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Expression And Identify Of Lac In Pichia Pastoris And Studies On Its Fermentation Strategy Optimization And The Downstream Process Of Fermentation Iquors

Posted on:2008-06-07Degree:MasterType:Thesis
Country:ChinaCandidate:Q SuFull Text:PDF
GTID:2120360218959667Subject:Prevention of Veterinary Medicine
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β-D-galactoside galactohydrolase (EC 3.2.1.23)is usually named lactase which can hydrolyze lactose into galactose and glucose, and also can transfer galactoside.Lactase is mainly used in producing low lactose dairy products, also lactase can be used for treatment of"lactose intolerance". Main problems of lactase production are low yield, high-cost and narrow applied range. The development of the genetic engineering technology supplies effective approach for producing high yield, high quality, simple craft, low cost lactase.This study using genetic engineering technology to construct a eukaryotic expression vector named pPIC9-cDNA. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems,and generally gives higher expression levels. To date, many heterologous protein have been expression in the Pichia pastoris successfully。In our experimentation, The expression level of secreted lactase protein in recombinant P.pastoris was 6mg/mL with enzymatic activity of 7800U/mL in the 3L fermenter,which was the highest among that of all kinds of recombinant strains reported now.The results show that 1#,7# had the same style of integration, and had different style of integration compared with 25#,this established rationale for looking for the relationship between high expressed strain and insertion site.We created a new method to large-scale ferment lactase. In conclusion, Explorated s- uccessfully of lactase Ferment optical conditions Inoculating amount was ten percent,TM was 30℃, pH was 5.2,the growth fine of bacteria was 24-28 hours ,the added methanol quantity was concrolled by DO,the induced type was that the expressed bacteria was indu ced firstly by the mixture of Glucose and methanol during Transition period ,then induce- de followed by methanol, the style of induction was mixture of glucose-methanol at tour studies established significant foundation for the further study the principle of membrane separation technology and its application situation in the downstream process of lactase and other fermentation liquor are reviewed. In this paper, we emphasize on the best ma- nipulation parameters of microfiltration membrane and nanofiltration membrane. To the Super filtration function, Enzyme live was losed leastly and the membrane was washed eastly when TM was 25℃,the operation pressure was lowe than 0.12MPa and concentr- ation was 10 fold .
Keywords/Search Tags:Lactase, Pichia pastoris, Polymerase chain reaction, Southern Blot, Fermentation, Membrane separation technology
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