Study On The Fermentation Technology Of The Gene Engineering Strain Producing NcarbamylDanimoacidamidohydrolase And Immobilization

D-hydroxyphenylglycine(D-pHPG) was the most important precursor used for the synthesis of Semisynthetic penicillin and cephalospor and was a produce competed furiously in the field of pharmacy.Making D-pHPG,enzyme method was better than chemical method which was poisonous.In enzyme method, P-hydroxyphenyldantoin (p-HPH) was transformed into N-Carbamoyl-D-P-hydroxyphenyldantoin(NC-D-pHPG) by Dhydantoinase(DHase), then NC-D-pHPG was transformed into D-pHPG by Ncarbamyl Danimo-acidamidohydrolase (DCase). But now the activity of strain was low,it was very significative that the activity of strain was improved.The strain of spMF0507 could produce DHase and DCase, but the activity of DHase was higher than the one of DCase.Controlling pH DHase had no activity but DCase for producing NC-D-pHPG.However the Espf7 was gene engineering strain of DCase's genes cloned in E.coli.DCase expressed by Espf7 could transform NC-D-pHPG into D-pHPG.But DCase was in cell membrance,Dialysing cell membrance was investigated.Moreover by orthogonal optimization the culture condition and the culture medium were investigated,then the result of optimization was used to fermentor.In end under of detemining protein and enzyme activity the result of immobilizing DCase was conformed.The results of all experiments were followings:(1) The pre-inoculation time of spMF0507 was 20 24h,under pH9.0-9.5 DCase had no activity but DHase 0.2 u ? ml^-1 .Otherwise NC-D-pHPG which was oxidated into red matter was prevented by N₂ in the course of transformation. Appending 2% acetone increased crystallization , the crystallized rate was 97.9% and the pure degree of NC-D-pHPG was 99% by HP LC.(2) The cell membrance of Espf7 was dialysed by 0.02% CHCl₃ the pre-inoculation time of 16²0h,the inoculum ratio at 2%,initial pH of 7.0,temperature of 30°C,arabinose 0.01%; glycerin 0.30%;Corn steep liquor 3%;NaCL 0.2%;KH₂PO₄ 0.05%;MgSO₄ 0.05%;MnCL₂ 0.02%;CoCL₂ 0.01%,the activity of DCase was increased from 1.12 u ? ml^-1 to 2.1 u ? ml^-1, increased by 87.5%.The activity of DCase reached 3 u ? ml^-1 under 5L fermentor conditions including: temperature 30°C, the shaking speed of 500r ? min^-1 fed in pH of 7.0.(3) DCase was dialysed via pressure of 70MPa,time of 15min. TJS was used as carriers for immobilization of DCase,the optimized conditions were the amount of TJS of...