Feeding Strategies And Large-scale Production Of Alkaline Polygalacturonate Lyase By Recombinant Pichia Pastoris

Polygalacturonate lyase (PGL or PL for short) could maintain a high-level activity under the alkaline environment. As a natural additive, PGL can be used in refining treatment process of textiles and fiber products. It has a removal effect on the surface of the fiber crops, or some other impurities, while the damage of cellulose is not very obvious. Therefore, if this novel scouring process could replace the traditional high temperature and high alkali chemical process, it will significantly reduce environmental pollution and alleviate the high energy consumption and other serious problems. Also it could improve the quality of the textile texture and cotton fabrics.We have got some significant results during our previous studies: First we screened a high-production strain Bacillus sp.WSHB04-02 for PGL in the nature. Second, the PGL gene from Bacillus sp.WSHB04-02 was extracted and transformed to Pichia pastoris GS115. Through the high-density fermentation and low temperature induced fermentation strategy, the PGL production has been significantly enhanced. Based on these studies, this paper through a detailed analysis studied the effects of mixed carbon sources in cultivation of recombinant P. pastoris GS115 for PGL production in the period of induction phase. And we found sorbitol co-feeding with methanol could significantly enhance the PGL production. Furthermore, this strategy could not only decrease the cell mortality, but also alleviate the proteinase degradation. On the other hand, in order to realize the industrial production of PGL, we successfully completed the fermentor amplification studies, from 3 L, 30 L, and 1000 L, up to 5000 L. The major findings are as follows:1. In order to increase the production and productivity of alkaline polygalacturonate lyase (PGL), we compared the mixed carbon sources feeding strategies during the induction phase by recombinant Pichia pastoris GS115. Glycerol, sorbitol or lactic acid co-feeding with methanol all enhanced the PGL production. Among all the feeding strategies, the sorbitol co-feeding strategy was most significant. By using this strategy, the PGL activity and productivity reached 1593 U/mL and 16.7 U/ (mL·h). Compared to the control, PGL activity and productivity increased by 84.6% and 45.2 % respectively, when the sorbitol feeding rate was set at 3.6 g/(h·L).2. In this study, in order to improve the expression of PGL in the host, sorbitol, which had been confirmed to be a non-respressive carbon source in a phenotype of Muts, was added together with methanol in the induction phase. Results demonstrated that the PGL activity was improved to 1593 U/ml with the sorbitol addition at a constant rate of 3.6 g/h within an induction period of 100 h, which was 1.85-fold compared to the control fermentation (863 U/ml). And it was found that sorbitol could enhance the expression of PGL. First, at the end of induction phase, cell mortality reduced from 25% to 9% by sorbitol co-feeding with methanol. Secondly, an appropriate sorbitol co-feeding strategy will not inhibit the expression of promoter, and even promote the activity of AOX of 6.9 U/g (control is only 5.7 U/g). Thirdly, due to the decrease of cell mortality had a certain extent to weaken the release of proteases from intracellular to extracellular, thereby reducing proteinase degradation on PGL. So sorbitol co-feeding with methanol has a significant role for improving heterologous protein production in Pichia pastoris expression system.3. To achieve the production of PGL on industrial scale, fermentations were scaled up from 3L, 30L, 1000L, to 5000L bioreactors. After overcoming some restrictive problems such as dissolved oxygen ad mass transfer, etc., the production of PGL and the repeatability of the fermentation process were improved. In this study, the PGL activity was up to 1425 U / ml in the 30 L fermentor, while it was 503 U/ml in 5000 L fermentor. As in the pilot-scale fermentation process, the induction temperature could not be controlled at a lower level, so that the PGL production could not be further improved. So how to improve the heat transfer capacity of the bioreactor and reduce the heat produced by the high-density fermentation need to be dealt with in future research.