CHARACTERIZATION OF THE RECOMBINANT XYLANASES IN ESCHERICHIA-COLI FROM AN ALKALIPHILIC THERMOPHILIC BACILLUS SP NCIM-59

The extracellular xylanases produced by recombinant Escherichia coli c arrying the 4,5-kb DNA fragment from an alkaliphilic thermophilic Baci llus (NCIM 59) were purified to homogeneity. The M(r) of the enzymes w ere estimated to be 35,000 (xylanase I) and 14,500 (xylanase II) by so dium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified xylanases had similar temperature (60 degrees C) and pH (6) optima, an d the isoelectric points were 4 and 8, respectively. The enzymes retai ned 100% activity at 50 degrees C for 24 h; however, at 60 degrees C t hey showed a half-life of 2 h The apparent K-m values of xylanase I an d II were 5.8 and 8.3 mg ml(-1) and V-max values were 0.010 and 0.520 mu mol min(-1) mg(-1), respectively. The recombinant xylanases showed reduced ability to bind xylan and had lower specific activity and K-m values than those of the xylanases from the parent Bacillus reported p reviously. Both enzymes yielded different hydrolysis patterns when inc ubated with oat spelt xylan. The hydrolysis pattern of the recombinant xylanases was found to be distinctly different from that of the origi nal xylanases, which may be due to differential binding of the cloned enzymes to the substrate.