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Journal of Biochemistry Advance Access originally published online on July 23, 2007
Journal of Biochemistry 2007 142(3):317-324; doi:10.1093/jb/mvm134
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© 2007 The Japanese Biochemical Society.

Effects of Neutral Salts and Alcohols on the Activity of Streptomyces caespitosus Neutral Protease

Kuniyo Inouye*, Takashi Shimada and Kiyoshi Yasukawa

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

*To whom correspondence should be addressed. Tel: +81-75-753-6266, Fax: +81-75-753-6265, E-mail: inouye{at}kais.kyoto-u.ac.jp

Received May 9, 2007; Accepted June 12, 2007


  Abstract

Streptomyces caespitosus neutral protease (ScNP) is one of the smallest metalloproteinase with a molecular mass of 14 kDa. Effects of solvent composition on ScNP activity were examined using a peptide substrate. The kcat/Km values of ScNP exhibited bell-shaped pH-dependence with the optimal pH of 6.4–7.0 and the pKa values of 5.0 ± 0.1 and 8.3 ± 0.1. ScNP activity increased in an exponential fashion with increasing [NaCl]. The relative kcat/Km value at 3.6 M NaCl to that at 0 M NaCl was 3.7, and the degree of the activation at x M NaCl was expressed as 1.2 x (x < 2.0) and 1.4x (x > 2.0). On the other hand, ScNP activity decreased with increasing concentrations of LiCl, KCl, NaBr, LiBr, KBr and NaClO4. Alcohols inhibited ScNP activity with the IC50 values, the concentration required for decreasing the activity at 50% of the maximum, of 0.77–6.54 M. The order of the inhibitory potency was 1-butanol, 2-methyl-1-propanol, 2-methyl-2-butanol > 2-methyl-2-propanol, 2-butanol, 1-propanol > 2-propanol >> ethanol >> methanol. The activities recovered completely by the dilution of alcohols, suggesting that the ScNP inhibition by alcohols is reversible. These characteristics of ScNP are compared with those of human matrix metalloproteinase 7 and thermolysin.

Key Words: alcohol, metalloproteinase, pH-activity profile, salt effect, Streptomyces caespitosus neutral protease

Abbreviations: FAAFA, N-[3-(2-furyl)acryloyl]-Ala-L-Phe-NH2; FAGLA, N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH2; FALAA, N-[3-(2-furyl)acryloyl]-L- Leu-L-Ala-NH2; MOCAc-PLGL(Dpa)-AR, N-(7-methyoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropioyl]-L-Ala-L-Arg-NH2; ScNP, Streptomyces caespitosus neutral protease


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