Five chitinases were isolated from culture filtrates of Bacillus licheniformis B-6839 R and S variants by combination of hydrophobic, ion-exchange, and gel permeation chromatography. The enzymes had molecular masses of 66, 62, 53, 49, and 42 kDa. The chitinases revealed two activity optima against colloidal chitin at pH 4.5–5.5 and 9.0–9.5 and they were rather stable at pH 4.0–9.5. The temperature optimum of activity was 90 °C for the 62-kDa chitinase and 70 °C for the other enzymes. The 66-, 53-, and 42-kDa chitinases showed pronounced similarities in their N-terminal sequences and apparently belonged to the same group, which might be related to Bacillus circulans chitinase A1. The 49- and 62-kDa enzymes did not reveal structural similarities with other chitinases produced by the studied B. licheniformis strain. No relationship was found with the 89- and 76-kDa chitinases isolated earlier from B. licheniformis X-7u.Key words: Bacillus licheniformis, chitinase, multiplicity.
A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or delta-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.
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