Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene.
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 alpha-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.
Chen
Zhiyu
Z
Laboratory of Yeast Molecular Genetics and Breeding, Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Beijing, PR China.
Wang
Zhaoyue
Z
He
Xiuping
X
Guo
Xuena
X
Li
Weiwei
W
Zhang
Borun
B
D018533:Biomass; D002175:Candida / Q000201:enzymology*; D018929:Cell Culture Techniques / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014503:Urate Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014503:Urate Oxidase