Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted -1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.The arthropod gut is a differentiated organ harboring a complex biotope comprising both resident and transient members from protozoal, bacterial, and archaeal genera. Many of these organisms are symbionts that contribute in a concerted way to a complex chemical cycle sustaining the metabolic competence of the host. These symbiotic relationships help to define the metabolic traits of the insect, contributing to efficient sharing of the derived nutrients. In addition to carbon metabolism, data have demonstrated that the microbial consortia contribute to nitrogen cycling, methano-and acetogenesis, and prevention of foreign microbial pathogenesis (29).A complete understanding of the complexities of the gut biotopes is lacking, however, due to the difficulties encountered in culturing the myriad of contributing microbes and in describing the physiologies of the individual species. Studies aimed at a description of microbial complexity have recently been undertaken in termites by using 16S ribosomal DNA analysis and have shown that a number of unique lineages of microorganism inhabit the hindgut (28). These data support the idea that the insect gut represents a contained biome wherein unique species and chemistries evolve.Polysaccharide hydrolysis is a key element in insect nutrition. Since the diet of most arthropods comprises primarily plant matter, digestion of the structural polysaccharides cellulose and hemicellulose is essential for energy metabolism and the ability to obtain carbon from these sources contributes materially to the success of the order. These polysaccharides are resistant to degradation, and the insects themselves do not secrete all of the digestive enzymes to hydrolyze -linkages in the polymer. Rather, much of the hydrolysis of these polysaccharides is carried out by enzymes produced by the microbial symbionts (4,5,36,41).Hemicellulose consists primarily of xylan and it is the second most abundant polymer type in plant materi...
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