Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1

Singh, Sanjay Kumar; Singh, Santosh Kumar; Tripathi, Vinayak Ram; Garg, Shweta

doi:10.1016/j.procbio.2012.05.021

Cited by 58 publications

(24 citation statements)

References 33 publications

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“…Kim et al, (2001) [12] reported that the character protease from Bacillus cereus KCTC 3674 were studied excellent precipitated with ammonium sulfate concentration of 80% as seen from the amount of sediment and enzyme activity were obtained. Similar to the results of research Singh et al, (2012) [13] stated that the concentration of ammonium sulfates 80%, which is best used to precipitate proteins from enzymes Bacillus cereus SIU1.…”

Section: Appropriate (Nh4)2so4 Concentration For Purification Of Alkasupporting

confidence: 81%

“…Prakash el al., (2005) [24] conducted a study of Bacillus cereus with the results of studies suggest that the molecular weight of alkaline protease enzyme origin of Bacillus cereus is 30 kDa. Singh et al, (2012) [13] also reported that the alkaline protease enzyme produced by Bacillus cereus SIU1 has a proteins molecular weight of 22 kDa. So it can be assumed and drawn a conclusion that the enzyme alkaline protease produced by Bacillus cereus strain LS2B is by weight 34 kDa.…”

Section: Characterization Of the Molecular Weight Of The Alkaline Promentioning

confidence: 98%

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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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Section: Appropriate (Nh4)2so4 Concentration For Purification Of Alkasupporting

confidence: 81%

Section: Characterization Of the Molecular Weight Of The Alkaline Promentioning

confidence: 98%

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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“…The presence of inhibitors reduced the β-sheet content and also the proteolytic activity. Similar observations were reported for B. cereus SIU1 serine protease in which the decrease in the β-sheet content was correlated to decrease in the enzyme activity (6). High β-sheet content was reported for serine protease isolated from halophilic Virgibacillus and the β-sheet content was reduced in the absence of NaCl which in turn resulted in a poor activity (23).…”

Section: Analysis Of the Secondary Structure Of The Protease In Thsupporting

confidence: 79%

“…Study of the enzyme conformation using far UV circular dichroism (CD) and activity measurement as a function of pH, metal ions, and other additives will help us in establishing the structure-function relationship of the enzymes (6). With proteases gaining considerable importance in the industrial market, efforts on optimizing enzyme activity has been focused with the use of different statistical software packages (7).…”

mentioning

confidence: 99%

Optimization of Parameters that Affect the Activity of the Alkaline Protease from Halotolerant Bacterium, Bacillus acquimaris VITP4, by the Application of Response Surface Methodology and Evaluation of the Storage Stability of the Enzyme

2016

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A great deal of efforts is focused on meeting the increasing demand for novel microbial catalysts that are capable of functioning under extreme conditions, and therefore, the production of these novel microbial enzymes with appreciable activity and stability in the harsh environments (1). Enzymes from organisms which can grow in 0-15% of the NaCl will have great potential for the use in the industries because of their inherent ability to be active and stable both in the presence as well as in the absence of salt. However, most of these enzymes require metal ions for the maintenance of the structure and/or stability (2). These enzymes are active and stable under conditions that cause in a lower activity, which include high salt concentrations or organic solvents (3). In most of the cases, measurable proteolytic activity is achieved upon supplementation of cofactors like divalent metal ions in the assaying buffer. Also loss of enzymatic activity can be restored by incubating the enzyme with excess metal ions (4). 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 Ca 2+ , 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.

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“…), B. cereus SIU1 with MW of 22 kDa (Singh et al . ), B. cereus with MW of 28 kDa (Doddapaneni et al . ) and B. megaterium with MW of 28 kDa (Asker et al .…”

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of Extracellular Gelatinolytic Protease from B acillus Amyloliquefaciens H11

Sai‐Ut

Benjakul

Sumpavapol

et al. 2015

Journal of Food Biochemistry

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Extracellular gelatinolytic enzyme from Bacillus amyloliquefaciens H11 was purified by gel filtration chromatography on Sephacryl S‐200 and ion exchange chromatography on diethylaminoethyl‐cellulose with 35% yield and 14‐fold increase in purity. Based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was estimated to be 21 kDa. The optimum gelatinolytic activities of purified enzyme toward porcine gelatin were 50C and pH 8.0. The inhibitor study revealed that the purified enzyme was a metallo‐serine protease and activated by Ca2+ and Mg2+ but resistant to Triton X‐100 and methanol at a concentration of 10% (v/v). Among all gelatins, that from unicorn leatherjacket fish skin was the most preferred for hydrolysis by the purified enzyme, in comparison with porcine and tilapia counterparts. Thus, the enzyme from B. amyloliquefaciens H11 could be used as a potential protease for production of gelatin hydrolysate. Practical Applications Extracellular protease from Bacillus amyloliquefaciens H11 plays a specific catalytic role in the hydrolysis of gelatin and can be used for production of gelatin hydrolysate with bioactivities. It can also be a potential alternative for commercial protease in conversion of marine processing by‐products to generate high value‐added products.

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Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1

Cited by 58 publications

References 33 publications

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

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

Optimization of Parameters that Affect the Activity of the Alkaline Protease from Halotolerant Bacterium, Bacillus acquimaris VITP4, by the Application of Response Surface Methodology and Evaluation of the Storage Stability of the Enzyme

Purification and Characterization of Extracellular Gelatinolytic Protease from B acillus Amyloliquefaciens H11

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