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Study On The Strategies For High Cell Density Fermentation Of Alkaline Polygalacturonate Lyase In Recombinant Pichia Pastoris

Posted on:2009-02-11Degree:MasterType:Thesis
Country:ChinaCandidate:Y WangFull Text:PDF
GTID:2120360272456602Subject:Fermentation engineering
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Alkaline polygalacturonate lyase (PGL, E.C.4.2.2.2), is a kind of microbial pectinases with high-activity in alkaline environment, which cleaves theα-1,4-galacturonosidic linkages of polygalacturonic acid (PGA) and releases unsaturated soluble oligogalacturonates via a trans-elimination mechanism. It can be applied in degumming pretreatment of textile fibers and used as scouring assistant due to its fine removal effect on fibroglia and relatively small damage to natural cellulose base fiber. Therefore, a novel and mild biotechnological degumming not only presents an ecofriendly and economic alternative to the problems in textile industry, but also improves the quality of cotton fabrics.In previous studies, the coding sequence of PGL gene, extracted from a high-yield strain WSHB04-02, was successfully amplified and then expressed in Pichia pastoris GS115 by previous study. In order to enhance the production of PGL by recombinant P. pastoris GS115, the effect of cell and methanol concentration on the PGL production was carefully investigated by single factor experiment so as to determine the optimal ratio of methanol and cell concentration. The optimized ratio of methanol and cell concentration could be succesfully controlled by exponential glycerol feeding strategy in cell growth phase and multiphasic methanol feeding strategy in heterologous protein production phase. Moreover, the effect of induction temperature on degradation of heterologous protein in Pichia pastoris expressing system was disscussed. The major results were presented as follows:1) The key factors on high-level PGL production in recombinant Pichia pastoris GS115 were investigated. In 250 mL shake flask, the optimal glycerol concentration, initial methanol concentration, methanol supplementation quantity, duration of induction, initial pH, medium volume were 40 g/L, 9%, 1.5% (in every 24 h), 72 h, pH6.0 and 30 mL, respectively. Finally, the dry cell weight was 40 g/L, and the maximum yield of PGL was achieved to 130.6 U/mL.2) In 3 L fermentor, cells utilized methanol for growth when initial cell concentration were 62.5 g/L and 90 g/L, resulted in limited production of PGL due to methanol shortage. However, cells were not able to utilize methanol for growth when initial cell concentration reached 122 g/L, so methanol was mainly consumed for PGL production. At this point, highest PGL activity (376 U/mL) and highest PGL productivity (4.06 U/mL/h) were achieved at the methanol concentration 20 g/L. Moreover, it was found that properly increasing the ratio of methanol to cell concentration was beneficial for enhancement of PGL production.3) It was observed that the application of exponential glycerol feeding strategy in cell growth phase could achieve the maximum cell growth with high-efficiency. Dry cell weight could reach 140 g/L by a 19-h exponential glycerol feeding, gained the productivity 2.4-fold higher than that of DO-stat.4) A reasonable methanol-fed profile was proposed to control the ratio of methanol to cell concentration at the range of 0.163 to 0.171 g/g. Finally, the highest PGL activity (430 U/mL) and highest PGL productivity (4.34 U/mL/h) were achieved, which were 1.2-fold and 1.4-fold higher than that of DO-stat strategy.5) It was discovered that cell concentration remained constant till the end of cultivation with induction temperature at 30 oC, while the yeast obviously demostrated growth phenomenon during induction phase at 26 oC and 22 oC, with initial cell concentration increased to 147 g/L and to 148 g/L, respectively. Biphasic cultivation with temperature shifting from 30°C to 22°C in production phase led to 2.9-fold and 1.4-fold improvement of PGL actvity (931 U/mL) compared to 30°C (322 U/mL) and 26°C (657 U/mL), respectively.6) It was observed that the induction temperature was the key factor to influence cell viability. To seek the reason of PGL degradation, the exsistence of protease and cell viability were analyzed. At lower induction temperature, the cell mortality was greatly decreased, and there was almost no protease released into in culture supernatant, while at higher induction temperature, protease activity increased obviously, which may result in the degradation of heterologous PGL.7) The induction temperature had a remarkable effect on intracellular metabolites. The results indicated that intracellular AOX could be enhanced largely by decreasing the induction temperature, which may strengthen expression of down-stream desired gene of PGL. Meanwhile, ATP, ADP and AMP content was much higher, and the EC dropped significantly between 20 and 40 h after adaption under low temperature. It suggested that variety of EC may either signal a shift in carbon flux or may directly regulate recombinant protein synthesis.
Keywords/Search Tags:polygalacturonate lyase, recombinant Pichia pastoris GS115, methanol induction, feeding strategy, induction temperature, protein degradation
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