The Research Of Preparation Processing Of Immobilized2-deoxy-D-ribose-5-phosphate Aldolase

Statins are a group of main drugs best known for lowering cholesterol and warding off heart disease.2-Deoxy-D-ribose-5-phosphate aldose (DERA) has important application value because of its ability to stereoselectivity catalytic synthesis of statins intermediates. Great progress has been made to improve the performance of DERA about its activity and tolerability of substrate by powerful means of genetic engineering and protein engineering. However, DERA still has problems such as high cost, instability and difficult to recycle in practice. Immobilized enzyme technology provides an effective means to solve this problem and becomes a research hotspot in enzyme engineering research.This thesis focuses on the research on immobilization process of DERA and the process of catalazing the aldol reaction between n-benzyloxycarbonyl-3-amino-acetaldehyde and propionaldehyde to generate6R-[(Benzyloxycarbonyl)amino]ethyl-tetrahydro-2H-pyran-2,4-diol, which is an important statins intermediate, by obtained immobilized enzyme. The results from this study suggest that the immobilized DERA may be useful for industrial production of atorvastatin calcium.The main research contents and conclusions are as follows:1. Extraction of crude enzyme. After the fermentation of a high yield DERA inducible gene engineering strain E.coli BL21, the obtained cell pellet was resuspended in phosphate buffer(0.1M, pH7.5).Then the concentration of cell pellet was0.5g/ml.Cell disruption was performed by ultrasonication for different conditions in an ice-water bath, which determined the best time and crushing power. Release profile of the protein was detected by measuring the activity of supernatant and OD600of the sonicated cell suspension under different conditions. Comprehensive considering the restriction of the cell disruption and activity recovery in this experiment, the optimum power and time are600W and30min.The specific activity of crude enzyme was164.9U/ml.2. Preparation of immobilized enzymes. ESR-3ion exchange resin from Tianjin Nankai HECHENG Company was used to immobilize enzymes by the sieving of various regular resins. This amino resin, pretreated with glutaraldehyde, was combined with enzyme by forming schiff base. Through studying the effect of enzyme concentration、 buffer solution and pH value、ambient temperature and immobilized time on activity recovery (the total activity of immobilized enzyme/the total activity of crude enzyme used to immobilize) of immobilized DERA, experimental result showed that the optimal immobilization conditions were as follows:phosphate buffer (0.1M, pH8.0) system, load capacity was6mL free enzyme solution/lg carrier, the temperature was30℃, vibration time was45h.3. Comparison of enzymatic properties of the immobilized before and after. By studying the effect of buffer solution、pH value、temperature and enzyme concentration on immobilized enzyme activity, laboratory finding the optimal temperature of the E. coli BL21cells and immobilized enzymes was25℃, and the optimal pH value was7.0in phosphate buffer system (0.05M). Both of them showed good stability when pH value ranged from6to8, but when pH value beyond this area immobilized enzyme was more stable. Theremore immobilized DERA showed better resistant to high temperature and acetaldehyde than E. coli BL21. When temperature surpass50℃both of immobilized enzymes and E. coli BL21showed decreased enzyme activity, but immobilized enzymes had a relatively slow attenuation. When the temperature reaches90℃The activity of immobilized enzyme could still maintain about20%while E. coli BL21was inactivated. In the investigation process of acetaldehyde tolerated test, after both of them were treatment in the30mm concentration of acetaldehyde in2h the immobilized enzymes remained65%activity but E. coli BL21remained only23%.4. Stability of immobilized enzymes. Based on the investigation of storage stability and reuse stability of immobilized enzyme, we found out that immobilized enzyme sealed in4℃refrigerator for a month maintained at more than85%of the initial enzyme activity. Moreover, the immobilized enzymes had a stable operation property that still remained60%of the initial enzyme activity until the operation for4times.5. Immobilized enzyme optimized catalytic conditions.Under the above conditions, prepared immobilized enzyme was used to catalyze n-benzyloxycarbonyl-3-amino-aceta ldehyde and propionaldehyde to generate6R-[(Benzyloxycarbonyl)amino]ethyl-tetrahydro-2H-pyran-2,4-diol. The results showed that:by means of studying the effect of differe nt amount of immobilized enzyme and contact time on the percent conversion of n-be nzyloxycarbonyl-3-amino-acetaldehyde, when the optimal ratio of immobilized enzym es and substrate materials reached2.8:1and catalyzed reaction time reached100min, t he conversion rate of the substrate N-benzyloxycarbonyl-3-amino propionaldehyde reac hed75%.