Recombinant Expression And Characterization Of The Uricase From Meyerozyma Guilliermondii

The development of uricase bearing high stability and activity at neutral pH is the key issue for the application of uricase in treatment and diagnosis.Bacillus fastidious uricase(BFU)contains 322 amino acid residues,its active form is tetramer.By characterizing the thermoinactivation processes of BFU and its different mutants,it was found that there was no plateau of BFU under physiological condition,and its half-life was about 26 days in the low-concentration system.The thermostability of the double mutant AER/V144A/Y319R was significantly enhanced.The longest plateau was up to 42 days and the half-life time was147 days under physiological condition.It is the uricase of the best thermostability under physiological condition at present.Unfortunately,the optimal pH of the mutant AER/V144A/Y319R is still alkaline;it is urgent to change the optimal pH from alkaline to neutrality by molecular engineering.The strain C.G.M.C.C2.1008 was identified as a strain of Meyerozyma guilliermondii.The optimum pH of the MGU(Meyerozyma guilliermondii uricase)induced by uric acid was neutral in borax borate buffer.MS/MS analysis of the partial peptide sequences after trypsin digestion supported its high homology to a putative uricase A5DFP1 deduced from the genomic sequence of Meyerozyma guilliermondii ATCC 6260 and its induced expression by uric acid was further verified by RNA-seq.The target gene was amplified by PCR through precise primers designed according to the coding sequence of A5DFP1,and the template of cDNA from C.G.M.C.C2.1008.The cloned coding sequence was inserted into the expression vectors pDE1 and pDE2 to construct the plasmids pDE1-MGU and pDE2-MGU bearing 6His tags.After the deletion of 6His tag and the linking peptide,the vector for the non-tagged uricase was constructed and denoted R-MGU.The plasmids were transformed into E.coli BL21(DE3)for induced expression.R-MGU had the peptide weight of about 35KDa by SDS-PAGE,but of 17.43 KDa that was consistent with that of MGU wildtype by MALDI-TOF-MS,with a difference from that of 33.98kDa derived from the amino sequence,supporting the unidentified chemical modification of MGU.Meanwhile,for R-MGU,the K_m and K_i of representative inhibitors had no significantly differences from those of the wildtype.The maximum specific activity of the recombinant uricase bearing6His tags was about 6.0U/mg.However,it is difficult to purify R-MGU,and the thermostability of R-MGU was slightly worse than the wildtype,due primarily to the low purity of the sample.In summary,the excellent thermostability of the mutant AER/V144A/Y319R makes it an attractive starting enzyme,but its optimum pH is alkaline and its activity is lower under physiological condition.It is important to optimize the optimum pH of this mutant to be neutral.MGU has the characteristic that the optimal p H is neutral,and its clone and recombinant expression is conducive to the further analysis of the determinants of the optimal pH,whose involved interaction network can be transplanted to mutant AER/V144A/Y319R to optimize its optimal p H to near neutrality.