[Construction of a double functional recombinant strain of Pichia pastoris co-expressing phytase and mannanase and the enzymatic analyses].

Co-expression of phytase and mannanase in Pichia pastoris is a useful way to reduce the production cost in feedstuff industry. Based on the published DNA sequences of phytase gene and mannanase gene, primers were designed and genes phyA and man were cloned by PCR from Aspergillus terreus and the plasmid pHBM1201, respectively. Then the two fragments were treated and inserted into the same expression vector pHBM907C, which contains both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pHBM907C-phyA and the plasmid pHBM907C-man. The phyA expression cassette was combined to the expression vector pHBM907C-man which contains the expression cassette of mannanase, so pHBM907C-phyA-man was obtained. The recombinant expression plasmid pHBM907C-phyA-man was digested by Sal I and introduced into the chromosomes of Pichia pastoris GS115 by using the LiCl/PEG method. Following transformation, several parameters that demonstrated the expression of phytase and mannanase were measured. Firstly, the two different Petri dishes that contain enzymatic substrates such as calcium phytate and mannose were screened simultaneously, thus 100 clones were found to be positive on both of these plates. Secondly, 6 clones among them were chosen for induced expression at shakeflasks showing the probability of high expression. After that, some relative enzymatic properties were measured. At 72 hours' induction in the condition of shake cultivation, the enzyme activity of phytase in supernatant was 120.6 U/mL, while the enzyme activity of mannanase in supernatant was 39.7 U/mL. The expression product phytase was active under pH 2.0-6.5, and the activity was up to the highest under pH 5.5. And the other expression product mannanase was active under pH 5.5-10.5, and the activity was up to the highest under pH 7.5. The optimal temperatures for the two enzyme were both around 52 degrees C: the optimal temperature for phytase activity was 50 degrees C, and that for mannanase was 55 degrees C. At last, stability test of the engineered yeast was taken, and the engineered yeast still showed an excellent stability even after 10 generation growth in the absence of selective pressure. The stable double functional engineered yeast simultaneously expressing extracellular phytase and mannanase is obtained. It will satisfy the demand for industrialized production in some degree.