Characterization of a neuraminic acid synthase from the psychrophilic organism Moritella viscosa
ForfatterBerg, Tor Olav
Sialic acids are family of nine carbon α-keto acids found to have important roles mammals and bacteria. They are negatively charged sugars which have found to play critical roles in the cognitive development, as well as being a part of the innate immune system and being targets for pathogens. Mammalian cells express sialic acids to confer self to the immune system, a trait being exploited by some pathogens that use sialic acid on their cell surface to hide themselves from the hosts immune system. Moritella viscosa is a psychrophilic bacteria found to a causative agent for winter ulcer in salmon. This bacteria have been found to have the genes necessary to synthesise sialic acids. The neuramininc acid synthase responsible for the reaction that synthesises neuraminic acid from ManNAc and PEP have been successfully cloned and expressed in two constructs NHis and CHis which coded for a TEV cleavable His-tag and a non-cleavable His-tag. The constructs were successfully expressed in two different expression strains and purified based on affinity chromatography. The activity was tested with the TBA assay and both NHis and CHis were found to be active. Both were expressed and purified in large scale and a purification table was made to analyse the process. The NHis construct was found to precipitate during purification preparations and only a low amount of the protein was obtained. CHis was found to give high amounts of protein. The NeuB1 enzyme was characterized in regards to pH, temperature, metals and stability. It was found that the optimum pH was 8.0. The optimum temperature was 30°C and cobalt was found to bind strongly at low concetrations. The stability of NeuB1 was assayed at the three temperatures 25°C , 37°C and 45°C and it was found the residual activity after 2 hours, 1 hour and 30 minutes was 70, 20 and 5% respectively. Km and kcat was found for ManNAc and PEP. Km was 18.1254±5.2537 and kcat was 222.997 for ManNAc and Km was 0.7646±0.1674 and kcat was 225.438 for PEP. Crystallization conditions were found for NeuB1 to be PEG 3350 and sodium citrate, no crystals were obtained. Using HPLC-MSMS to detect sialic acids in Moritella viscosa gave positive results for several compounds.
ForlagUniversitetet i Tromsø
University of Tromsø
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