Construction And Fermentation Of Recombinant Pichia Pastoris To Express Dextranase And Its Applications

Dextran, a kind of bacterial polysaccharides, has been recognized as a serious problem in sugar processing over 100 years. Dextran (C6H10O5)n is synthesized by microorganisms such as Streptococcus, Lactobacillus and Leuconostoc mesenteroides. The glucose monomers are predominantly linked by a(1,6) bonds in their major chains with a variable percentage of a(1,3) and occasional a(1,2) or a(1,4) branched linkages. The presence of dextran is associated with the processing problems from sugar cane juice to refined products. The dextran in sugar factories leads to a falsely high polarization, increased viscosity, lower evaporation rates, elongated crystals and increase of sugar loss to molasses.The aim of this work was to determine and eliminate the dextran level during sugar production. A monoclonal antibody against dextran was developed and an assaying kit base on the antibody for the dextran detecting was established. Then engineering strains of Pichia pastoris highly expressing an extracellular a-dextranase were constructed and screened. After fermentation in the optimal conditions, the activity of a-dextranase can reach 1048U/mL. The application of the recombinant dextranase was identified in the removal of dextran from sugar products and also in the production of lower molecular dextran in the medical and pharmacologic field.The main work is listed below:1. Construction and screening of engineered strains with recombinant plasmid encoding dextranaseThe a-dextranase gene from Penicillium minioluteum was cloned into vector pPICZa A and was expressed in Pichia pastoris under the control of AOX promoter. The engineered strain with recombinant plasmid X-33/pPICZaA-DEX was induced to express a-dextranase by methanol. We also constructed a recombinant plasmid of KM71H/pGAPZa A-DEX, with GAP promoter. The P. pastoris containing this plasmid expressed a-dextranase under the control of GAP promoter, without inductors in the glycerol or glucose medium. It was found that the yield of the a-dextranase is relative to the density of the density of engineered strains.2. Optimization of fermentation conditions for the expression of a-dextranaseThe fermentation conditions were optimized for P. pastoris with X-33/pPICZaA-DEX. Impact of temperature, dissolved oxygen, initial pH, inoculation size, methanol level in the induction step and interval time of feed supplement on the production of a-dextranase was analyzed. The dextranase activity from P. pastoris with X-33/pPICZaA-DEX culture and KM71H/pGAPZa A-DEX was determined to be 1048U/mL and 398 U/mL, respectively.3. Development of anti-dextran monoclonal antibody and establishment of assaying kit for dextranDextran was conjugated with BSA and then injected into Balb/c mice for the immunization. Cells of spleens and SP2/0 were fused by the hybridoma technology and the growing hybridomas were screened by indirect ELISA for their ability to secret high titer antibody against dextran. After cloning and subcloning by the Limiting Dilution method,7 hybridomas were selected, all of which exhibit titers higher than 1×106 in the culture supernatant. After purification of the culture supernatant, the antibody was analyzed to be specific against dextran, without cross-reacting with BSA, OVA, sucrose and glucose. Based on the newly-generated antibody, an immunonephelometric kit for quantitative detection dextran was developed. The kit passed the inspection and acceptance of the State Quality Inspection Administration of China in July,2010. The kit was proved be a rapid, correct and convenient tool for detecting dextran in raw material, by-products and end-products of sugar. This kit also can be applied to quantify dextran in medical and pharmacological field. The immunonephelometric assay is also respected to be a national standard detecting method, even a unitive analytical approach across the world. 4. Characterization of the a-dextranaseDextranase precipitated from the fermentation broth using ammonium sulfate was analyzed. Results showed that the optimal temperature for dextranase was 47-50℃and the optimal pH was 4.5-5.0. The stability of the a-dextranase was tested and results showed better stability in pH of 4-5. Half time of the dextranase was approximately 35 days when stored at 4℃.5. Application of a-dextranase in the sugar-making industryThe evaluating parameters for the clarification efficiency of a-dextranase on the sugar juice were viscosity reducing rate, color removal rate and dextran removal rate. Sugar juice showed better purity and color when treated with a-dextranase during various clarification process. Content of dextran and value of color of sugar cane juice in low purity decreased significantly after treated with a-dextranase. This indicated that a-dextranase was remarkably efficient on sugar cane juice in low purity.6. Application of dextranase in the production of low molecular mass dextranHere we also put forward the original technology of producing low molecular mass of dextran from fermentation of sucrose using Leuconostoc mesenteroides. Dextranase was added to hydrolyze the large polysaccharides into smaller ones. The molecular weight of dextran can be controlled by adjusting the a-dextranase adding level and adding time. Dextran with molecular weight of 3000-8000Da can be synthesized in one step when 0.8-1.2U/mL a-dextranase were added into the culture which had been fermented for 16h. The dextran was separated from the broth by ultra-filtration membrane and the obtained dextran meets the criterion of dextran in "Pharmacopoeia of the People’s Republic of China". The new technology solved the problems of uncontrolled molecular mass of dextran, severe pollution of wastewater, and large amount of organic solvent used when purifying.