Screening Of New Strains With D-hydantoinase Activity And Study On The Properties And Immobilization Of Whole Cells

The new strains with D-hydantoinase activity have been screeded by use of DL-p-hydroxy-phenylhydantoin (DL-p-HPH) as a single nitrogen source from nature. Totally, three strains which had higher enzyme activity were obtained by double-plate and rapid micro-bacteria methods. The one showed highest enzyme activity was selected to do further study.Next, the strain screened above was fermented, and the preliminary fermentation products were identified by TLC and HPLC. Compared with the standard samples of D-p-hydroxyphenyIgIycine (D-p-HPG), a conversion product had the same Rf value as the standard sample of D-p-HPG. Then transformation products were purified after treated with NaNO2 by ion-exchange resin 732 in the following conditions:the flow rate of sample solution is about 2ml/min, and the eluted solution was 0.2 mol/L NaOH at a rate of 0.4 ml/min. The effluent was concentrated and crystallized. After that the specific rotation and IR spectrum of the pure product was measured. Then we could confirm the product is D-p-HPGOn the basis above, the bacteria strain was identified as Alcaligenes sp. based on 16S rRNA result.Culture conditions of the strain were optimized and the optimal cultivation conditions:the optimum carbon source was glycerol, the nitrogen source was the yeast extract, optimum growth temperature was 28-30℃, the best primary pH was pH 7.0. The best culture volume was 50 ml/250ml flask. The cell biomass reached to the highest when cultured for 48 hours, but the enzyme activity had reached to the highest after 24 hours.Afterwards, bacteria were immobilized by PVA-boric acid method. The optimal concentration of PVA for the immobilization was 0.1 g/ml. During the immobilization process, adding Mg2+ in boric acid solution had better result than adding other cations (Na+,Zn2+,Ca2+). The optimal pH was 8.0 for both of immobilized and free cells. The variation of pH (from 6.5 to 9.5) has little effect on activity of immobilized cells while had dramatic effect on free cells. The immobilized cells can tolerate higher temperature than free cells. The optimal temperature of the immobilized cells was 47℃compared with 37℃of the free cells. Under such conditions, the activity of the immobilized cells can reach about 3 times of the free cells. After 7 batches of reaction, the remained activity of the immobilized cells was still more than 80% of initial activity. After 30 days storage at 4℃the immobilized cells kept more than 80% of its initial activity. For free cells, it kept only 30% of initial activity. In short, the immobilized cells of Alcaligene sp. prepared by PVA entrapment has better pH and temperature tolerant, higher activity and stability compared to free cell and has potential application in the future.