Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674

Kim, Seongseop; Kim, Young; Rhee, In‐Koo

doi:10.1007/s002030100282

Cited by 37 publications

(21 citation statements)

References 8 publications

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“…cereus KCTC 3674 vigorously excretes proteases into the extracellular environment (Kim et al, 2001). Thus, it is very difficult to prepare the functional inverted membrane vesicles for energy measurement experiment.…”

Section: Resultsmentioning

confidence: 99%

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Enzymatic Properties of the Membrane-bound NADH Oxidase System in the Aerobic Respiratory Chain of Bacillus cereus

Kim

Kim²

2004

BMB Reports

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Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K m value of approximately 65 µM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 µM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 µM and the activity was also highly sensitive to Ag + .

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Section: Resultsmentioning

confidence: 99%

“…The bacterial strain used in this work was B. cereus KCTC 3674 (Kim et al, 1998;Kim et al, 2001). The bacterium was grown aerobically at 37 o C in a liquid medium which contained 0.5% polypeptone and 0.5% yeast extract in 50 mM Tris-HCl buffer (pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Properties of the Membrane-bound NADH Oxidase System in the Aerobic Respiratory Chain of Bacillus cereus

Kim

Kim²

2004

BMB Reports

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“…Similar observations were reported by Kobayashi et al [22] and Singh et al [33] on SDS-PAGE analysis of the serine alkaline proteases of Bacillus KSM-K16 and B. sphaericus, respectively. Generally, the molecular weights of the alkaline proteases of various Bacillus species lie within the range 15-36 kDa [6,16,21,28,32,33,35]. However, the presence in some Bacillus species of distinctive alkaline proteases with molecular weights of 42 and 68 kDa has also been reported [33,36].…”

Section: Enzyme Purificationmentioning

confidence: 98%

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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“…Microbial alkaline proteases constitute one of the most important groups of hydrolytic enzymes, accounting for about 50% of the total industrial enzyme market, since industrial applications of alkaline proteases such as detergents, food, leather and silk has increased remarkably (Gessesse, 1997;Horikoshi, 1999;Kumar and Takagi, 1999;Kim et al, 2001;Ghorbel et al, 2003;Manczinger et al, 2003;Joo and Chang, 2005;Patel et al, 2005;Amoozegar et al, 2006;Hadj-Ali et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Isolation and identification of alkaline protease producer halotolerantBacillus licheniformis strain BA17

et al. 2007

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An alkaline protease producer Bacillus licheniformis strain was isolated from Van Lake in Turkey. The strain is Gram positive, aerobic, motile, sporulating rod-shaped bacterium. Spores were ellipsoidal and positioned central in nonswollen sporangium. The cells were able to grow well at a pH range of 5.7-10. The optimal growth temperature was found to be 37 °C. Growth at a wide range of NaCl concentration (from 0 to 20%) showed that BA17 is halotolerant. Main fatty acid composition of BA17 was anteiso-C15:0 and iso-C15:0. The strain was presumptively identified as B. licheniformis according to 16S rDNA gene sequence analysis. The most appropriate medium for the growth and protease production is composed of 0.5% yeast extract, 0.5% NaNO 3 , 0.02% MgSO 4 ·7H 2 O, 0.1% K 2 HPO 4 and 0.5% maltose. The optimum temperature and pH of the alkaline protease of strain BA17 were found to be 60 o C and pH 11, respectively. The activity was completely lost in the presence of PMSF, suggesting that the preparation contains serine-alkaline protease(s).

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Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674

Cited by 37 publications

References 8 publications

Enzymatic Properties of the Membrane-bound NADH Oxidase System in the Aerobic Respiratory Chain of Bacillus cereus

Enzymatic Properties of the Membrane-bound NADH Oxidase System in the Aerobic Respiratory Chain of Bacillus cereus

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

Isolation and identification of alkaline protease producer halotolerantBacillus licheniformis strain BA17

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