Isolation and characterization of Pichia stipitis mutants with enhanced xylanase activity

Pichia stipitis strain NRRL Y-11,543 was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) to improve xylanolytic activity. A total of 20,000 mutants were screened for xylanase overproduction by observing the clear zones around the colonies on remazol-briliant-blue-xylan (RBB-xylan)-containing agar. Of 94 mutants isolated 11 of them were found to have enhanced xylanase activity compared to the parental strain. The most active mutant NP54376 had superior properties to the wild type which included: double the enzyme activity of wild type, a shorter generation time of 2.22 h compared to 3.13 h when grown on xylan, and an enhanced growth and yield of xylanase when low levels of xylose were added to the medium. Zymogram analysis of the crude enzyme preparations from both NP54376 and the wild type by isoelectric focusing showed multiple bands ranging between pI 4.2 and 7.4. No significant difference was observed in the K_m and V_max values of the parental strain and NP54376. K_m and V_max values of xylanase for birchwood xylan were 4.2 mg ml^-1 and 0.08 μmol min^-1 mg^-1 of protein, respectively.