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...

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90°C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61°C to as high as 96°C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).

The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. TheP. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.

High throughput screening of microbial DNA libraries was used to identify ␣-amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95°C without addition of endogenous Ca 2؉ . After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca 2؉ and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one ␣-amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.Endo-1,4-␣-D-glucan glucohydrolase (␣-amylase, 1 EC 3.2.1.1) is currently used in a broad array of industrial applications. These include starch hydrolysis for the production of ethanol and high fructose corn syrup, starch soil removal in laundry washing powders and dish-washing detergents, textile de-sizing, the production of modified starches, baking, hydrolysis of oil-field drilling fluids, and paper recycling. Since 1980, the most widely used enzyme for these applications has been the ␣-amylase isolated from the ubiquitous mesophilic soil bacterium Bacillus licheniformis (1-3). This enzyme operates optimally at 90°C and pH 6, and it requires addition of calcium (Ca 2ϩ ) for its thermostability (4), conditions that are substantially different from those encountered in the various industrial processes where the enzyme is utilized. The disparity between these industrial requirements and the native environment for the ␣-amylase results in sub-optimal enzymatic performance in many applications.Corn wet milling is an example of a multistep industrial process where there is considerable scope for enzyme performance improvement. Initially, whole corn kernels are fractionated into semi-purified streams of protein, fiber, oil, and starch. The resulting starch fraction has a pH of 4.5. The next process step involves liquefaction of the semi-purified starch to glucose oligomers by the B. licheniformis ␣-amylase, ideally at a pH of ϳ4.5 and a temperature of 105°C. However, because the enzyme is unstable under these conditions (5), the pH mu...

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