Walter Callen

Unusual Microbial Xylanases from Insect Guts

ABSTRACT 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 β-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.

An evolutionary route to xylanase process fitness

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).

αa-D-Galactosidases from Thermotoga species

Publisher Summary Based on similarities in primary structure and hydrophobic cluster analyses, αGals have been grouped into three well-conserved families in the general classification of glycosylhydrolases Those from bacteria have been grouped into families 4 and 36 and those of eukaryotic origin into family 27. To date, only α Gals of the hyperthermophilic bacteria Thermotoga maritima ( Tm GalA) and T. neapolitana ( Tn GalA) have demonstrated activity and prolonged stability above 75°. These two enzymes are therefore of considerable interest from a biotechnological standpoint. Potential applications include the high temperature hydrolysis of galactomannans used for well stimulation in the oil and gas industry and oligosaccharide synthesis via glycosyltransferase reactions. Genes encoding both Tm GalA and Tn GalA have been cloned and expressed in Escherichia coli . Although these enzymes are structurally related, they exhibit different biochemical properties in terms of pH optima, activity, and thermostability. This chapter discusses the purification, cloning, and expression of recombinant α Gal from T. neapolitana and T. maritima , in addition to some of their biochemical properties.

Carboxylesterase from Sulfolobus solfataricus P1.

Publisher Summary To date, relatively few investigations regarding the purification and characterization of thermostable (T opt > 60 °) esterases have been conducted. Thus far, esterases from Bacillus acidocaldarius, Pyrococcus furiosus , Bacillus stearothermophilus , Sulfolobus shibatae , Thermoanaerobacterium sp, Pyrococcus abyssi , and Sulfolobus acidocaldarius have been studied to varying extents. Those thermostable esterases that have been evaluated vary significantly with respect to molecular mass, although their biochemical properties, such as pH optimum and substrate specificity, are quite similar. This chapter describes the cloning, expression, purification, and characterization of a recombinant carboxylesterase (Sso P1 carboxylesterase) from the extreme thermoacidophile Sulfolobus solfataricus P1. S. solfataricus , which is a member of the Crenarchaeota, can be found in sulfurous caldrons and volcanic muds.

β-Endoglucanase from Pyrococcus furiosus

Publisher Summary Glycosylhydrolases have been isolated from a variety of heterotrophic hyperthermophiles and include glucanases, hemicellulases, and cellulases. Pyrococcus furiosus , a hyperthermophilic heterotroph isolated by Fiala and Stetter from geothermal regions of Vulcano Island, Italy, grows on a wide range of α - and β -1inked carbohydrates, a property supported by the enzyme inventory revealed in its genome sequence. This chapter describes the approaches used for the cloning and expression in Escherichia coli of the eglA gene, which encodes the P. furiosus endoglucanase, and protocols used to study the biochemical properties of the recombinant enzyme. It is also interesting to know that P. furiosus and other heterotrophic hyperthermophilic archaea do not seem to produce enzymes for the hydrolysis of nonglucan polysaccharides, such as mannan or xylan, even though Thermotoga maritima, a hyperthermophilic bacterium, does. Whether this is a distinguishing feature of archaeal growth physiology remains to be seen.