| Ginsenosides, the main active ingredients of ginseng, play a unique role in anti-radiation, anti-inflammatory, anti-tumor and treatments of diseases in cardiovascular, nervous system. The content of ginseosides is extremely low in the nature, while ginsenoside Compound K even doesn’t exist. Due to its special chemical structure, ginsenoside Compound K is mainly prepared mildly by microbial transformation with specificity. Microbial transformations can increase the yield of active products or generate new structural compounds through the catalytic reactions by the enzymes produced in the microbial metabolic processes. Therefore, regulations on the biotransformation both in the synthesis and activity of enzyme level is of great significance in theory and industrial application.We have screened a strain named Paecilomyces bainier sp.229from the soil planted with wild ginsengs, which could transform ginsenoside Rb1to ginsenoside Compound K with a conversion rate of72.9%, but the concentration of substrate is only0.5%. In order to increase the substrate concentration, we carried out a mutation with sp.229, screening the strains with high tolerance to substrate, and obtained mutants sp.229-6and sp.229-7.The end-production of sp.229-6is still ginsenoside Compound K. The optimal culturing and transformation conditions were achieved by a series of studies on metabolic regulations of biotransformation through investigating the mebolic pathway of sp.229-6and regulatory factors, controlling the cell membrane infiltrates and substrate induction:take an inoculation in250mL flasks with30mL liquid medium after36h-cultivation and adding4%o Tween80. After being cultured for24h at28℃at200r-min-1,0.5%substrate was added as an inducer. Then being cultured for another24h,1.5%or1.0%substrate was added, and the pH value was kept at5.0during the72hours transformation time. When the substrate concentration was1.5%, the bioconversion rate was43.63%; when it was1.0%, the bioconversion rate reached61%. Under the same fermentation system, the output of ginsenoside Compound K can be increased by67-80%by sp.229-6compared to sp.229, which has a promising prospect in industry. The only production of sp.229-7is ginsenoside Rd, and it’s demonstrated thatcalcium ion could significantly promote the producing of ginsenoside Rd when weoptimized its fermentation conditions. Therefore, we placed an emphasis on theeffects of calcium signaling transduction on biotransformation of ginsenoside Rb1toRd by sp.229-7through adding calcium ions and CaM antagonist, and found thatcalcium and calcium singnaling transduction could effectively enhance the activity ofβ-glucosidase and promote the output of ginsenoside Rd. The study revealed theregulatory role played by calcium ion on biotransformation of ginsenosides for thefirst time. During the process of biotransformation of ginsenoside Rb1to Rd insp.229-7, calcium ions stimulate the signaling transduction mechanism, and in the45mM extracellular calcium level, the output of ginsenoside Rd and the activity ofP-glucosidase reached maximum. CaM was also demonstrated to be involved in theregulation of synthesis of β-glucosidase gene, and we speculated a preliminarycalcium signaling transduction way in sp.229-7. In addition, we purified aβ-glucosidase from sp.229-7, and its specific activity was169.91. Takeing crudeenzyme as100%, the activity recovery of the purified β-glucosidase was1.42%, andthe protein revovery was0.009%, the purification fold is163.37. The effects of pHand temperature on enzyme activity and stability were studied. The basic results were:the p-glucosidase showed its optimal activity at pH4.0, and was stable within pH5.0-8.0; the p-glucosidase showed its optimal activity at55℃and was stable attemperatures blow55℃. In a certain range of concentration, calcium ion can activatethe p-glucosidase at different pH and temperatures. It can also enhance the stability ofthe enzyme at different temperatures, but only can enhance the stability of the enzymeat pH<6. |
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