Gene Cloning Of Aspergillus Niger 9891 Endoinulinase And Its Expression In Pichia Pastoris, And Design Of Intermediate Pilot For Inulin-oligosacharides (IOSs) Prodcution

Inulin is a kind of natural fructose polymer, Endoinulinase can catalyse the hydrolysis of inulin into oligo-fructose, which is regarded as a kind of functional food ingredient. It plays an pretty important role in healthy aspect, such as its ability to decrease blood sugar level, to improve the balance of micro-ecology of in human and animal digestion tract and to increase the immunity to disease and so on. It can be obtained at yield level of 80% oligo-fructose in one step by utilizing microbies' endoinulinase. Only few microorganism produce endoinulinase, however it exists some problems, that is, lower quantity of oligo-fructose and difficulty of being isolated and purified, to sum up, it is urgent to solve these problems to benefit the industrial product. The object of the present study is to construct the recombinant yeast for the high-level expression of the endoinulinase, in order to provide an efficient way to solve the problems in the produce of oligo-fructose.In this paper , the gene encoding endoinulinase is amplified by PCR with the template of genomic DNA of Aspergillus niger 9891 and then it is cloned into expression vector pPIC9. After verified by restriction ,PCR and sequencing ,the vector pPIC9-Endoinu after being linearized with BglⅡ is transformed into the eukaryotic host (yeast Pichia pastoris strain GS115) with electronic pulse. The recombinant yeast obtain through culturing on nutrition deficient medium. The recombinant endoinulinase was highly expressed and the optimization of the expression in a 7 liter of fermentor has been investigated. During fermentation, the concentration of protein secreted is 2.15 mg/ml. SDS-PAGE analysis suggested that the size of protein is about 59KD. The activity of endoinulinase is 1501 U/ml with sucrose as substrate and 291 U/ml with inulin as substrate, 105 and 273 folds higher than that from the original strain respectively and through the research of properties of enzyme activity ,it shows that the optimal condition for enzyme reaction are pH5.6 and 55 ℃. At the same time, the advanced structure of endoinulinase is predicted with software. According to the result of SDS-PAGE and the enzyme functional assay ,the target protein is expressed successfully in the Pichia pastoris. Additionally, the yeast transformant--Pichia pastoris GS115/9891(I1-27) showed the quite good genetic stability. In addition, the design project of middle experimentation product line was accomplished, that is the downstream work of the production of inulin oligo-fructose hydrolyzed by endoinulinase, which included that the technical design of the production of inulin oligo-fructose hydrolyzed by endoinulinase;selecting the model of equipment and installations;the plane design of workshop and the pilot study of constructing the middle experimentation workshop.