Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli.

AIMS: To assess the influence of sporulation media on heat resistance, and the use of stress recovery media to measure preservation injury of spores of five representative spoilage bacilli. METHODS AND RESULTS: Bacillus spores prepared on nutrient agar supplemented with Ca2+, Mg2+, Mn2+, Fe2+ and K+ were more heat-resistant than spores obtained from nutrient agar with Mn2+. This increased heat resistance correlated with a decrease in the protoplast water content as determined by buoyant density sedimentation. The degree of preservation injury severity could be assessed on media containing NaCl at moderate pH and organic acids at acid pH. Ca-DPA, K+ or proline were added to the recovery media to demonstrate that heat probably caused injury to both spore germination and the outgrowth system. SIGNIFICANCE AND IMPACT OF THE STUDY: The metal content of sporulation media can strongly effect the validity of preservation resistance studies. The distinctive recovery media developed here can be relevant for assessing and comparing new preservation technologies.

Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae.

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.

A beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae.

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.

Physiological Role of Chlorinated Aryl Alcohols Biosynthesized De Novo by the White Rot Fungus Bjerkandera sp. Strain BOS55.

The white rot fungus Bjerkandera sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dichlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H(2)O(2) production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus Bjerkandera sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.