ABSTRACT
In order to discover enzymes having potential for wood fibre modification, bacteria (fourteen strains designated MMB1 to MMB14) were isolated from a decomposing stump from a resinous tree. Phylogenetic analysis and biochemical characterization indicated that these isolates were related to Microbacterium, Chryseobacterium, Lysinibacillus, and Bacillus gene; although most demonstrated phenotypic differences compared to previously characterized relatives. Only the Bacillus strains showed cellulolytic activity (as CMCase detected with Congo red) and only Bacillus subtilis strains (MMB10 to MMB14) displayed cellulolytic and secreted xylanase activity. Phenotypic characterization of two strains (MMB8 and MMB9) related to a previously characterized isolate (Bacillus sp. JU2), supported their reassignment to the genus Lysinibacillus. The Microbacterium strain MMB1 produced a green pigment when grown in the presence of light. Some microbes from the consortium were devoid of wood polymer modifying enzymes, and may be dependent on other organisms for their survival in this biotope.
ABSTRACT
Protein design is currently used for the creation of new proteins with desirable traits, which include a superior nutritional value. One of the challenges of protein design in this area is to achieve the production of stable native-like proteins that resist the proteolytic pressure of the organism used for its production (the bioreactor). We report here the identification of a specific peptide bond sensitive to E. coli proteolysis in the designer protein MB-1Trp. In an attempt to reduce proteolysis, we have created a MB-1TrpHis gene library in which the two amino acids surrounding the peptide bond, N44 and L45, were randomized using degenerated oligonucleotides. The initial characterization of MB-1TrpHis N44E/L45V and MB-1TrpHis N44E/L45M, 2 variants of the library that were more resistant than the parent protein, was performed in order to investigate the nature of the mutants' resistance. Our results suggest that the mutants behaved like MB-1Trp regarding folding and thermal stability, and that proteolytic resistance is due to the elimination of the protease recognition site.