Microbial Production Of 2,3-butanedion From Inulin

2,3-Butanediol is one of remarkable bulk chemicals,which exhibits a wide range of utilizations in manufacture of printing ink,cosmetics,explosives,plastics and so on.By means of bio-refinery,the defects of chemically producing procedure from crude oil can be avoided,catering to the current sustainable development in China.It is being attached more and more importance to explore an environmental technology to produce 2,3-BD.Jerusalem artichoke is an inexpensive and renewable non-grain raw material.It is an excellent carbon source widely used in microbial production of alcohol,lactate etc,however immature in 2,3-BD.Klebsella pneumoia and Bacillus polymyxa is considered the potential industrial production of 2,3-BD,but the former is conditioned pathogen and the latter is lower-yield.In addition,the commercially available inulinase increases the cost of production.The main objective of this study was to develop cheaper and more systemactic methods to realize the production of 2,3-BD or other high-value chemicals from inulin substrate.Firstly,an exo-levanase existed in Bacillus licheniformis is cloned and expressed in E.coli and the induced condition was optimized in consideration of the low level of heterogenous expression.The optimal induction shows at 20? and the suitable dosage of IPTG was 0.6 mM.To purify the levanase,an efficient process which includes various purification protocols such as His-trap Butyl FF,gel filtration were used.After purification,special activity of the levanase reached 981.6 U/mg protein and 60%of total activity was obtained.The characterization of the levanase produced by recombinant E.coli was performed in order to verify the optimum temperature and pH for inulin.The results for these assays,where the optimum temperature was 60? and the optimum pH was 6.5 specify the proper utilization of levanase in later fermentation experiments.The levanase is stable in range of pH 5?11 and temperature below 55?.It is suitable for the process in this paper.Secondly,five competent strains stored in lab was cultured and the final fermentation results indicated that B.licheniformis DSM 13 was better than any other strains.Pondering the dosage of loading levanase,the consumption and production rate of reducing sugar during SSF process was determined and the optimal dosage of levanase was between the range of 20?40 U/g substrate.SSF and SHF is compared in flasks scale and the SSF is eminent in comparison.Fed-batch simultaneous saccharification and fermentation was effectively performed and 106.7 g/L target products(2,3-butanediol plus acetoin)was obtained in 30 h by a stage-shift aeration strategy.As we known,it is the highest concentration of products in utilization of inulin.To simplify the procedure and pare the expenditure,a new strategy for SSF is considered as a more promising method than mentioned above.An exo-inulinase existed in Bacillus polymyxa is cloned and expressed in E.coli.The heterogenously expressed inulinase was purified and the optimal condition of hydrolysis was determined.The optimium temperature is 37? and the optimium pH is 6.Subsequently,the ice-nucleation protein from Pseudomonas syringae was artificially synthesized and then fused to the levanase from B.licheniformis and inulinase from Bacillus polymyxa respectively.When the fused proteins was overproduced in E.coli,the whole-cell catalysis activity was too low to provision the sufficient carbon source for the production of 2,3-BD.The feasibility of this method is expected to continue,but it realized the surface display of the two enzymes in E.coli.