The Construction Of Genetically Engineered Strains Of Producing Urate Oxidase

Urate Oxidase(EC 1.7.3.3)can catalyze the degradation of uric acid to produce allantoin with a solubility of more than ten times that of uric acid.It is an important enzyme in the metabolic process of pruine.Due to the mutation of the urate oxidase gene in the human body during the process of biological evolution,the uric acid oxidase has no activity,so the uric acid content of human has been high,and too much uric acid can cause a series of diseases such as gout.As an enzyme that can rapidly reduce uric acid content,urate oxidase has great potential in medicine.However,due to the low yield,the purification is complicated,and its high cost makes it not widely used in China.Therefore,it is very meaningful to construct a genetically engineered strain with high-yield urate oxidase.E.coli BL21(DE3)and P.pastoris GS115 were used as hosts to overexpress the uric acid oxidase gene(uox)derived from Aspergillus flavus,and the culture conditions and codons were optimized to improve the yield of urate oxidase.In addition,the yield of urate oxidase is further increased by expressing molecular chaperone proteins,transport factors,folding factors and transcription factors.The main work of this paper is as follows:(1)We has introduced the urate oxidase gene(uox)derived from Aspergillus flavus into the E.coli BL21(DE3)to construct the genetically engineered strain E.uox,and the yield of uric acid oxidase was 2.09 mg/L.The urate oxidase gene was codon optimized and the production of urate oxidase was increased to 2.66 mg/L.Furthermore,some fermentation conditions such as the time of fermentation,the amount of the inducer,the induction temperature has been optimized,and after the fermentation for 11 hours,4.03 mg/L of uric acid oxidase is obtained,and the urate oxidase purified by the nickel column has the enzyme activity of 8.11 U/mg.In order to further promote the expression of urate oxidase,the molecular chaperones tig,dnaK-dnaJ-GrpE and GroES-GroEL were introduced into the strain E.uox to promote post-translational folding,and the yield was increased to 6.07 mg/L,6.66 mg/L and 4.82 mg/L,respectively.But the yield of the strain introduced with the molecular chaperone tig-GroES-GroEL decreased to 3.15 mg/L.Finally,the fermentation conditions were optimized by orthogonal experiment,and the yield under the optimal fermentation conditions was 8.77 mg/L,and the soluble protein of interest accounts for 62%of total protein.(2)The uric acid oxidase gene uox was introduced into Pichia pastoris GS115,and the high-copy strains were screened to obtain P.uox-11 and P.uox-16 strains.After the fermentation for 6 days,the enzyme activity in the fermentation broth was 0.746 U/mL and 0.793 U/mL,respectively.The enzyme activities after purification were 9.32 U/mg and 9.41 U/mg,and the yields were 80.04 mg/L and 84.12 mg/L,respectively.By verifying the copy number,a rough relationship between the copy number and the amount of enzyme expression in the screening range was obtained.(3)P.uox-11 was used as the original strain,which was regulated from three aspects of transport,folding and transcription to improve the enzyme expression.There was no significant increase in yield after the introduction of the folding factor Sec53.In the aspect of folding,the enzyme expression was increased to 96.1 mg/L by expressing Kar2p folding factor.And for the transcription,the enzyme expression was increased to 100.3 mg/L by expressing HAC1 transcription factor,and the enzyme expression was increased by 22.8%and 28.6%,respectively.A strain with high yield of UOX was constructed.Finally,the optimization of fermentation conditions was carried out on the strains expressing PDI folding factor by orthogonal experiment optimization.After the optimization of fermentation conditions,the final enzyme expression reached 104.5 mg/L,which was increased by 13.1%.