Authors : Wen Du, Qian Wang, Jia-Kun Wang and Jian-Xin Liu
Abstract: The catalytic activity of a xylanase retrieved from a Fosmid Library of rumen microbiota in Hu sheep was improved by directed evolution using error-prone PCR. After the first round of random mutagenesis, one (C12) was selected from 1450 clones. And E12 with the highest activity was acquired from about 2500 clones in the second round. The specific activity of purified mutant C12 and E12 was 69.67 and 118.50 U mg-1, 1.2 and 2.0 times as that of its parent enzyme HA2, respectively. The optimum temperature of Wild Type (WT) and mutants were 50°C while the mutants displayed lower thermostability than WT. The optimum pH for HA2, C12 and mutant E12 was 5.0, 5.0 and 6.0, respectively. Both WT and mutants retained about 85% of initial activity at pH range from 5.0-9.0. Sequencing analysis revealed that C12 was mutated at two amino acid positions (Q14H and K57R) and E12 was mutated at Q14H, V20A, M53I and K57R. Compared to HA2, the mutation of Q14H and K57R increased the affinity of xylanase to substrate (km was changed from 11.22-2.42 mg mL-1) decreased catalytic efficiency (kcat from 42.82-9.82 sec-1). Nevertheless, further mutation of V20A and M53I reversed the km to 45.14 mg mL-1 and kcat to 198.75 sec-1 which suggested that V20A and M53I probably contributed to the enhanced activity.
Wen Du, Qian Wang, Jia-Kun Wang and Jian-Xin Liu, 2014. Enhancing Catalytic Activity of a Xylanase Retrieved from a Fosmid Library of Rumen Microbiota in Hu Sheep by Directed Evolution. Journal of Animal and Veterinary Advances, 13: 538-544.