| Leptoxyphium fumago is one of the main strains which are used to produce CPO by fermentation. The production has been developing for more than 20 years abroad. CPO is a kind of chiral biocatalyst which has wide-ranging substances applicability. Nowadays, CPO has been utilized on the synthesis of enantiomers and medicine industry. It can reduce the loss of chiral isomer, get rid of the chiral enantiomers's toxicity in drug, and relieve the pollution caused by waste. However, the yield of CPO's fermentation is not steady while the concentration of CPO is too low in the medium. Instability and low percent recovery are the barriers of CPO's development and utilization.We aimed to raise the stability during the production and the purity of CPO during the purification. Then we arranged four sections of experiment. The results are as follows:1. We collect numerical values of fermentation and analysis the relativity between numerical values and stability of CPO's yield. And then found out the factors that affect reptation behavior in biosynthesis and instability of CPO. The consequence showed us:the stability of CPO's yield was influence d by multiple factors. When pH value of fermentation broth was above 6, enzyme activity dropped quickly. This reflects that they have relativity between them. The temperature during fermentation can affect not only the yield but also the peak of CPO. The data showed that the yield was largest(180 U/ml) while the peak came earliest(6th-7th day) under 25℃. The yield was much steadier while the inocula age was 15-20 days. The smaller volume of medium is, the peak came earlier. Inoculums concentration, shearing force, metal ion and additives didn't influence the stability too much.2. We used 6 channels feeding shaker to control the fermentation condition online. Experiment finds that: curve of enzyme yield was steady while pH value stayed at 6. Feeding during pH rising can control the pH value, and avoid enzyme inactivation caused by high pH value, and extents the period of enzyme producing. Feeding acid, carbon source, nitrogen source raised the stability of fermentation. Enzyme concentration can reach 364 U/ml.3. After separation liquids and solids in the fermentation liquor, decolorization, salting out, ion-exchange chromatography by DEAE-Cellulose 52, gel filtration chromatography by Sephadex G-75, the concentration of CPO has reached 4594 U/mg, which is 12.19 times of the initial concentration. But, percent recovery is only 9%. We also use the characteristic that PEG6000 can entrain protein while precipitate under high ionic strength to get high concentration enzyme protein by aqueous two-phase system extraction. Then we get high purity enzyme by gel filtration chromatography by Sephadex G-75. (NH4)2SO4 solution whose saturation is 60% can make PEG6000 entrain 95% of totally CPO activity. The distribution rate k in PEG/(NH4)2SO4 aqueous two-phase system can be less than 0.383, as the concentration of purified CPO can be higher than 2973.68 U/mg, percent recovery reached 42.48%. Degree of purity rose 21.57 times and rose again after gel filtration chromatography. The research applied a new method to efficiently extract trace quantity bioactive substance.4. The study of immobilization has showed that:Immobilization with composite carrier made by sodium alginate and carrageenin can make the optimum reaction temperature rise from 27℃in liquid to 30℃after immobilization; CPO's high performance temperature range was widen(27～30℃) while its high performance pH value range was widen too(2.0～3.0); The stability of CPO stored under room temperature was advanced a lot; all these improvement above have made the enzyme much steadier.In general, this research has solved the problem of CPO's instability and low percent recovery during fermentation, provides reliable technical support for large scale production.
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