Influence Of Various Saccharides On The Synthesis Of Acarbose And Its Structural Analogs With Actinoplanes Sp. ZJB-03852 Employing Proteomics Technologies

Acarbose is a common drug for typeⅡdiabetes treatment,which is industrially produced by Actinoplanes sp.The fermentation level of wild type Actinoplanes sp.is always low.Although a mass of researches involving strain breeding and fermentation controlling have enhanced the production of acarbose,there is much more we can do to improve strain performance.Besides of acarbse,some analogues containing acarviosyl can be produced during fermentation of Actinoplanes sp.These analogues are difficult to separate from acarbose,whose contents are strictly limited by European pharmacopoeia.In order to investigate the influence of different saccharides on acarbose fermentation,9 kinds of saccharides involved glucose,maltose,maltotriose were used as carbon source.Moreover,the differences in intracellular and extracellular proteins expression by Actinoplanes sp.ZJB-03852 between different carbon source fermentation were analyzed by using 2D-DIGE.The different proteins were further identified by MALDI-TOF/TOF-MS.The results of acarbose fermentation by different saccharides could be summarized as follows.Except xylose,all saccharides can be utilized by Actinoplanes sp.ZJB-03852,producing acarbose only in the culture media containing maltose or maltotriose.The formation of component C is always accompanied with the formation of trehalose.Hence,we speculate that component C may be formed with trehalose.Actinoplanes sp.ZJB-03852 tends to synthesize component D,acarbose and component F respectively when cultuitivated in the media containing glucose,maltose and maltotriose.Before the analysis of diferent proteins of Actinoplanes sp.ZJB-03852 between different carbon sources,the sample preparation procedure method suitable for 2D-DIGE was evaluated.Cells disruption method using a FastPrep-24 cell disrupter with disruption buffer(50mmol/L Tris-HCl,pH7.4)involved protease inhibitor cocktail and protein phosphatase inhibitor is preferred for the the extraction of intracellular proteins.The phenol extraction and methanol precipitation method is suitable for both intracellular and extracellular protein sample preperation.Proteins can be separated well by IEF using a 24 cm immobiline drystrip with a non-linear pH range of 3-10 and SDS-PAGE using a 10.5% polyacrylamide gel.The difference between protein expression between different carbon sources was analyzed by 2D-DIGE method with sample cultultivated in glucose containing medium as controlling group,and samples cultultivated in maltotriose,maltose,glucose-maltose containg media as experimental group.The protein identification results indicated that the acarbose cluster protein AcbV,AcbN and AcbL are up-regulated when Actinoplanes sp.ZJB-03852 grown on maltose or maltotriose,while AcbK was down-regulated.Besides the acarbose cluster proteins,4 proteins participating in sacchrides transport and a glutamate synthetase have varying expression levels.Among the 4 saccharides transport proteins,AglE are up-regulated in maltose containing medium,while MalE,MstE and ChvE are down-regulated.The glutamate synthetase GlnA is up-regulated in the culture media containg maltose or maltotriose.We concluded that AcbV,AcbN,AcbL and GlnA are the essential enzymes to acarbose synthesis.The down-regulated of AcbK by maltose or maltotriose contributed to the formation of component D,acarbose and component F by Actinoplanes sp.ZJB-03852 when grown on glucose,maltose and maltotriose,plus the different expression levels of saccharides transport proteins in different medium.