Modification of Highly Active Xylanase by Directed Evolution

An Orpinomyces xylanase gene (XynA) has been cloned, sequenced, and overexpressed in a readily fermentable production host (E. coli). Fermentation and down-stream processing have been established. XynA is most active and stable at temperatures of 50/o/C to 60/o/C and at a pH ranging from 5.8 and 7.5. The enzyme works well for industrial bleaching of paper pulp when the temperature and pH are within these ranges. Furthermore, the enzyme significantly improves animal (swine & poultry) weight gain and feed conversion when the feed has NOT undergone high temperatures and moisture pelleting. Most pulp bleaching processes are run at high temperature and pH, and a substantial percentage of chicken feeds are pelleted at high temperature (85-100/o/C); conditions that are not favorable to XynA. To fully capture the potential of the current xylanase, we propose to genetically alter the XynA gene through accelerated directed molecular evolution/breeding to produce mutant enzyme derivatives with higher thermostability and tolerance to more alkaline pH's. During Phase I of the project, we will focus on development and fine-tuning of mutation and screening procedures. In Phase II, mutant XynA enzymes with improved thermostability, with improved pH profiles, and isolates with both improved properties will be identified. Mutant enzymes will be tested for their compatibilities with fermentation and down-stream processes and their field performance under industrial conditions.