Purification of xylanase from Bacillus pumilus using Eudragit S-100 and optimization of conditions

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-1and 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.