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Water Miscible Mono Alcohols’ Effect on the Proteolytic Performance of Bacillus clausii Serine Alkaline Protease

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Abstract

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V m, k cat and k cat/K m values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG ≠ and ΔG ≠ E − T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ≠ ES value of the enzyme remained almost the same. The constant K m and ΔG ≠ ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG ≠ and ΔG ≠ E − T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

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Abbreviations

Log P :

Solvent hydrophobicity

x water :

Mol fraction of water

D :

Dielectric constant

T [K]:

Absolute temperature

C [%]:

Mono alcohol concentration

a, b, m, n :

Empirical constants

[S] [M]:

Substrate (casein) concentration

v [U min−1]:

Initial reaction rate of casein hydrolysis

V max [μmol ml−1 min−1]:

Maximum casein hydrolysis rate of enzyme

K m [M]:

Michaelis–Menten constant

k cat [min−1]:

Catalytic rate constant (turnover number) of enzyme

k cat/K m [min−1 M−1]:

Catalytic performance of enzyme

[I] [M]:

Inhibitor (mono alcohol) concentration

K I [M]:

Inhibitor (mono alcohol) concentration

α, β :

Fold changes in K m and V m, respectively

ΔG ≠ [kJ mol−1]:

Activation free energy of enzyme for casein hydrolysis

ΔG ≠ ES [kJ mol−1]:

Free energy of substrate binding

ΔG ≠ E − T [kJ mol−1]:

Free energy of transition state formation

R [J K−1 min−1]:

Gas constant (8.314)

h [J s]:

Plank constant (6.63 × 10−34)

k B [J K−1]:

Boltzmann constant (1.38 × 10−23)

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Acknowledgments

This research was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) by the Project No: TUBITAK TBAG-U/123 104T262.

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Authors and Affiliations

  1. Faculty of Arts and Sciences, Department of Chemistry, Section of Biochemistry, Kocaeli University, Umuttepe Campus, 41380, İzmit-Kocaeli, Turkey

    Yonca (Avcı) Duman & Altan Erarslan

  2. Faculty of Engineering, Department of Bioengineering, Marmara University, Göztepe Campus, 34722, Kadıköy-Istanbul, Turkey

    Dilek Kazan

  3. The Scientific and Technological Research Council of Turkey (TUBITAK), Genetic Engineering and Biotechnology Institute (GEBI), Marmara Research Council (MRC), P.O. Box 21, 41470, Gebze-Kocaeli, Turkey

    Aziz Akın Denizci

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  1. Yonca (Avcı) Duman
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  2. Dilek Kazan
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  4. Altan Erarslan
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Correspondence to Altan Erarslan.

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Duman, Y.(., Kazan, D., Denizci, A.A. et al. Water Miscible Mono Alcohols’ Effect on the Proteolytic Performance of Bacillus clausii Serine Alkaline Protease. Appl Biochem Biotechnol 172, 469–486 (2014). https://doi.org/10.1007/s12010-013-0525-3

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