Susanne Wieland

Generation of readily transformable Bacillus licheniformis mutants

A set of mutants was generated by targeted deletion of the hsdR loci of two type I restriction modification systems (RMS) identified in Bacillus licheniformis DSM13. Single as well as double knock-outs resulted in strains being readily transformable with plasmids isolated from Bacilli. Introduction of shuttle plasmids isolated from Escherichia coli was routinely possible when the double mutant B. licheniformis MW3 (ΔhsdR1, ΔhsdR2) was used in transformation experiments. Growth and secretion of extracellular enzymes were not affected in any of the mutants. Thus, along with an optimized transformation protocol, this study makes available an urgently needed transformation system for this industrially exploited species.

Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases.

In the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non‐redundant subtilase sequence tags with degenerate primers. Furthermore, an activity‐ as well as a sequence homology‐based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full‐length protease genes were recovered, sharing only 37–85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin‐like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications.

C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli.

Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40°C and pH 7-11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40°C and pH=9.5 resulted in K(M)=0.23 ± 0.01 mM and k(cat)=167.5 ± 3.6s(-1). MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.

Targeted deletion of the uvrBA operon and biological containment in the industrially important Bacillus licheniformis.

From a Bacillus licheniformis wild type as well as a defined asporogenous derivative, stable UV hypersensitive mutants were generated by targeted deletion of the uvrBA operon, encoding highly conserved key components of the nucleotide excision repair. Comparative studies, which included the respective parental strains, revealed no negative side effects of the deletion, neither on enzyme secretion nor on vegetative propagation. Thus, the uvrBA locus proved to be a useful deletion target for achieving biological containment in this industrially exploited bacterium. In contrast to recA mutants, which also display UV hypersensitivity, further strain development via homologous recombination techniques will be still possible in such uvr mutants.