| Lipstatin is produced by Streptomyces toxytricini.A chemically synthesized hydrogenated derivative of it is Orlistat,which is the first gastrointestinal lipase inhibitor approved by the FDA for the treatment of obesity.The aim of this study is to investigate the production of Lipstation by strain breeding and optimizing fermentation process.Results of the paper could lay a foundation for industrial production of Lipstatin.Firstly,The Streptomyces toxytricini LIPS-352,a producer of lipstatin of 4125mg/L,was mutated with UV and nitrosoguanidine.A mutant S.toxytricini LIPS-N-27-46-42 was obtained,which yielded 8 122.9 mg/L lipstatin in shake flask culture,an increase by 96.9% compared with that of the parent S.toxytricini LIPS-352.Secondly,the culture conditions and medium of S.toxytricini LIPS-N-27-46-42 was optimized.The optimal culture conditions were as follow,slant culture time 7~8d,initial pH of fermentation medium 7.5,seed age 28 h,inoculation amount 10%,culture time 7~8d.One-factor-at-a-time method showed that carbon sources and nitrogen sources had a significant effect on lipstatin yield.And the fermentation medium was further optimized by Box-Behnken design of response surface methodology.The optimal medium were as follow,soybean meal 3.35%,corn gluten 1.01%,sunflower oil7.56%,glycerol 2.5%,soybean lecithin 2.5,anti-oxidant 0.05,FeSO4·7H2O 0.01,Tween-80 0.005,CaCO3 0.08.Then,yield of Lipstain was 9859.8mg/L,which was21.4% higher than the without optimized medium and had a relative error of 7.27% to the predicted value(10633.1mg/L).Finally,pH parameters of 10 L fermenter of S.toxytricini was maintained in the normal range by adjusting carbon source and nitrogen source of culture medium.And the production of Lipstatin reached 6904mg/L.In order to improve the utilization of oxygen,a recombinant strain S.toxytricini LIPI-42-VHb was constructed,which could express the biologically active VHb,but yield of Lipstatin in 10 L fermentor was only2788mg/L. |
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