Physicochemical properties of alkaline serine proteases in alcohol.
The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1 micrograms/ml and that of alcalase was 48.1 micrograms/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters, L-Ala-L-Ala-(D- or L-)Pro-L-Phe-OMe and L-Ala-L-Ala-(D- or L-)Ala-L-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.
Chen
S T
ST
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Chen
S Y
SY
Tu
C C
CC
Chiou
S H
SH
Wang
K T
KT
D000595:Amino Acid Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004795:Enzyme Stability; D000431:Ethanol; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D012995:Solubility; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D014867:Water / Q000378:metabolism
D014867:Water; D000431:Ethanol; D013381:Subtilisins