Catalytic Profile and Amylolytic Studies of Toluene-Tolerant Enterococcus faecalis str. nov mercadA7

This work pivots around screening a toluene-tolerant bacterium with concomitant alpha-amylase production ability. Eighteen alpha-amylase-producing bacterial strains were isolated from samples collected from Tokat province of Turkey among which strain A7 showed maximum alpha-amylase production titers (968 U/ml). The strain A7 was identified as Enterococcus faecalis mercadA7 (KX298857) with 54% GC content and 98% similarity to the closest strain, Enterococcus faecalis NBRC100480 through 16srRNA typing studies. The amylase production was improved 2.6-folds compared to the basal medium at optimal conditions recorded: 37 degrees C, pH 7.0, 18 h incubation, 1% v/v inoculum content and 20% toluene (carbon) supplementation. The amylase activity was demonstrated to increase upon exposure to toluene by a yet to be determined mechanism. The various growth kinetic parameters recorded are specific growth rate, mu(max) of 0.318 h(-1); constant, K-s 10.48 mg/ml; yield coefficient, Y-x/s of 0.1157 mg/g; and cell doubling time, t(d) of 51 min. The enzyme was partially purified with a threefold increase in specific activity; it was found to be active at pH 8 and 40 degrees C; stable from 30 to 50 degrees C and 7-10 pH range; maintaining stability up to 40% of toluene. The purified enzyme's maximum velocity, V-max of 3.33 mmol/h ml with constant, Km of 5 mg/ml were determined by Line Weaver Burke plot. Both Enterococcus faecalis mercadA7 and the purified alpha-amylase might find applications in several industrial processes due to the catalytic promiscuity triggered by the presence of toluene, and augmented stability of the cell-free purified enzyme as well as the organism to high alkaline/toluene levels.