Abstract:
Soluble-insoluble polymers have considerable applications in the enzyme purification and these
polymers precipitate from the solution by changing the pH. Eudragit S-100, nontoxic polymer of
methacrylic acid and methyl methacrylate, has been used in affinity precipitation. The study
describes the purification of xylanase from Bacillus pumilus by three phase partitioning (TPP)
method and precipitation (PT) method using Eudragit S-100 and to optimize these methods to
improve the purification fold. In the TPP method, 14.6 U mL-1
of xylanase activity was obtained
with a specific activity of 31.74 U mg protein-1
. The recovery of the enzyme was 52.24 % with 1.79-
fold purification. In the PT method, 15 U mL-1
of xylanase activity was obtained with the specific
activity of 33.4 U mg protein-1
. The recovery of the enzyme was 52.18% with 1.72-fold purification.
Since there is no significant difference in the purification folds of both these methods and to
improve the purification fold, the conditions of these methods were optimized. When different
concentrations of (NH4)2SO4 were used, xylanase with significantly higher specific activity (33.74
U mg protein-1
) was precipitated with 50% of (NH4)2SO4 saturation. Among the different Eudragit
concentrations used, 40gL-1 Eudragit S-100 yielded significantly higher xylanase activity (18.9
UmL-1
) than the other Eudragit concentrations. When the spent medium was treated with 40gL-1
Eudragit S-100 and Eudragit bound xylanase was eluted with different concentrations of NaCl,
significantly higher activity of xylanase (17.9UmL-1
) was eluted with of 0.3 M NaCl. When TPP
method of purification was done under optimized conditions (with 50% (NH4)2SO4 saturation and
usage of 0.3 M NaCl for elution), 17.87U mL-1
of xylanase activity was obtained with a specific
activity of 38.1 U mg protein-1
and the xylanase enzyme yield and purification fold were
significantly increased to 63.41% and 2.01 than the non-optimized TPP method. When PT method
of purification was done under optimized conditions (with 40gL-1 Eudragit S-100 and usage of 0.3
M NaCl for elution), 18.96 U mL-1
of xylanase activity with the specific activity of 43.09 U mg
protein-1 was obtained and the xylanase yield and the purification fold were significantly increased
to 69.67% and 2.31 respectively than the non-optimized PT method. Since the purification fold of
the optimized PT method is significantly higher than that of optimized TPP method, optimized PT
method could be recommended for the purification of xylanase from Bacillus type bacteria. When
the purified xylanase was subjected to poly acrylamide gel electrophoresis, it gave a single band
and the molecular weight of the purified xylanase was determined as 55.4 kDa.