Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Abstract

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.

Uric acid in plants and microorganisms: Biological applications and genetics - A review

“…The highest production of recombinant uricase reached 52.3 U/mL (about 2.1 g/L of protein) extracellularly and 60.3 U/mL (about 2.4 g/L of protein) intracellularly in fed-batch fermentation after 58 h of incubation, which are much higher than those expressed in other expression systems [134].…”

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris

“…According to Chen et al, the only appropriate amount of urate oxidase expression in yeast has been achieved in H. polymorpha (Chen et al 2008). In their report, the Candida utilis urate oxidase was expressed by H. polymorpha and α-MF signal sequence of S. cerevisiae was used to secret the protein.…”

Hyperproduction and Comparison of Wild and Mutated Urate Oxidase from Bacillus S

“…It was studied that 3% inoculum was used for the optimum production of parent and mutant derived enzyme. Reference [3] optimized the parameters and showed that inoculum size had great influence on the production of urate oxidase.…”

“…The substrate (urate) tightly binds to the one subunit of the enzyme by interaction with arginine (Arg180), leucine (Leu222) and glutamine (Gln223) while to the other subunit with threonine (Thr67) and aspartate (Asp68) [1] , [2]. The solubility of uric acid in the body is considered to be poor as compare to the allantoin [3] , [4]. According to previous studies, it was considered that urate oxidase has copper at its active site but later on it was proved that when this enzyme was isolated from Bacillus subtilis and Aspergillus flavus, contain no copper [5].…”