Abstract
Xylolytic hydrolases are generally applied in many kinds of fields, including food industry, animal husbandry, paper and pulp industry and are considered still very potential for application. In industry endo-1, 4-β-xylanase is mostly applied enzyme among the xylolytic hydrolases, and its potential is desirously expected. In our last research, we have isolated a thermo-alkaline Paenibacillus sp. (Isolate BL11) from black liquor (58℃, pH 9.83). The bacterium can secret an endo-1, 4-β-xylanase, named xylanase X. At optimal reaction condition (60℃ pH 7), xylanase X demonstrates an enzyme activity of 2392 IU/mg. The research goal of this experiment is to create a mutagenesis library of xyl X using error-prone PCR and isolate mutants of improved thermostability by screening the library for industry application. At the end of this experiment we have got two activity-improved mutants, an amino acid substitution mutatant pXcm-H1(T44A) and a downstream deletion mutant pXcm-L1(amino acid sequence 270-290 from TSTHSFSLRGASNNANMARVD changed to LALIVSHCGGRRTMPTWPGWT by a nucleotide deletion). Between 67.5℃ and 70℃ pXcm-H1 showed twice activity then xylanase X whereas pXcm-L1 had triple activity. Besides, we have got a thermostability-decreased mutatant pXcm-M2(T44M). The mutant carries a different amino acid substitution at the site of pXcm-H1, but its thermostability decreased distinctly. According to this result, we suggest that the Thr-44 is a critical amino acid affecting the thermostability of xylanase X.