PARTIAL-PURIFICATION AND PROPERTIES OF THERMOSTABLE INTRACELLULAR AMYLASES FROM A THERMOPHILIC BACILLUS SP AK-2

  • 1 January 1984
    • journal article
    • research article
    • Vol. 33  (1) , 57-+
Abstract
Intracellular thermostable amylases from a thermophilic Bacillus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68.degree. C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the Three reactions. The optimum temperature for the three fractions (FI, FII and FIII) was 65.degree. C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both .alpha. and .beta.-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the .alpha.-amylase type and FII of the .beta.-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 .times. 10-3 and 1.13 .times. 10-3 g/ml for .alpha.-amylase and .beta.-amylase, respectively.