Cloning And Characterization Of Sphingolipid Ceramide N-deacylase And Its Application In Enzymatic Synthesis Of Glycosphingolipids

Glycosphingolipids （GSLs） are amphipathic molecules that consist of a ceramidelipid anchor （consisted of Fatty acid and Sphingosine） linked to an oligosaccharidechain of variable length and complexity. GSLs are of extreme significance in cellsignaling and appear to play a critical role in virtually every stage of the cell life cycle,including growth, proliferation, differentiation, adhesion, senescence, and apoptosis.GSLs are potential therepy for many neurodegeneration diseases （such asAlzheimer’s disease, Parkinson’s disease and Huntington’s disease） as well as cancerand stroke, among which GSLs have already been administrated in clinical.Sphingolipid ceramide N-deacylase （SCDase） is an enzyme that cleaves the N-acyllinkage of ceramides in various GSLs as well as sphingomyelin to produce their lysoforms. It could catalyze the reverse reaction of hydrolysis, by which it exhibites agreat value in modification and enzymatic synthesis of GSLs. Up tp date, only twoSCDases which are from Pseudomonas sp. TK4and Shewanella alga G8werereported. The full length gene of SCDase from Shewanella alga G8（SCDase-G8） wascloned and expressed in E.coli. Full length SCDase-G8became mature protein afterthe splicing of C-terminal region, however the heterogenous expression of matureprotein has not reported and its property has not characterized yet.We cloned the C-terminal truncated mutatant of SCDase-G8（SCDase-G8_del）and successfully expressed functionally in E.coli. Recombinant enzyme with purityabove95%was purified. A novel SCDase assay method based on HPLC withO-phthalaldehyde derivation was established due to the low reproducibility, lowresolution and low quantitative accuracy of the time-costing conventional methoddased on TLC analysis of radioisotope labled GSLs substracte. Compared with the conventional method, the novel time-saving one has advantages of safty, goodsensitivity, high veracity, and suitable for high-throughput analysis.With systematic characterization, we found the recombinant enzyme has aspecific activity of125.2U/mg; its optimal tempeture is40℃and pH is8.0.5mMZn²⁺、Cu²⁺strongly inhibite its activity, other divalent metal ions could activate theenzyme among which Mn²⁺、Co²⁺exhibit the strongest activation by0.4foldincreasing in activity at the same concentration. Triton X-100has a remarkableenhancement on enzyme activity; the specific activity could increase14fold in thepresence of0.3%Triton X-100. Polar organic solvents like DMSO, DME, DMF andTHF inhibite enzyme activity at low concentration, but the inhabitation decreasedwith higher concentration, and enzyme activity could increased up to130%in thepresent of20%DME.We enzymatically synthesized ganglioside GM1and its precursor Glu-Cer forthe first time by applying SCDase-G8_del and Glycosynthases engineered fromendoglycoceramidase II from Rhodococcus sp. strain M-777together. A one-potenzymatic synthetic system was explored for GM1with product yield of41.5%.Structure-defined GM1（d18:1/18:0） was successfully obtained in this system.This study established a fast, sensitive and precise SCDase assay method,furnishing a powerful tool for systematic investigation of SCDase; cloned, expressedand systematic characterized the mature proein of SCDase from Shewanella alga G8,roughly explored its application in enzymatic synthesis of ganliosides which layfoundations for enzymatic synthesis of GLSs.