Purification and characterization of moderately halophilic alkaline serine protease from marine Bacillus subtilis AP-MSU 6

Maruthiah, Thirumalai; Esakkiraj, Palanichamy; Prabakaran, Ganesan; Palavesam, A.; Immanuel, G.

doi:10.1016/j.bcab.2013.03.001

Cited by 41 publications

(30 citation statements)

References 22 publications

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“…have been reported (Haddar et al 2009b;Jain et al 2012;Annamalaia et al 2014a). However, it was higher than that reported for proteases from several haloalkaliphilic bacteria (Maruthiah et al 2013;Raval et al 2014). The thermal stability of the NPST-AK15 protease was investigated by pre-incubation of the enzyme at different temperatures for different time intervals before the measurement of residual activities.…”

Section: Effect Of Temperature On Protease Activity and Stabilitymentioning

confidence: 93%

“…Proteases (EC 3.4.21) are a large group of hydrolytic enzymes that catalyze the hydrolysis of the proteins by cleavage of the peptide bonds between the amino acid residues in other proteins (Shankar et al 2011). Proteases are classified into acid, neutral and alkaline proteases based on their optimum activity corresponding to pH (Maruthiah et al 2013). Alkaline proteases, with high activity and stability in alkaline range, are interesting for several bioengineering and biotechnological applications.…”

Section: Introductionmentioning

confidence: 99%

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Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization

et al. 2015

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Alkaline protease produced by the halotolerant alkaliphilic Bacillus sp. strain NPST-AK15 was purified to homogeneity by the combination of ammonium sulfate precipitation, anion-exchange and gel permeation chromatography. The purified enzyme was a monomeric protein with an estimated molecular weight of 32 kDa. NPST-AK15 protease was highly active and stable over a wide pH range, with a maximal activity at pH 10.5. The enzyme showed optimum activity at 60 °C and was stable at 30-50 °C for at least 1 h. Thermal stability of the purified protease was substantially improved by CaCl2 (1.1- to 6.6-fold). The K m, V max and k cat values for the enzyme were 2.5 mg ml(-1), 42.5 µM min(-1) mg(-1), and 392.46 × 10(3) min(-1), respectively. NPST-AK15 protease activity was strongly inhibited by PMSF, suggesting that the enzyme is a serine protease. The enzyme was highly stable in NaCl up to 20 % (w/v). Moreover, the purified enzyme was stable in several organic solvents such as diethyl ether, benzene, toluene, and chloroform. In addition, it showed high stability and compatibility with a wide range of surfactants and commercial detergents and was slightly activated by hydrogen peroxide. These features of NPST-AK15 protease make this enzyme a promising candidate for application in the laundry and pharmaceutical industries.

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Section: Effect Of Temperature On Protease Activity and Stabilitymentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization

et al. 2015

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“…For example, the protease from B. subtilis GA CAS8 was inhibited by Cu 2+ and Zn 2+ (Sathishkumar et al, 2015). In contrast, the hydrolytic activity of the protease from B. subtilis AP-MSU6 was stimulated by Cu 2+ and Hg 2+ (Maruthiah et al, 2013). As shown in Figure 5, the hydrolytic activity was increased when surfactants like Triton X-100 and Tween-20 were added to the hydrolytic reaction, which was similar to other proteases from the different Bacillus species (Had-Alij et al, 2007;Haddar et al, 2009;Shah et al, 2010).…”

Section: +mentioning

confidence: 79%

“…Furthermore, the optimal temperature for this protease to catalyze hydrolysis was higher than that of the other proteases from various Bacillus species, such as B. subtilis CFR3001 (40°C) (Bhaskar et al, 2007), B. pumilus BA06 (50°C) (Wan et al, 2009), B. proteolyticus AP MSU6 (40°C) (Maruthiah et al, 2013), and B. subtilis GA CAS8 (50°C) (Sathishkumar et al, 2015). Consequently, this protease exhibited better thermostability in comparison with the other bacterial serine proteases (Haddar et al, 2009;Maruthiah et al, 2013), as the half-life of thermal denaturation at 60°C approaches 72.2 min (Figure 4). Regarding pH, the puriðed protease was active over a wide range (6.0 to 12.0) and the optimal pH was 8.0, which was similar with the other proteases from various Bacillus strains (Wan et al, 2009;Sathishkumar et al, 2015).…”

Section: Discussionmentioning

confidence: 94%

Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4

et al. 2016

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ABSTRACT. The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries.

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“…Increased purity HiTrap continued by using ion exchange, the enzyme flowed in a column filled with a matrix sepharose. The matrix in the column for has a charge so that the purified enzyme based cargo of cations and anions [18]. Alkaline protease enzyme has wet properties so positively charged cations thus equilibration buffer used have a pH of 8.…”

Section: Purification Of Alkaline Protease Enzyme Bacillus Cereus Ls2mentioning

confidence: 99%

Semi Purification and Identifications Molecule Protein Weigh of Alkaline Protease Enzyme from Bacillus cereus LS2B

Junaidi¹,

Pertiwiningrum

Erwanto

et al. 2017

IJBSBT

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Purification and characterization of moderately halophilic alkaline serine protease from marine Bacillus subtilis AP-MSU 6

Cited by 41 publications

References 22 publications

Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization

Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization

Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4

Semi Purification and Identifications Molecule Protein Weigh of Alkaline Protease Enzyme from Bacillus cereus LS2B

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