Study On Fermentation Of Carbonyl Reductase Producing Engineering Strain And Properties Of Recombinant Carbonyl Reductase

The importance of optically active compounds has been gradually recognized in the pharmaceutical, food and fine chemistry industry. For the preparation of biologically active compounds, chiral building blocks are extremely valuable synthons. To explore the mechanism of the reaction catalyzed by enzyme, and to gain enough information forimproving the practical process, purification and characterization of the enzyme in the stereoinversion is extraordinary important. In the paper, a recombinant bacteria E.coli BL21(pET30a-cr318) with the highest expression of carbonyl reductase was selected from 3 recombinant bacteria by fermentation experiment in shaking flask. On the basis of preliminary optimized culture condition, the high density cultivation of E.coli BL21(pET30a-cr318) was investigated. In addition, the properties of carbonyl reductase was studied. The main research results are as follow:(1) The fermentation condition in the shaking flask was optimized by comparing the specific activity of carbonyl reductase in different culture condition. The optimized culture medium compositions were as follow:glucose 1%, peptone 1.5%, yeast powder 0.5%, pH= 7.5, trace element concentration 1 ml/L. The optimized culture conditions were that the medium volume was 50mL in the shaking flask of 250mL, and that the inoculum volume was 5%. The optimized induced conditions were that carbonyl reductase biosynthesis was induced by supplementation of IPTG(final concentration was 1 mmol/L) at the early stage of logarithmic growth, and that the induction was conducted for 8h at 28 ℃. The specific enzyme activity was 0.78 U/mg after optimization, which was 1.6 times of that without optimization.(2) The batch fermentation of E. coli BL21(pET30a-cr318) was carried out in a 15L automatic fermentor and the corrsepongding fermentation kenetic equations were set up. On the basis of batch fermentation, the pH-stat feeding batch fermentation was practiced. The higher enzyme activity and cell density were obtainted with 22.7 g/L (DCW) and 39.34 U/mL, which were 2.15 times and 3.7 times more than those under batch fermentation.(3) The two cell cracking methods were compared and optimized. The high pressure craking obtained the enzyme activity of 0.65 U/mg and its cell disruption rate was 97.5% under its optimized process condition of the craking time 6 min and the cell concentration for cracking 0.083 g/mL. The optimized process condition of ultrasonic cell craking was as follow:the power 900 W, the craking time 12 min, and the cell concentration 0.083 g/mL, but its enzyme activity was only 0.48 U/mg and its cell disruption rate was 82.3%, which was just 74% and 84% of those that take the method of high pressure cracking.(4) The properties of carbony reductase were studied after purification by Nickel column chromatography. SDS-PAGE analysis showed that the recombinant carbony reductase had a molecular mass of around 30 kDa. The optimal reaction temperature and pH were 35℃ and 7.0 for the carbony reductase. The results of stability tests indicated that carbony reductase was stable during pH 5.5-7.5 and sensitive to temperature. The recombinant carbony reductase could be activated by K+、Ca2+、 Na+、Mn2+ and inhibited by EDTA. It was very unstable in some organic solvents, such as pyridine、acetonitrile and tetrahydrofuran.(5) The carbony reductase catalytic reaction mechanism was ordered mechanism. The apparent Km of NADPH was 10.21 mmol/L, and the apparent Km of ATS-6 was 1.52 mmol/L. The conversion process of ATS-6 catalyzed by carbonyl reductase was determined, and the reaction started to achieve balance after 4 h. The value of d.e. remained at 100% and the yield got to 94%.