Alkaline-resistance model of subtilisin ALP I, a novel alkaline subtilisin.
The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule.
Maeda
H
H
Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. maeda@biochem.tohoku.ac.jp
Mizutani
O
O
Yamagata
Y
Y
Ichishima
E
E
Nakajima
T
T
D001329:Autolysis / Q000139:chemically induced; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation; D020860:Subtilisin / Q000737:chemistry*; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*
D001426:Bacterial Proteins; D014364:Tryptophan; C094600:AprS protein, Bacillus; D012697:Serine Endopeptidases; D013381:Subtilisins; C435719:subtilisin ALP I; D020860:Subtilisin; C032923:microbial serine proteinases; C053771:nattokinase