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| Expression of Trehalose Synthase Gene in Pichia pastoris |
| LI Meng-yue, WANG Teng-fei, WANG Jun-qing, ZHAO Yi-jin, CHENG Cheng, WANG Rui-ming |
| Department of Biology Engineering, QILU University of Technology, Jinan 250353, China |
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Abstract Trehalose synthase can be used to transform the production of trehalose in one step, and its substrate specificity is higher, the production process is simple, and can not be influenced by the concentration of substrate maltose, which is the first choice for industrial production of trehalose. To obtain the surface display vector of Pichia pastoris with the ability of producing trehalose synthase, trehalose synthase gene(tres, 2064 bp) was amplified by PCR from genome of Pseudomonas putide P06(gi=1042893, NCBI), then linked to the plasmid pPICZαA to get the recombinant plasmid pPICZαA-tres. Pir series protein Pir1p which is covalently linked cell wall of Saccharomyces cerevisiae, was used as the anchoring protein, and the pir1p(847 bp) was amplified by PCR technique, then linked to recombinant plasmid pPICZαA-tres, and the recombinant plasmid pPICZA-tres-pir1p was obtained. The recombinant plasmid was transferred into Pichia pastoris GS115 by electroporation, and the protein was directed to the cell wall by a-factor signal peptide and then was displayed on the surface of Pichia pastoris. The positive clones were selected by Zeocin resistance screening. Centrifuging, crushing and using laminarinase hydrolysis the fermentation products, SDS-polyacrylamide gel electrophoresis analysis showed obvious fusion protein bands. The trehalose synthase successfully anchored in Pichia pastoris. The Pichia pastoris strains was hang up using pH 7.5 buffer suspension and the concentration of substrate for 30% of the maltose in 30℃ to 60℃ water bath roling 2 h. Reaction products were analyzed by HPLC and the enzymatic activity can be detected. In the optimized condition, pH 7.5, 50℃, the activity of trehalose synthase reached 300.65U/g. The enzyme activity was stable in the range of 40℃ to 50℃, holding 60 min, the residual activity was more than 75%. The optimum pH was 7.5, and in alkaline environment enzyme activities remained stable.
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Received: 15 September 2015
Published: 19 November 2015
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