Directed Evolution Of Urate Oxidase From Bacillus Subtilis

Urate oxidase is an important enzyme in purine degradation.It catalyzes uric acid degradation and produces allantoin,which is much more soluble than uric acid.Urate oxidase activity seems to have been lost in humans,so the level of urac acid in humans is high,which is of significance during the evolutionary process.However,if the level of uric acid in the body is over high for a long time,it will lead to the occurrence of many diseases including gout.Allopurinol is a drug used to treat gout.It inhibits xanthine oxidase enzyme and blocks the formation of uric acid.However,it increases the burden on the kidneys and may cause allergic reaction.Urate oxidase as a new drug to degradate uric acid can not only decrease the level of uric acid in blood quickly,but also degrade the deposit of uric acid crystals in body without perhaps minor any side effects.It is also used in diagnostic kit which can quckily detect the level of uric acid.This is very meaningful in clinical diagnosis.In this study we use the urate oxidase from Bacillus subtilis(BSUO)as the starting material for directed evolution.Though error-prone PCR and DNA shuffling to construct mutants libaray.After several rounds of mutagenesis and screening,we obtained two mutants 6E9 and 8E279 with high enzyme activity at 9.09U/mg and 10.44U/mg,which is 3 times of wild type BSUO and similar to that of urate oxidase from Aspergillus flavus.We also characterize the properties of mutants 6E9,8E279 and wild type BUSO.It turns out that the mutants have lower optimal reaction temperature and optimal pH but better thermal-stability.We constructed 17 mutants with single mutantion site in the gene sequence through site-directed mutagenesis.D44V,Q268R and K285Q were identified as the three most beneficial amino acid substitutions which exhibited the highest activities.D44V/Q268R,which was obtained through random combination of the three mutants,displayed the highest catalytic activity.The Km,kcat/Km and enzyme activity of D44V/Q268R increased by 68%,83%and 129%respectively,compared to that of wild-type urate oxidase.Structural modeling indicated that mutations far from the active site can have significant effects on activity.For many of them,the underlying mechanisms are still difficult to explain from the static structural model.We also compared the effects of the same set of single point mutations on the wild type and on the final mutant.The results indicate strong effects of epistasis,which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.