Effect Of Mpeg Modification On Candida Utilis Uricase And Candida Sp. Uricase

Uric acid as the end-product of purine metabolism in human body is passed out mainly by kidney. It has so low solubility. And serum uric acid increases significantly when the production of uric acid is faster than renal excretion, which results in hyperuricemia that is danger to human health. Uricase catalyzes uric acid into products that can be tolerated by human body or have no obvious pathophysiological roles. Therefore, exogenous uricase is a potential protein drug in treating diseases associated with hyperuricemia because human is short of uricase in vivo. However, any exogenous uricase has immunogenicity and very short circulation half-life in vivo, but upon its modification by mPEG, its immunogenicity can be reduced and its circulation half-life can be prolonged. Thus, PEGylated uricase is a plausible formulation.we modified uricases of Candida utilis and Candida sp. uricase with ss-mPEG and compared their features including the first dosing half-life. The following results were obtained.1 Preparation of Candida utilis uricase and Candida sp. uricase.Candida utilis (AY 204007) from China Center For Type Collection was used. The gene of uricase was amplified by PCR with its genomic DNA and cloned into PET-28a. Following verification by sequencing, the vector PET28a-Uricase was transformed into BL21 (DE3). Uricase was induced in BL21 with IPTG. After being induced by IPTG, cells were harvested through centrifugation and lyzed with ultrasound. The lysate is fractioned with 30%~90% ammonium sulfate and then passed through DEAE-cellulose equilibrated at pH 8.0 to remove most unwanted proteins.Candida sp. (CGMCC 2.1008) from China General Microbiological Culture Collection Center was used. After being induced by 0.5% uric acid, cells were harvested through centrifugation and lyzed with ultrasound. The lysate is fractioned with 30%~90% ammonium sulfate and then passed through DEAE-cellulose equilibrated at pH 8.0 to remove most unwanted proteins. The highest specific activity reached 10.0 U/mg after twice DEAE-cellulose chromatography.2 The kinetic parameters of two uricaseThe uricase from Candida utilis (China Center For Type Collection, AY 204007) had Michaelis-Menten constant of (36±5)μmol/L(n = 3) and the inhibition constant of xanthine was (11.3±0.4)μmol/L(n = 3).The uricase from Candida sp. (China General Microbiological Culture Collection Center CGMCC 2.1008) had Michaelis-Menten constant of (33±3)μmol/L(n = 3), and the inhibition constant of xanthine was(4.8±0.5)μmol/L(n = 3). 3 Enzymological features and the first dosing half-life These two uricases were modified by PEG (molecular weight was 5kD) after being activated to N-Hydroxysuccinimide ester. the optimum pH, optimum temperature, thermostability and circulation half-life were compared after modification.After mPEG modification, the optimum temperature of the uricase from Candida sp. was increased from 30℃to 35℃and the optimum pH was still 8.7. Following incubation in 37℃for 24h, the retained activity was improved from 40% to 85% and the first dosing circulation half-life in rats was prolonged from 45 min to 11h.The optimum temperature and optimum pH of the uricase from Candida utilis were 40℃, pH 8.5, after mPEG modification, the optimum temperature and optimum pH were changed into 35℃and pH 8.0. However, the uricase of Candida utilis was unstable at 37℃.PEG-modified uricase from the Candida sp. is promising to be a biodrug for treating hyperuricemia.