Isolation, Expression and Characterization of Family 10 and 11 Xylanases from an Extremely Thermophilic Bacterium, Dictyoglomus Thermophilum Strain..., 1997 Biological Sciences Symposium Proceedings

A xylanase gene, xynA, coding for a family 10 enzyme from Dictyoglomus thermophilum strain Rt46B. 1 was isolated from a genomic library, sequenced, characterized and over-expressed in Escherichia coli. The enzyme has a single domain structure and is extremely thermostable and active over a broad pH range (over 50% activity between pH 5.5 to 9.5). Recombinant XynA was found to release reducing sugars from kraft pulp at a rate which matched closely an increased A280 of the supematant. A second gene (xynB) was also isolated by genomic walking PCR from the same organism which encodes a family 11 xylanase comprising an N-terminal catalytic domain and a C-terminal binding domain. Seven genetically engineered xylanases produced from different xyn B constructions were expressed to different levels and each had pH optima of 6.5. They retained 50% activity at pH8.5 and had temperature optima ranging between 70 and 85°C. Although ideal as a host for preliminary characterisation, E. col i produces insufficient recombinant enzyme for mill-trials. Accordingly we have transferred our genes to the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei, because these organisms are capable of far greater extra-cellular production of recombinant enzymes.