The development of new analytical techniques and the commercial availability of new substrates have led to the purification and characterization of a large number of xylan-degrading enzymes. Furthermore, the introduction of recombinant DNA technology has resulted in the selection of xylanolytic enzymes that are more suitable for industrial applications. For a successful integration of xylanases in industrial processes, a detailed understanding of the mechanism of enzyme action is, however, required. This review gives an overview of various xylanolytic enzyme systems from bacteria and fungi that have been described recently in more detail.

A total of 26 proteolytic moderate halophiles were isolated and characterized. Most isolates were members of the genus Salinivibrio (16 strains), while others were identified as Bacillus (4 strains), Salinicoccus (2 strains), or members of the gamma-Proteobacteria (4 strains). Strain CP76 was selected as the best producer of an extracellular protease, designated CP1, and was used for further studies. Sequence analysis of the 16S rRNA gene in addition to phenotypic tests led to the placement of this organism in the genus Pseudoalteromonas. Maximal protease production was detected at the end of the exponential growth phase. This CP1 protease was purified and biochemically characterized, showing optimal activity at 55 degrees C, pH 8.5, and high tolerance to a wide range of NaCl concentrations (0 to 4 M NaCl). The most interesting features of this enzyme are its moderate thermoactivity, its activity at a range of pH values (6-10), and, especially, its salt tolerance (optimal activity at 7.5% total salts). The purified protease has a molecular mass of 38 kDa, and the N-terminal amino acid sequence determined showed similarity to metalloproteases previously described. The protease activity was strongly inhibited by EDTA, PMSF, and Pefabloc. No significant inhibition was detected with E-64, bestatin, chymostatin, or leupeptin. These results suggest that Pseudoalteromonas sp. strain CP76 produces an extracellular metalloprotease moderately thermotolerant and stable at high salt concentrations.

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO 2 , was determined by the application of [1-13 C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatographymass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.The naphthalene metabolism of mesophilic microorganisms under aerobic conditions has been intensely investigated, and detailed information has been presented on degradation rates, metabolic pathways, and the involved enzymes (7,8,11,25). In contrast, little is known about the metabolism of naphthalene or other polycyclic aromatic hydrocarbons (PAH) by thermophilic bacteria. Several studies on the growth of thermophilic microorganisms on aromatic compounds such as benzoic acid, cresols, or phenols have been carried out; however, respective degradation pathways are largely unresearched (1,4,19,20). The degradation of xenobiotics by thermophilic microorganisms provides crucial advantages compared to mesophilic organisms, especially when they are applied in biotechnological processes. Limited bioavailability as a result of the low water solubility of hydrophobic contaminants may be overcome due to a higher water solubility at elevated temperatures. The water solubility of naphthalene, for example, rises from 30 mg liter Ϫ1 at 20°C to 130 mg liter Ϫ1 at 60°C (26). Moreover, diffusion rates increase at higher temperatures with an additional positive impact on bioavailability.The bacteria applied in this degradation study were isolated from a compost consisting of wooden ties treated with lignite tar. They were able to utilize naphthalene as a sole source of carbon and energy. Stable isotope labeled [1-13 C]naphthalene was used as a model contaminant. The fate of naphthalene was traced by means of the technique of 13 C isotope analysis, which has been successfully applied to trace metabolic pathways (3,18,22). Stable isotope labeling enabled us to trace quantitatively the transformation of the xenobiotic carbon into specific fractions such as CO 2 , biomass, and metabolites. Moreover, the incorporation of the xenobiotic carbon into the bacterial fatty and amino acid fraction was determined on a molecular level. We describe here str...

The immobilization of an endoglucanase, benzoylformate decarboxylase (BFD) from Pseudomonas putida, as well as of lipase B from Candida antarctica (CALB) onto the carrier supports Sepabeads EC-EP, Sepabeads EC-EA, and Sepabeads EC-BU was accomplished. It is shown that via these immobilized biocatalysts the synthesis of both fine and bulk chemicals is possible. This is illustrated by the syntheses of polyglycerol esters and (S)-hydroxy phenyl propanone. The benefit of immobilization is illustrated by repetitive use in a bubble column reactor as well as in a stirred tank reactor. High stability of two biocatalysts was achieved and reusability up to eight times was demonstrated. The comparison of CALB immobilized on Sepabeads EC-EP to Novozym 435 shows similar activity.

The capability of secreting thermoactive enzymes exhibiting α‐amylase and pullulanase with debraching activity, seems to be widely distributed amongst anaerobic thermophilic bacteria. Interestingly, pullulanase formed by these bacteria displays dual specificity by attacking α‐1,6‐ as well as α‐1,4‐glycosidic linkages in branched glucose polymers. Unlike the enzyme system of aerobic microorganisms the majority of starch hydrolysing enzymes of anaerobic bacteria is metal indepedent and is extremely thermostable. This enzyme system is controlled by substrate induction and catabolite repression; enzyme expression is accomplished when maltose or maltose‐containing carbohydrates are used as substrates. By developing a process in continuous culture we were able to greatly enhance enzyme synthesis and release by anaerobic thermophilic bacteria. An elevation in the specific activities of cell‐free amylases and pullulanases could also be achieved by entrapping of bacteria in calcium alginate beads. The unique properties of extracellular enzymes of thermophilic anaerobic bacteria makes this group of organisms suitable candidates for inductrial application.

Thermophilic and hyperthermophilic microorganisms are found as normal inhabitants of continental and submarine volcanic areas, geothermally heated sea-sediments and hydrothermal vents and thus are considered extremophiles. Several present or potential applications of extremophilic enzymes are reviewed, especially polymer-hydrolysing enzymes, such as amylolytic and hemicellulolytic enzymes. The purpose of this review is to present the range of morphological and metabolic features among those microorganisms growing from 70 o C to 100°C and to indicate potential opportunities for useful applications derived from these features.

A ND C. D RA IN A S. 2000. An extracellular a-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1·4-kb protein-coding sequence is identical to that of the corresponding a-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei a-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal a-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant a-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0·7-2·5 mol l −1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei a-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements.

Aims:  The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Methods and Results:  Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. Conclusions:  This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. Significance and Impact of the Study:  The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro‐organisms within a mixed population.