Study On Pseudonocardia Autotrophica CGMCC 7935 Biocatalytic Synthesis Of 1α,25（OH）₂VD₃

la,25(OH)2VD3 is mainly used for treating the diseases like rickets,osteoporosis,idiopathic,pseudo,hypoparathyroidism,renal osteodystrophy,osteomalacia,etc,and possesses an extensive market prospect in aging society.Currently the low production of 1α,25(OH)2VD3 makes it expensive,in order to ease the burden of using the medicine,it is significant to increase its production and reduce the cost of the production.The two ways of preparing la,25(OH)2VD3 are chemical synthesis and biocatalysis,but with the hardness to synthesis,easiness to generate isomers,complication to separate and purify and multiple synthetic steps of the chemical synthesis,the biocatalysis,which has fewer reaction steps,strong specificity of the enzyme,mild conditions,shows the potential to substitute the chemical synthesis.In this paper,the process of the 25(OH)VD3 convert to la,25(OH)2VD3 by Pseudonocardia autotrophica CGMCC 7935 has been investigated,and the feasibility of preparing la,25(OH)2VD3 by whole-cell catalytic system,free enzyme catalytic system,and immobilized enzyme catalysis has been explored,meanwhile the process parameters were further optimized.The whole-cell catalytic system and its optimization of conditions were investigated.The optimal conditions were determined as follows:0.04 g/L Fe3+and 0.5 g/L Mg2+in the medium,125 mg/L 25(OH)VD3,5 g/L hydroxypropyl cyclodextrin,240 mg/L tween 80.The conversion rate of 25(OH)VD3 was 18.1%,and the yield of 1α,25(OH)2VD3 was 22.63 mg/L.The crude enzyme solution was precipitated with ammonium sulfate for fractionation.Under the support of H2O2,1α,25(OH)2VD3 was successfully synthesized with 25(OH)VD3 catalyzed by the P450 in vitro.By optimizing the hydrogen peroxide shunt reaction conditions of the free enzyme catalytic system,The best conditions were:240 mg/L Tween 80,100 mg/Lβ-cyclodextrin,100 mg/L H2O2,pH 7.5,and the conical flasks was shaken for catalysis at 30℃.When the concentration of 25(OH)VD3 was 150 mg/L,the conversion rate was 9.3%,and the reaction reaches the equilibrium time of 6 h.Compared to the whole cell catalytic system,the free enzyme catalytic system could significantly shorten the reaction time from 72 h to 6 h.The synthesis of la,25(OH)2VD3 by the immobilized enzyme was investigated.The P450 enzyme in the bacteria was embedded in calcium alginate micelles,and then la,25(OH)2VD3 was synthesized under the support of H2O2.Optimizing the preparation conditions and reaction conditions of the micelles,the results showed that when the density of 25(OH)VD3 was 50 mg/L,the optimal H2O2 density of the immobilized enzyme was 150 mg/L,and the conversion rate was 10.4%.In terms of stability,the tolerance of the immobilized enzyme to H2O2 density was increased by 50 mg/L,and the conversion rate was increased by 1.8%compared with the free enzyme.Stronger stability to pH and temperature changes exhibited,After leaving free enzyme at 45℃ for 4 h,its enzyme activity has lost 65%,while the enzyme activity of immobilized enzyme reduced by 40%.In the buffer solution at pH 9.0,the enzyme activity of the free enzyme was reduced by 60%,and the enzyme activity of the immobilized enzyme was reduced by 40%under the same condition.