Overexpression of Thermal and pH Stable Alginate Lyase of P. aeruginosa ۲۹۳ and In silico Study of algL Gene

P. aeruginosa is an opportunistic bacterium that produces a capsule-like polysaccharide called alginate in response to various stimuli. The mucoid strain of Pseudomonas aeruginosa produces alginate which is an exopolysaccharide and is involved in the pathogenicity and persistence of these bacteria in infections. The alginate lyase gene is required for alginate synthesis. The enzyme can also degrade this polymer. This enzyme has a polymorphism in different bacteria even in one species and finding an enzyme with tremendous characters is very important. In this study, alginate lyase from P. aeruginosa strain ۲۹۳ which was previously isolated from the sputum, and the encoding gene was characterized, and thermal and pH stability, as well as the substrate specificity of the partially purified alginate lyase, were determined. The amount of ۷۰% activity of the enzyme was maintained after incubation at ۸۰ ˚C for ۶ hrs and ۵۰% activity retained after incubation in alkaline and acidic pH. Moreover, it showed activity towards guluronic acid blocks, mannuronic acid blocks, and alginate blocks with both of them. Due to the unique properties of the alginate lyase that are useful in medicinal, and industrial applications, the gene encoding the enzyme was expressed in pET-۲۸a (+)/E. coli BL۲۱ (DE۳) system to produce ۳۷۱ -amino acid alginate lyase protein, the molecular weight of which was estimated by Sodium Dodecyl Sulfate- Polyacrylamide gel electrophoresis to be about ۴۰ kDa. Bioinformatic analysis of P. aeruginosa strain ۲۹۳ algL gene revealed that G۲۲۵A point mutation can improve its thermostability. Therefore, the P. aeruginosa ۲۹۳ alginate lyase is proposed as an appropriate candidate for the evaluation of potential therapeutic and industrial applications.

Keywords:

Alginase , AlgL , computational study , pH stability , thermostability