Purification and characterization of SDS stable protease from Bacillus safensis strain CK

Jalkute, Chidambar B.; Waghmare, Shailesh R.; Nadaf, Naiem H.; Dhanavade, Maruti J.; Jadhav, Deepak B.; Pendhari, S.I.; Patil, Rahul; Sonawane, Kailas D.

doi:10.1016/j.bcab.2017.02.012

Cited by 11 publications

(4 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The molecular weight of purified protease from B. licheniformis LBA 46 was estimated as 40 kDa by SDS-PAGE (Figure 4). Jalkute et al (2017) purified the protease from Bacillus safensis CK about 7-fold by DEAEcellulose column chromatography and estimated the…”

Section: Biochemical Characterization Of Purified Proteasementioning

confidence: 99%

ALKALINE PROTEASE PRODUCTION BY Bacillus licheniformis LBA 46 IN A BENCH REACTOR: EFFECT OF TEMPERATURE AND AGITATION

Aguilar

Castro

Sato

2019

Braz. J. Chem. Eng.

View full text Add to dashboard Cite

The production of protease from Bacillus licheniformis LBA 46 was studied in a 6 L reactor using the experimental design tool. The higher protease production was obtained in the exponential phase of growth reaching maximum activity (~3,000 U/mL) after 48 h of fermentation at 30 ºC and 300 rpm in a culture medium made of agroindustrial by-products. In the thermostability study, the semi-purified enzyme retained about 78% of the initial activity after 120 min at 50 ºC. The protease was purified 3.33 times by ammonium sulfate precipitation and DEAE-Sepharose column chromatography and had a molecular mass estimated at 40 kDa by SDS-PAGE. The purified protease showed optimum activity at 50 and 60 ºC, optimal activity in pH 8.5 and stability in the range between pH 5-10 after 24 h of incubation at 4 ºC, presenting more than 86% of the initial activity.

show abstract

Section: Biochemical Characterization Of Purified Proteasementioning

confidence: 99%

ALKALINE PROTEASE PRODUCTION BY Bacillus licheniformis LBA 46 IN A BENCH REACTOR: EFFECT OF TEMPERATURE AND AGITATION

Aguilar

Castro

Sato

2019

Braz. J. Chem. Eng.

View full text Add to dashboard Cite

show abstract

“…In contrast, the SDS that is cited in the literature as an enzyme inhibitor [54] stimulated protease activity (Table 2). Proteases from Bacillus safensis CK [55] and Bacillus pumilus D3 [56] in the presence of SDS also showed an increase in the values of enzyme activity, demonstrating that the inhibitor did not have a negative effect on these enzymes. The Tween-20 (5%) caused a 42.86% residual loss in B. rmus protease activity.…”

Section: Characterization Of the Alkaline Protease Of B Rmus Obtained In Fed-batchmentioning

confidence: 99%

Production, Purification, and Characterization of Extracellular Alkaline Protease From Bacillus Firmus Var. Arosia NCIB 10557

Dias

Silva

Santana

et al. 2021

Preprint

View full text Add to dashboard Cite

The most important alkaline proteases from the commercial standpoint are produced by bacteria of the genus Bacillus and used mainly in the formulation of detergents. The aim of the present study was to evaluate the production, partial purification, and characteristics of alkaline protease obtained by Bacillus firmus var. arosia NCIB 10557 in fed-batch fermentation with constant feeding profile and carbon source restriction. Firstly, it was carried out on batch fermentation and after 6.5 h of fermentation, glucose became limiting, and then the fed-batch was started with a flow rate of 0.0802 mL/min. Maximum activity (998.1 U/mL) was reached after 10.5 h of fed-batch, with a subsequent 60.91 % drop in activity after two hours. The purification steps resulted in a 1.65-fold increase in the value of the specific activity. The protease showed optimum activity at 37°C and pH 9 and residual activity above 80 % at pH 11 and 12. Residual activity was greater than 70 % at temperatures ranging from 30 to 70 °C and 90 % of this activity was maintained for 30 minutes at 70 °C until the occurrence of complete inactivation. Enzyme activity was estimated using SDS. The organic solvents Triton X-100, Tween-20, EDTA and β-mercaptoethanol and the ions Zn2+, Fe2+, Cu2+ and Ni2+ partially inhibited the activity of the protease. Ca2+, Mn2+ and Mg2+ had no stimulating action on the enzyme.

show abstract

“…As shown in Fig 8, the recombinant protease 1147 was detected in the soluble form at approximately 18 kDa position and verified by zymography and Western blot analysis. The majority of bacterial proteases have a molecular weight ranging between 15 and 45 kDa [47]. The produced protein was purified using His-tag affinity column chromatography and a nonchromatographic purification method named single-step thermal shock.…”

Section: Plos Onementioning

confidence: 99%

Structural and biochemical characterization of a novel thermophilic Coh01147 protease

et al. 2020

View full text Add to dashboard Cite

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, K m , and k cat , k ca t/K m values of 13.72 mM, 3.143 × 10 −3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60˚C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t 1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100˚C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

show abstract

Purification and characterization of SDS stable protease from Bacillus safensis strain CK

Cited by 11 publications

References 23 publications

ALKALINE PROTEASE PRODUCTION BY Bacillus licheniformis LBA 46 IN A BENCH REACTOR: EFFECT OF TEMPERATURE AND AGITATION

ALKALINE PROTEASE PRODUCTION BY Bacillus licheniformis LBA 46 IN A BENCH REACTOR: EFFECT OF TEMPERATURE AND AGITATION

Production, Purification, and Characterization of Extracellular Alkaline Protease From Bacillus Firmus Var. Arosia NCIB 10557

Structural and biochemical characterization of a novel thermophilic Coh01147 protease

Contact Info

Product

Resources

About