Cloning,Expression And Enzyme Activity Detection Of Cytochrome 450 Vitamin Dhydroxylase

Cytochrome P450?CYP450?is a prolease superfamily,whose active region is noncovalent-bound ferroheme.After it binds with CO,the complex gives absorption maximum peak at 450 nm.CYP450 participates in endogenous and exogenous compounds metabolism,containing multiple oxidation and reduction reaction.As a essential fat-soluble prohormone in human body,vitamin D₃ plays a significant role in many fields,such as calucm and phosphorus metabolism,immunoregulation,cell reduplication and differentiation.Vitamin D₃ is inactive,while its hydroxylate posseses physiological function.Vitamin D hydroxylase is distinct kind of CYP450,which can hydroxylate some special sites of vitamin D,making some specially functional derivative with stronger bio-activity,of which one example is 1?,25?OH?2 vitamin D₃,which participates in many regulating and metaboliting processes.Although it is available by chemistry synthesis,it has multi-steps and many by-products,makeing it impossible for producing enlargement.It has double huge mense for economic returns and environmental protection that vitamin D transformation by microorganism.CYPvdh was cloned and expressed,which catalyzing la,25?OH?2 vitamin D₃ production.Genome DNA extraction method has benn optimized,and the ORF is cloned by PCR amplification,whose GC content reaches up to 73%.Cloning reconstruction plasmid pMD-18T vector was established;with subcloning method CYPvdh ORF was obtained and inserted in a expression vector pET-30.It was translocated into the host cell E.coil BL21?DE3?,which was then induced at the condition of 25? with 0.8 mmol/L IPTG and 4 h.SDS-PAGE result indicated the specific band?about 44KD?.Rarefied recombinant protein was obtained by purification kit.MS analysis showed that the autoploidy of the induced protein was CYP450 hydroxylase.Analysis of bioinformatics software shows that,CYP450 hydroxylase is a soluble protein,containg a conservative P450 Domain from 136 to 367 aa,without transmenbrane structure with molecular weight 44.4 KD and isoelectric point 4.73.Evolution alignment shows the similar and different function of CYP450 hydroxylase with other musculomyces.NADPH detecting method showed biocatalytic activity of heterologous expression of vitamin D hydroxylase.The results of analysis of Enzyme property by NADPH detection showed that enzyme activity was affected by different pH,temperature and concentration of potassium ion and sodion.Catalytic action reached the highest when pH7.5,30? and 10 mmol/L potassium ion and sodion by mono-factor experiments.