Improving catalytic efficiency of endo-β-1, 4-xylanase from Geobacillus stearothermophilus by directed evolution and H179 saturation mutagenesis

Endo-β-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (kcat/Km) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.