ...
... It lost > 75 % of its activity at pH ≤ 3 and ≥ 12 (Fig. 4a). The effect of pH on the stability and activity of KB-SP was similar to the findings of previous studies on proteases from other bacteria having a pH of 7-12 for halo-alkaline proteases (3,27,32,40) and the alkaline protease belonging to B. ...
... The activity of control was determined after dialysis against 10 mM EDTA. These results are similar to those reported for B. licheniformis MP1 and BA17 for each metal ion at 5.0 mM(32,38). In addition, the enzymatic activityof B. licheniformis RP1 and P003 and halophilic proteases from Bacillus spp. ...
Research background. Haloalkaline proteases are one of the most interesting types of commercial enzymes in various industries due to their high specific activity and stability under extreme conditions. Biochemical characterization of enzymes is an important requirement for determining their potential for application in industrial fields. Most of microbial proteases have been isolated from Bacillus spp. In this study, the purification and characterization of an extracellular haloprotease produced from Bacillus sp. KB111 strain, which was previously isolated from mangrove forest sediments, are investigated for industrial applications.
Experimental approach. The whole genome of KB111 strain was identified by DNA sequencing. Its produced protease was purified by salting out and anion-exchange chromatography, characterized based on protease activity and stability using a peptide substrate, and identified by LC-MS/MS.
Results and conclusions. The strain KB111 was identified as Bacillus licheniformis. The molecular mass of its extracellular protease, termed KB-SP, was estimated to be 70 kDa. The optimal pH and temperature for the activity of this protease were 7 and 50 °C, respectively, while the enzyme exhibited maximal activity in the broad salinity range of 2–4 M NaCl. It was fully stable at an alkaline pH range of 7–11 at 50 °C with a half-life of 90 min. Metal ions such as K+, Ca2+ and Mg2+ could enhance the enzyme activity. Therefore, this protease indicates a high potential for the applications in the food and feed industry, as well as the waste management since it can hydrolyse protein at high alkaline pH and salt concentrations. The amino acid profiles of the purified KB-SP determined by LC-MS/MS analysis showed high score matching with the peptidase S8 of B. licheniformis LMG 17339, corresponding to the mature domain of a minor extracellular protease (Vpr). Amino acid sequence alignment and 3D structure modelling of KB-SP showed a conserved catalytic domain, a protease-associated (PA) domain and a C-terminal domain.
Novelty and scientific contribution. A novel extracellular haloprotease from B. licheniformis was purified, characterized and identified. The purified protease was identified as being a minor extracellular protease (Vpr) and this is the first report on the halotolerance of Vpr. This protease has the ability to work in harsh conditions, with a broad alkaline pH and salinity range. Therefore, it can be useful in various applications in industrial fields.
... Protease with optimum pH at 8.0 and 12.0 could be potentially applied in detergent formulations. Previous works have characterized alkaline proteases with optimums between 8.5 and 11.0 from several Bacillus species such as B. licheniformis BA17 [31], B. pimulis CBS [32] and B. mojavensis A21 [33]. ...
The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses.
... Temperature profiles on enzyme activity are shown in Figure 4. Optimum temperature for the enzyme was found to be 60 ºC for both isolates. Similar results were reported by other researchers where optimum temperature of 60 ºC was recorded for proteases from other B. licheniformis strains (Öztürk et al.¸2009; Bezawada et al., 2011; El Hadj-Ali et al., 2007). Inhibition studies primarily give an insight of the nature of an enzyme. ...
A total of 379 B. licheniformis strains isolated from commercial milk powder were characterized genotypically and phenotypically. RAPD analysis yielded three different profiles, which include all isolates in this study, which could be assigned to strain F (n=375) or strain G (n=4), strain F also could be divided into two groups (group 1, n = 117; group 2, n = 258). Clustering by pairwise sequence similarity and phylogenetic relationships between isolates based on comparisons of the 16S rRNA gene sequence, showed two well defined groups. Group I contains all isolates tested belonging to genotype F, and Group II consists of three G genotype isolates. A total of 32 isolates, respecting the representation of each genotype, were randomly selected for extracellular enzymatic activity plate assays. Most isolates (25 out of 32) showed extracellular proteinase, lipase and amylase activity. Hydrolytic activities tested in this study are strain-dependent and none enzymatic activity could be linked to a defined group at genetic level. Preliminar characterization of proteolytic crude enzyme extract suggests the presence of a metal-activated serine protease active at an optimun temperature of 60 ºC. The exoenzymes production and its variation against different factors such as temperature, is isolate dependent so these results indicate that not all B. licheniformis strains may mean the same risk to process or product quality.
... Iran 2.38BD -2.75CD -8.44A 2 -9.81B 2 -5.69C 2 -3.31D 2 In the above equation the variables A, B, C, and D represents the coded levels of temperature, pH, CaCl 2 , MnCl 2 , respectively. ...
... The combined effect of CaCl 2 and temperature on protease activity can be depicted from ( Figure 2B) Table 3. Secondary structural composition (%) of the protease (0.2 mg.mL -1 ) probed by circular dichroism in the presence of different additives Figure 2. Response surface plots for the protease activity obtained from optimization studies. A: interaction of temperature and pH; B: interaction of temperature and CaCl 2 ; C: interaction of MnCl 2 and temperature; D: interaction of CaCl 2 and MnCl 2 The maximum protease activity (90.3 U.mL -1 ) was observed at 51°C and 6.8 mM CaCl 2 through response surface plot analysis. Increased concentration of CaCl 2 has resulted in a significant positive effect on protease activity at low temperature, but a reduction in the activity was observed as the concentration of CaCl 2 was further increased. ...
Background:
It was previously shown that the activity of a serine protease from a moderately halotolerant Bacillus aquimaris VITP4 strain is active in a wide range of pH and temperatures and could be modulated by the presence of the divalent metal ions.
Objectives:
In the present study, a quantitative analysis was done in order to explore the parameters that are contributing to the protease activity.
Materials and methods:
Changes in the secondary structure of the enzyme was determined by circular dichroism analysis. The conditions for the optimal activity was investigated by Response Surface Methodology. Stability of the enzyme was determined by thermal inactivation experiments.
Results:
The initial one-factor-at-a-time experiments have indicated that the activity of the enzyme could be enhanced not only by the presence of low concentrations of NaCl but also by divalent metal ions, such as Ca(2+), Mn(2+) and Cu(2+). A clear dependence of the activity to the secondary structure of the enzyme could be established using circular dichroism spectroscopy. In the next level of optimization, four factors; viz. pH, temperature, concentration of Ca2+, and Mn(2+) were used to optimize the conditions required for the maximal activity of the enzyme by Response Surface Methodology, and the data could be explained using quadratic model. Under optimal condition of 43°C, pH 8.0, 8.2 mM Ca(2+), and 4.3 mM Mn(2+) a 1.5 times enhancement in the enzyme activity could be achieved. The storage stability of the enzyme under these selected conditions has indicated a non-linear relation between the conditions for the enzymatic activity as well as stability. However, the condition for the maximal stability (267±18 min) has corresponded to that of the optimal conditions for the maximal activity.
Conclusions:
This study, for the first time, has explored the possibility of using statistical methods for identifying the optimal conditions for alkaline protease activity isolated from the halotolerant Bacillus aquimaris VITP4.
... However, Mn 2? ions at 10 mM concentration were reported as strong inhibitor of alkaline proteases of some Bacillus sp. [26,27]. ...
... These two metal ions were even reported to inhibit the activity of organic solvent, detergent, and thermal stable alkaline protease of B. licheniformis [26]. For most of the protease characterized from Bacillus sp. ...
Bacillus tequilensis hsTKB2, produced extracellular alkaline (9–10.5), thermostable (50–80 °C), halophilic (0–30 %), organic solvent tolerant keratinolytic protease. The maximum enzyme production was observed in high alkaline condition (pH 10) at 45 °C in sea water as supporting medium and chicken feathers as substrate with cell-immobilization
of chitin flakes. The molecular mass of purified enzyme was estimated to be about 59.89 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and MALDI-TOF analysis. The apparent Km for the enzyme activity on keratin was 1.9 mg mL−1. Optimum temperature and pH for keratinolytic protease activity were 70 °C and 10.5 respectively. The effect of organic solvents on the enzyme activity showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. Circular dichroism spectroscopy revealed that at pH 10.5 and temperature 70 °C, the enzyme upholds a typical secondary structure (α-helix 25.23 %, β-sheet 61.02 %, turn 11.97 % and coil 1.78 %). Mn2+ and toluene were influenced the enzyme activity by effecting structural changes. This enzyme folded into its active conformation inside the cell and secreted outside. Furthermore the tolerability with surfactants and commercial solid and liquid detergents appeared to be a favorable approach towards laundry and dishwashing detergents.
... These enzymes also find use in the resolution of the rasemic mixtures of amino acids, in the hydrolysis of gelatin layers of X-ray films for silver recovery, and in peptide synthesis123. In literature, there are a number of reports on alkaline proteases of different Bacillus strains, which are stable at elevated pH and temperature45678910. Savinase R is a commercially available alkaline protease from the facultative alkaliphilic Bacillus clausii, which is used as a laundry detergent additive [11]. ...
Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu2+) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45–65°C). Maximal residual activity was observed in the presence of 5 mM CuCl2. The enzyme was thermoinactivated according to first-order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that Ea,i, ΔGi, and ΔHi values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ΔSi values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower Km values and higher kcat and kcat/Km values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions Ea, ΔG≠, ΔH≠, , and values of enzyme were lower, but there was no change in ΔS≠ values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing.
... Alkaline proteases are of immense interest due to their wide application in detergent, tanning, textile, dairy industry, organic synthesis, peptide synthesis, instant recovery of silver and wastewater treatment (Gupta et al., 2002). Microbial proteases are extracellular and would simplify the downstream processing of the enzyme as compared to those obtained from plant and animal sources (Oztruk et al., 2009). Despite the long list of protease producing microorganisms, only a few microorganisms are considered as appropriate producers for commercial exploitation, being non-toxic and non-pathogenic. ...
The objective of this study is the production of an extra cellular alkaline protease by a new local isolate which was characterized and identified by 16S rRNA as Streptomyces griseus sub sp griseus and it was submitted in gene bank with accession number AB723782. The culture conditions for higher protease production by Streptomyces griseus were optimized with respect to carbon source, nitrogen source and pH. Maximum protease production was obtained in medium no. 5 supplemented with 3% glucose as a carbon source, 0.4 % NaNO 3 as a nitrogen source, the optimum pH was 8 at 30°C and the highest production was obtained after 6 days of incubation. Wheat bran proved as the best food waste for protease production (9208.5 U/mg protein) compared to other wastes, and finally the optimum gamma radiation was (1.6 kGy) led to increase the specific activity to 14450.5 U/mg protein. The protease profile of the isolate shows its potential as a good source for industrial application.
