| Polygalacturonate lyase is a kind of enzyme that is abundantly used in textile industry for cotton scouring. Previously we have reconstructed polygalacturonate lyase gene in Pichia pastoris for the expression of this enzyme. To improve the production of polygalacturonate lyase, a methanol induction strategy combined with pH was formulated, during which methanol was provided in two stages and pH was varied in growth and induction phase. For the two-stage pH control strategy during the growth phase the pH was controlled at 5.5 and in the induction phase different pH levels were investigated for the optimum enzyme production. During the online methanol control the different levels of methanol (v/v) were investigated online for the best enzyme production at pH 5.5. These two strategies were combined together for enhanced PGL productivity, the induction phase was divided into two stages in which methanol levels were maintained online at different levels. The transition phase was introduced 40 hours after induction phase instead of introducing it after the growth phase. The two stage combination strategy was formulated on the bases of methanol consumption of cells, optimum pH, cell viability and the production of polygalacturonic acid lyase by Pichia pastoris. By using this strategy, the production improved 100% compared with common conditions and the highest polygalacturonate lyase activity reached 1631 U/mL. This strategy proved to be very useful for enhanced polygalacturonic acid lyase productivity with higher cell viability, AOX activity and phosphate related compounds of the cell.Previously the coding sequence from a high yield strain Bacillus WSHB04-02 was extracted and amplified in Pichia Pastoris GS115 (2008). Initially the methanol to cell concentration had been achieved successfully and a yield of above 800 U/mL was achieved. In our work we studied some other parameters and the results are shown below.1) In the 3 L fermentor a single parameter was modified to investigate the polygalacturonic acid lyses productivity in Pichia pastoris cultures. Six different pH levels were investigated from a range of pH 6.5 to 3.5. It was observed that at pH 4.5 the PGL productivity was at 1362 U/mL, the dry cell weight achieved was 144 g/L, by the end of the fermentation 80% of the cells were viable, the residual methanol was constant at 17 mL/L during the induction phase. But with other pH levels it was not appreciable. All other parameters were kept constant during this two stage pH control strategy.2) During the online methanol control strategy the pH was kept at 4.5 based on the results obtained from our previous strategy. Five different residual methanol levels were investigated; 8 mL/L, 12mL/L, 16 mL/L, 20 mL/L and 24 mL/L. Among all 16 mL/L was found to be the best for PGL production, but it was less than at a methanol rate of 10 mL/h. PGL was enhanced to 1260 U/mL, with a biomass at 140 g/L. The ATP activity was determined at 1.2 g/L, ADP at 0.04 g/L and AMP at 0. 28 g/L. The cell viability was 77 % by the end of the fermentation.3) A third strategy was formulated on the basis of the results obtained from the above two strategies. During this strategy the transition phase was shifted from after the growth phase to the induction phase. It was introduced 40 hours after induction phase had been initiated. It was the first time to report such a strategy. The lower level of residual methanol 12 mL/L was combined with three high levels i.e. 16 mL/ L, 20 mL/L and 24 mL/L. The combination of 12 mL/L with 16 mL/L was the best, the biomass was achieved as high as 162 g/L, PGL produced was 1634 g/L, cell viability was 89 % by the end of the fermentation, protease activity was 0.08 U/mL compared to 20mL/L and 24 mL/L respectively all at pH 4.5.
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