RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.
CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.
MATERIALS AND METHODS: Biofilm yield of 32 Helicobacter pylori strains (standard strain and 31 clinical strains) were determined by crystal-violet assay and grouped into poor, moderate and good biofilm forming groups. Whole genome sequencing of these 32 clinical strains was performed on the Illumina MiSeq platform. Annotation and comparison of the differences between the genomic sequences were carried out using RAST (Rapid Annotation using Subsystem Technology) and SEED viewer. Genes identified were confirmed using PCR.
RESULTS: Genes identified to be associated with biofilm formation in H. pylori includes alpha (1,3)-fucosyltransferase, flagellar protein, 3 hypothetical proteins, outer membrane protein and a cag pathogenicity island protein. These genes play a role in bacterial motility, lipopolysaccharide (LPS) synthesis, Lewis antigen synthesis, adhesion and/or the type-IV secretion system (T4SS). Deletion of cagA and cagPAI confirmed that CagA and T4SS were involved in H. pylori biofilm formation.
CONCLUSIONS: Results from this study suggest that biofilm formation in H. pylori might be genetically determined and might be influenced by multiple genes. Good, moderate and poor biofilm forming strain might differ during the initiation of biofilm formation.