2010
DOI: 10.1016/j.jbiotec.2010.02.024
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Effects of the mutational combinations on the activity and stability of thermolysin

Abstract: We have previously indicated that three single mutations (Leu144-->Ser, Asp150-->Glu, and Ile168-->Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53-->Asp and Leu155-->Ala) and one triple (Gly8-->Cys/Asn60-->Cys/Ser65-->Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The com… Show more

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Cited by 23 publications
(24 citation statements)
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“…We have reported several thermolysin variants, L144S, D150E, and I168A, with higher FAGLA-and ZDFM-hydrolyzing activities than WT. 13,27,28) On the other hand, G117E is the first thermolysin variant with lower FAGLA-hydrolyzing and higher ZDFM-hydrolyzing activities. Our strategy appeared to be effective in altering the substrate specificity of thermolysin.…”
Section: Discussionmentioning
confidence: 99%
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“…We have reported several thermolysin variants, L144S, D150E, and I168A, with higher FAGLA-and ZDFM-hydrolyzing activities than WT. 13,27,28) On the other hand, G117E is the first thermolysin variant with lower FAGLA-hydrolyzing and higher ZDFM-hydrolyzing activities. Our strategy appeared to be effective in altering the substrate specificity of thermolysin.…”
Section: Discussionmentioning
confidence: 99%
“…8,14) The amount of FAGLA hydrolyzed was evaluated using the molar absorption difference due to hydrolysis, Á" 345 ¼ À310 M À1 cm À1 , at 25 C. 8,14,28) The reaction was carried out in 40 mM acetate-NaOH buffer at pH 4.0-5.5, 40 mM MES-NaOH buffer at pH 5.5-7.0, 40 mM HEPES-NaOH buffer at pH 7.0-8.5, and TAPS-NaOH buffer at pH 8.0-9.0, each of which contained 10 mM CaCl 2 , at 25 C. Hydrolysis was carried out under pseudo first-order conditions, where the substrate concentration is much lower than the Michaelis constant (K m ) (>30 mM) 14) because of the slight solubility (<6 mM) of FAGLA. 8,14,28) Under the conditions, the kinetic parameters, K m and the molecular activity (k cat ), cannot be determined separately, and enzyme activity was evaluated by the specificity constant (k cat =K m ). The intrinsic k cat =K m , (ðk cat =K m Þ o ), and the proton dissociation constants (K e1 and K e2 ) for the bell-shaped pH-dependence of the activity (k cat =K m ) were calculated from eq.…”
Section: 18)mentioning
confidence: 99%
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“…The concentration of thermolysin was 1.0 mM in 5 mM Tris-HCl, 10 mM CaCl 2 , and 0 or 4.0 M NaCl at pH 7.5. 23,24) Because HEPES has high absorbance at 180-210 nm, Tris was used as buffer system. 25) The control baseline was obtained with solvent and all other components without thermolysin.…”
Section: )mentioning
confidence: 99%