The Structure-thermo-Stability Relationship Of Recombinant Expression Of Uricase From Bacillus Fastidiosus ATCC29604

Uricase cloned from Bacillus fastidiosus ATCC29604can be used asan analytical tool to determine serum uric acid by kinetic uricase method,and a recombinant mammalian uricase has been approved by FDA as abiodrug for the treatment of hyperuricemia. Kmwas (0.22±0.01)mM for arecombinant uricase with N-terminus sepuence AERTMFYGKGDV fromBacillus fastidiosus Uricase. The recombinant AER had a half-life timeabout6hours at pH7.4. In this paper, firstly，method for the evaluation ofkinetic parameters recombinant was investigated, structures of therecombinant were studied under different conditions. The series ofN-terminus and C-terminus mutants were designed by analyzing the activesite and its static network. Finally, the relationship between the sequenceof N-terminus, C-terminus and the thermostability were investigated.1. Method for the estimating the kinetic parametersThe interfere effects of intermediate HIU was measured by initialvelocity in Tris-Hcl and Boric acid-sodium borate buffer. The intermediateHIU accumulated more seriously in Tris-Hcl buffer than in Boricacid-sodium borate buffer with0.103U uricase and300μM uric acid, andMg2+accelerated the breakup of the HIU. The uric acid showed negativeinterference in Tris-Hcl buffer but positive interfere in Boric acid-sodiumborate buffer, which may be caused by the fention reaction. At pH7.4,8.2and9.2the Kmwere gradually tend to (0.17±0.02) mM,(0.34±0.02) mM and (0.35±0.01) mM, respectively. The optimal temperature ofuricase was around30℃and the optimal pHwas around9.0.2. Transformation of superb structure under different conditionsThe tetramer was unstable and gradually transformed into dimmer,meanwhile the specific activity was receded after freezed for3months,and the major components was dimmer after6months.3. Effect of ligand on thermostabilityTetramer, which still possess the catalyze activity, can be induced byfreezed dimer under the condition of400μM uric acid，120μM xanthineand30μM oxonate. And the oxonate can prolong the half life of tetramerto150hours.4. Relationship of structure and functionAccording to the result of the model of the variant with N-terminus ofAERTMFYGK, the activity site and its static network were analyzed,Series variants were constructed, and12mutants of N and C terminal werecloned. The sequence of N-terminus almost showed no effect on the Km,Kiand optimal temperature and pH. The first three amino acid playedimportant roles in specific activity and thermostability. The specificactivity of ADD, SQR, QQG mutants were only one-tenth of the nativeuricase, which were almost in the form of dimer on PAGE. While the halflife of QER, QQR, VQR were all longer than AER, and QQR possessedthe longest half life of450hours. Specific activity of truncated mutant CTerminus of ADD and ARR were only0.1%of native uricase; the CTerminus sequence played an important role in the catalyze site.