Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp.

Fujiwara, Nobuaki; Masui, Akihiko; Imanaka, Tadayuki

doi:10.1016/0168-1656(93)90117-6

Cited by 56 publications

(23 citation statements)

References 20 publications

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“…However, the optimal temperature of some mesophilic Bacillus species, e.g., Bacillus horikoshii, Bacillus mycoides and B. pumilus, is between 40 and 55°C [1,16,19,32], and that of some thermophilic Bacillus species, e.g., Bacillus cereus and Bacillus stearothermophilus, between 70 and 75°C [13,28,35,36]. On the other hand, an upward shift of 5-15°C was observed upon addition of 2-5 mM Ca 2+ to alkaline protease preparations of some Bacillus species [13,17,22,28,32,36]. The optimal temperature of the alkaline protease of B. clausii GMBAE 42 was estimated at 60°C, and a shift in the optimal temperature from 60 to 70°C was also observed after addition of 5 mM Ca 2+ .…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…Relatively higher purification levels and yields of alkaline proteases of various Bacillus sp. were obtained by Kobayashi et al [22] and Fujiwara et al [13] with procedures consisting only of ion-exchange chromatographic steps. On the other hand, 22-fold purification of a serine alkaline protease from Bacillus sphaericus with 52% recovery was achieved by Singh et al [33] by using phenyl agarose hydrophobic interaction chromatography (HIC) and QSepharose ion-exchange chromatography following ammonium sulfate precipitation.…”

Section: Enzyme Purificationmentioning

confidence: 99%

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Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

Kazan

Denizci²,

Öner

et al. 2005

J IND MICROBIOL BIOTECHNOL

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An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37 degrees C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH(4))(2)SO(4) precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60 degrees C; however, it is shifted to 70 degrees C after addition of 5 mM Ca(2+) ions. The enzyme was stable between 30 and 40 degrees C for 2 h at pH 10.5; only 14% activity loss was observed at 50 degrees C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0--12.2 range for 24 h at 30 degrees C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol(-1) (44.30 kJ mol(-1)). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30 degrees C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3'-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k (cat) value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K (m) and k (cat) values were estimated at 0.655 microM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21 x 10(3) min(-1), respectively.

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Section: Enzyme Purificationmentioning

confidence: 99%

Section: Enzyme Purificationmentioning

confidence: 99%

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

Kazan

Denizci²,

Öner

et al. 2005

J IND MICROBIOL BIOTECHNOL

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“…Casein is a highly stable and hydrophobic non-fibrous protein which does not coagulate or easily denatured by heat but can -9 -be hydrolysed by certain proteases (Fujiwara et al 1993;Cheng et al 1995). These characteristics informed our choice of the casein hydrolysis ring assay for the identification of proteolytic microorganisms that can hydrolyse casein and other structurally similar substrates such as keratins.…”

Section: Isolation and Identification Of Feather Degrading Bacteriamentioning

confidence: 99%

Identification and characterisation of a Bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather

Okoroma

Garelick

Abiola

et al. 2012

International Biodeterioration & Biodegradation

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Significant amount of keratins in the form of feather, hair, hoof and horn are generated annually by the livestock industry. Keratinases are increasingly important in the reprocessing and environmental pollution control of keratin wastes. The aim of this study is to isolate a microbial strain of high keratinase activity and to evaluate its feather degrading potential.Thirty-two keratin degrading microbial strains from farmyard wastes and primary effluent were isolated using a selective medium containing feather meal at 30, 37 and 50 °C. One of the isolates, which demonstrated the highest keratinolytic activity (11.00 ± 0.71 Uml -1 ) was identified as a species of Bacillus licheniformis based on the 16S rDNA analysis, designated as strain N22 and deposited in a culture collection. Optimum keratinase production by this bacterium was achieved in 32 h using a minimum growth medium containing 1.1% (w/v) feather meal at 50 °C and pH 8.

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“…Extracellular protease activity was determined using a modified procedure of Fujiwara et al [13] with 1.0% casein in 50 mM Tris-HCl buffer pH 8.0 as substrate. The assay mixture consisted of 0.4 ml of substrate and 0.1 ml of enzyme solution in 50 mM Tris-HCl buffer pH 8.0.…”

Section: Assay Of Protease Activitymentioning

confidence: 99%

Purification and partial characterization of serine alkaline metalloprotease from Bacillus brevis MWB-01

Olajuyigbe

Falade

2014

Bioresour. Bioprocess.

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Background: Proteases from bacteria are among the most important hydrolytic enzymes that have been studied due to their extracellular nature and high yield of production. Of these, alkaline proteases have potential for application in detergent, leather, food, and pharmaceutical industries. However, their usefulness in industry is limited by low activity and stability at high temperatures, extreme pH, presence of organic solvents and detergent ingredients. It is therefore very crucial to search for new alkaline proteases with novel properties from a variety of microbial sources. Results: In the present study, 21 Bacillus species isolated from organic waste sites were screened for proteolytic activity on casein agar. Bacillus brevis MWB-01 exhibited highest proteolytic activity with a clear zone diameter of 35 mm. Production of protease from B. brevis MWB-01 was investigated in optimized media after 48 h of cultivation with shaking (180 rpm) at 37°C. The protease was partially purified in a two-step procedure using ammonium sulphate precipitation and gel filtration chromatography on Sephadex G-200 column. The enzyme was purified 2.1-fold with yield of 4.6%. The purified protease had optimum temperature of 40°C with relative activity of about 50% at 50°C and was uniquely stable up to 60°C after 30 min of incubation exhibiting 63% residual activity. The enzyme had optimum pH of 8.0 and remarkably showed relative activity above 70% at pH 9.0 to 11.0 and 53% at pH 12.0, respectively and was very stable over a wide pH range (6.0 to 12.0). Ca 2+ and Mn 2+ increased protease activity with 9.8% and 3.5%, respectively; Hg 2+ and Zn 2+ strongly inhibited protease activity by 89% and 86%. The almost complete inhibition of the enzyme by phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA) confirmed the enzyme as a serine metalloprotease. The enzyme had highest compatibility with Sunlight, a commercial laundry detergent.

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Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp.

Cited by 56 publications

References 20 publications

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

Identification and characterisation of a Bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather

Purification and partial characterization of serine alkaline metalloprotease from Bacillus brevis MWB-01

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