Molecular characterization, catalytic, kinetic and thermodynamic properties of protease produced by a mutant of Bacillus cereus-S6-3

Abdel–Naby, Mohamed A.; El-Wafa, Wael Mohamed Abu; Salem, Gad Elsayed Mohamed

doi:10.1016/j.ijbiomac.2020.05.241

Cited by 14 publications

(6 citation statements)

References 33 publications

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“…Our results agree with Abdel-Naby et al [32], who indicated that maximum enzyme activity for B. cereus PP/S6-3 was detected at pH 8 and 40 • C, and pH 9 and 50 • C for B. cereus PM/UM90. Likewise, a significant increase in keratinase activity of S. radiopugnans KR12 was noticed at pH 8 and 40 • C [24].…”

Section: Effect Of Different Factors On Keratinase Productionsupporting

confidence: 93%

“…Surprisingly, D 137 N substitution was detected in KerS39, serine protease WP_000790934.1, and WP_061129616.1 (Figure 8). Abdel-Naby et al [32] demonstrated that combination of chemical/physical mutagenesis on B. cereus resulted in 19 amino acid substitutions that led to an improvement of the protease by about 31.17% compared with the wild type; nine of the amino acid substitutions include I 242 Y, K 244 R, D 245 A, K 246 R, G 248 N, R 253 V, T 260 H, W 279 R, and E 281 L and improved the catalytic efficiency of the enzyme. Furthermore, the introduction of amino acid substitutions by site-directed mutagenesis on the keratinase of Stenotrophomonas maltophilia (A218S and A218G), B. licheniformis (N122Y, N217S, A193P, and N160C), and Brevibacillus parabrevis (T218S, S236C, and N181D) exhibited high improvement in thermostability, enzyme production, and catalytic activity [49][50][51].…”

Section: Multiple Sequence Alignment and Phylogenetic Analysismentioning

confidence: 99%

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Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

et al. 2022

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In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51–3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants’ effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5–100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5–19.3 and aliphatic index between 74.7–75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: −7.17; S score: −6.54 kcal/mol) and seven mutants of KerS26uv (affinity: −7.43; S score: −7.17 kcal/mol) compared to the wild predicted structure (affinity: −6.57; S score: −6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.

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Section: Effect Of Different Factors On Keratinase Productionsupporting

confidence: 93%

Section: Multiple Sequence Alignment and Phylogenetic Analysismentioning

confidence: 99%

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

et al. 2022

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“…As known, ∆G* is the most suitable thermodynamic parameter to measure the feasibility and extent of a reaction, as the lower the ∆G* value the more feasible the reaction, i.e., the conversion of the reactant into the product is more spontaneous [35]. The value of ∆G* estimated for azocasein hydrolysis catalyzed by A. tamarii URM4634 protease (59.94 kJ mol −1 ) is lower than those of casein and azocasein hydrolysis catalyzed by other fungal (A. heteromorphus: ∆G* = 65.8 kJ mol −1 [20]) and bacterial (Bacillus stearothermophilus: ∆G* = 91.71 kJ mol −1 [32]; Bacillus cereus: ∆G* = 68.68-69.42 kJ mol −1 [36]) proteases, which indicates that the conversion of the transition state of enzyme-substrate complex into the product is more spontaneous.…”

Section: Kinetic and Thermodynamic Parameters Of Azocasein Hydrolysismentioning

confidence: 78%

Use of a Sequential Fermentation Method for the Production of Aspergillus tamarii URM4634 Protease and a Kinetic/Thermodynamic Study of the Enzyme

Oliveira¹,

Claudino²,

Converti

et al. 2021

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Microbial proteases are commonly produced by submerged (SmF) or solid-state fermentation (SSF), whose combination results in an unconventional method, called sequential fermentation (SF), which has already been used only to produce cellulolytic enzymes. In this context, the aim of the present study was the development of a novel SF method for protease production using wheat bran as a substrate. Moreover, the kinetic and thermodynamic parameters of azocasein hydrolysis were estimated, thus providing a greater understanding of the catalytic reaction. In SF, an approximately 9-fold increase in protease activity was observed compared to the conventional SmF method. Optimization of glucose concentration and medium volume by statistical means allowed us to achieve a maximum protease activity of 180.17 U mL−1. The obtained enzyme had an optimum pH and temperature of 7.0 and 50 °C, respectively. Kinetic and thermodynamic parameters highlighted that such a neutral protease is satisfactorily thermostable at 50 °C, a temperature commonly used in many applications in the food industry. The results obtained suggested not only that SF could be a promising alternative to produce proteases, but also that it could be adapted to produce several other enzymes.

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“…For casein hydrolysis, the K m and V max were determined to be 0.4 mg mL −1 and 2000 U mL −1 , respectively. K m and V max values in previous studies on proteases were reported as 2.94 mg mL −1 and 19.45 U mL −1 min for Staphylococcus xylosus A2 protease, [ 38 ] 0.706 mg mL −1 and 3000 μ m min −1 for B. subtilis M33 protease, [ 6 ] and 4.44 mg mL −1 and 555.55 U mg −1 protein for Bacillus cereus ‐S6‐3 protease, [ 39 ] respectively. Notably, K m of E. alkaliphilum VLP1 protease is lower than that of the others indicating that the enzyme has higher affinity for casein.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of a novel purified and characterized alkaline protease from the extremophile Exiguobacterium alkaliphilum VLP1 as a detergent additive

Hemsinli,

Gurkok

2023

Biotechnology Journal

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This study focused on the isolation and identification of a novel alkaline protease‐producing strain from Lake Van, the largest soda lake on Earth. The objective was to purify, characterize, and investigate the potential application of protease in the detergent industry. Through a combination of classical and molecular methods, the most potent protease producer was identified as Exiguobacterium alkaliphilum VLP1. The purification process, involving ammonium sulfate precipitation, ultrafiltration, and anion exchange chromatography, resulted in a 45‐fold purification with a yield of 6.4% and specific activity of 1169 U mg−1 protein. The enzyme exhibited a molecular weight of 69 kDa, a Km value of 0.4 mm, and a maximal velocity (Vmax) value of 2000 U mg−1. The optimum activity was observed at 40°C and potential of hydrogen (pH) 9, while the enzyme also exhibited remarkable stability in the ranges of 30–60°C and pH 9–12. Notably, this study represents the first report of an alkaline protease isolated and characterized from E. alkaliphilum. This study also highlighted the potential of the enzyme as a detergent additive, as it showed compatibility with commercial detergents and effectively removed blood and chocolate stains from fabrics.

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Molecular characterization, catalytic, kinetic and thermodynamic properties of protease produced by a mutant of Bacillus cereus-S6-3

Cited by 14 publications

References 33 publications

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

Use of a Sequential Fermentation Method for the Production of Aspergillus tamarii URM4634 Protease and a Kinetic/Thermodynamic Study of the Enzyme

Evaluation of a novel purified and characterized alkaline protease from the extremophile Exiguobacterium alkaliphilum VLP1 as a detergent additive

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