| Laccases(benze nediol: oxygen ox idoreductases, EC 1.10.3. 2) is a group of copper-containing oxidizing enzymes, which have the ability to oxidize many kinds of dyes with water as the only byproduct. So, they can used for the decolorization and detoxification of synthetic dyes. Recently, industrial use of fungal laccase is becoming more and more attention. But in some areas, such as high temperature, high pressure, high p H conditions,bacterial laccase has more advantage than fungal laccase. Lac TT is a novel bacterial laccase screened from Thermus thermophilus SG 0.5JP17-16 constructed in our laboratory, it have been successfully expressed in in Pichia pastoris. Lac TT can catalyze guaiacol and other substrates, ha ve a higher p H and thermal stability, but also has a strong halogen tolerated. And to congo red or many other deys has a good variety of decolorization. So Lac TT will play a huge industrial applications in dyestuff processing and degradation of textile dye wastewater.Because of the natural laccase low production,easy deactivation and higher cost, it has been limited greatly in industrial applications, the laccase production research working on raising the yield. Pichia expression system is an excellent eukaryotic expression system developed recently, use pichia as a host to express laccase, can effectively improve the laccase production. Foreign protein expression level related with factors such as the strain characteristics, exogenous gene copy number and stability. At while, the induction strategy optimization of fermentation process, is an important way to increase the production rateand reduce the production cost in downstream production process.In order to increase the expression of Lac TT, the use of genetic engineering techniques to build a Lac TT four copies of Pichia pastoris, the highest activity in shake flask cultures by 534.42 U / m L increased to 1206.53 U / m L. It is expected to further increase production optimization by improving culture conditions and fermentation processes. In this paper, through the research of affects of recombinant pichia induction conditions including inducing initial inoculation quantity, methanol content, p H, temperature in induction period, pichia foundation fermentation conditions were determined. Study found that the different induction of initial pichia densities affected the induction of seeds during the period of growth, which has a significant effect on production of the Laccase. Therefore in the process of fermentation, initial inducing inoculation amount with 8% advisably. In the process of inducing with methanol as the sole carbon source of heterologous protein expression, need to strictly control the amount of methanol, very high methanol content causing huge accumulation of methanol in the fermented liquid, toxicity to the cells of recombinant pichia, and inhibit the expression of laccase.The optimal amount of methanol supplementation is 2%.During the induction period, p H and temperature will affect the stabil ity of the foreign proteins and the activity of AOX. Pichia can grow in p H5.0-7.0, 25 and 30 ℃ environment, but enzyme production suitable conditions of heterologous protein expression is varied from exogenous gene. This study found that, when p H is 6.0, the temperature is 30 ℃ during the induction period, the highest laccase activity reached 2212.9 U/L in 3 L fermentor, compared to the original condition is improved by 23.3%.In addition, this study found mixed feeding strategy can significantly improve growth Lac TT four copies recombinant pichia in the induction period, especially with glycerol and methanol as mixing carbon source. Laccase expression due to the different proportion of glycerol in the mixed carbon sources have very big difference, decrease the proportion of glycero can improve AOX activity and ATP levels of the recombinant pichia, and also can reduce the protease activity in supernatant. It is good for the expression of laccase and improve its stability. This study found that when glycerol and methanol mixture ratio of 1:5, the expression of recombinant pichia laccase best.The highest enzyme activity have reached 6110.68U/m L, increased by 1.48 times and 10.43 times respectively compared with methanol induction alone and the shake flask level.In this paper for the first time the recombinant pichia laccase expressed fermentation conditions were optimized, explored suitable fermentation process. At the same time, through the detection of related parameters in the fermentation process, analysed the key factors affecting the mechanism of action, then solved difficult problems such as the recombinant multi-copy pichia growth slowly in inducing phase, low AO X expression level, high protease expression level will degrading purpose protein, protein purification. This study provide wider space for mass industrial production, technology and theoretical guidance for laccase. |
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