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Abstract:The phyA m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyA m -phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 °C and an optimal pH at 5.5~6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 °C to 95 °C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoH f ), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyA m .

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As a typical extreme environment, acid mine drainage (AMD) has been extensively studied for its biogeochemical cycle, but little is known about the quality of dissolved organic matter (DOM) in AMD. In this study, DOM molecules in an AMD lake were detected with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), and the change of DOM molecules in the stratified water column was analyzed with a multi-sample evaluation procedure. The results demonstrate that DOM quality is highly stratified and can be linked with severe biogeochemical gradients. In the surface layer, DOM molecules can be distinguished by low quantities and intensities, as well as potential photodegradation products. Oxygen-poor and oxygen-rich molecules alternately dominate the chemocline, which can be explained by the redox-dependent adsorption/desorption of DOM on metastable secondary minerals. A rich and abundant DOM pool with a high proportion of heteroatoms exists at the bottom which can be significantly influenced by material exchange with sediments. These findings emphasize the active role of DOM in extreme AMD environments and expand the understanding of the carbon cycle in the hydrosphere.

The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed and synthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering the protein sequence. The expression vector, pP9K-phyCs, was linearized and transformed in P. pastoris. The yield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium (BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale, respectively, improving over 90 folds compared with the wild-type isolate. Purified enzyme showed temperature optimum of 70 degrees and pH optimum of 7.5. The enzyme activity retained 97% of the relative activity after incubation at 80 degrees for 5 min. Because of the heavy glycosylation the expressed phytase had a molecular size of approximately 51 kDa. After deglycosylation by endoglycosylase H (EndoH(f)), the enzyme had an apparent molecular size of 42 kDa. Its property and thermostability was affected by the glycosylation.