A new look at xylanases: an overview of purification strategies.

Interest in xylanases from different sources has increased markedly in the past decade, in part because of the application of these enzymes in the pulp and paper industry. Purity and purification costs are becoming important issues in modern biotechnology as the industry matures and competitive products reach the marketplace. Thus, new paths for successful and efficient xylanase recovery have to be followed. This article reviews the isolation and purification methods used for the recovery of microbial xylanases. Origins and applications of xylanases are described, highlighting the special features of this class of enzymes, such as the carbohydrate-binding domains (CBDs) and their importance in the development of affinity methodologies to increase and facilitate xylanase purification. Implications of recombinant DNA technology for the isolation and purification of xylanases are evaluated. Several purification procedures are analyzed, taking into consideration the sequence of the methods used in each and the number of times each method is used. New directions to improve xylanase separation and purification from fermentation media are described.

The Oenococcus oeni genome: physical and genetic mapping of strain GM and comparison with the genome of a 'divergent' strain, PSU-1.

The physical and genetic maps of the Oenococcus oeni strains GM and PSU-1, which represent two genomic divergent groups on the basis of macrorestriction and ribotyping analysis, were compared. To achieve this comparison, the GM maps were constructed and the PSU-1 maps, already established, were improved. All the recognition sites of the restriction enzymes ASC:I, I-CEU:I, FSE:I, NOT:I and SFI:I were located in both chromosomes and the position of 26 genetic markers, including two rrn operons and 14 new putative oenococcal genes, were allocated to the restriction fragments generated by the five enzymes. The comparative analysis of O. oeni GM and PSU-1 genomes revealed extensive conservation of loci order. As for the differences encountered in the locations of restriction sites, they seem to be a reflection of the differences in restriction fragment sizes, explainable by insertion/deletion events and point mutations. No evidence for major genomic rearrangements was found. The genomic conservation between the two strains is in agreement and suggests homogeneity within the species, which was not unexpected in view of the restricted ecological niche of O. oeni. Further comparisons of physical maps, both of O. oeni strains and related species, will certainly help to assess whether O. oeni is really an homogeneous species and physical mapping is suitable for taxonomic purposes, both at the supra- and intraspecific levels.

Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers.

A physical map of the chromosome of Oenococcus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcription direction determined.