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 subtili sin, 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 e xamined as a control. ALP I gradually lost its activity, accompanied by pro tein degradation, but, on the contrary, Sendai was stable under alkaline co nditions. 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 fluorescenc e intensity of ALP I was greatly reduced under alkaline conditions; moreove r, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and o ptical activities of NAT were lost at high pH, indicating a lack of functio nal and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structu re of the enzyme molecule.