Wen  Du, Qian  Wang, Jia-Kun  Wang, Jian-Xin  Liu, 
    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,
    Volume 13,Issue 8,
    2014,
    Pages 538-544,
    ISSN 1680-5593,
    javaa.2014.538.544,
    (https://makhillpublications.co/view-article.php?doi=javaa.2014.538.544)
    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<SUP>-1</SUP>, 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&deg;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<SUP>-1</SUP>) decreased catalytic 
  efficiency (kcat from 42.82-9.82 sec<SUP>-1</SUP>). Nevertheless, further mutation 
  of V20A and M53I reversed the km to 45.14 mg mL<SUP>-1</SUP> and kcat to 198.75 
  sec<SUP>-1</SUP> which suggested that V20A and M53I probably contributed to 
  the enhanced activity.
    Keywords: Xylanase;rumen;catalytic activity;directed evolution;error-prone PCR