海藻糖合酶在毕赤酵母表面的展示

Expression of Trehalose Synthase Gene in Pichia pastoris

海藻糖合酶能够利用麦芽糖一步法转化生产海藻糖,其底物专一性较高,该酶体系生产工艺简单,不受底物麦芽糖浓度的影响,是工业生产海藻糖的首选。为获得具有生产海藻糖合酶能力的毕赤酵母表面展示载体,实验以筛选的Pseudomonas putide P06海藻糖合酶基因为模板,PCR扩增得到海藻糖合酶基因(tres,2 064 bp),连接至pPICZαA质粒中,获得重组质粒pPICZαA-tres。以来自酿酒酵母的共价连接细胞壁的Pir系列蛋白的Pir1p成熟肽蛋白作为毕赤酵母表面展示的锚定蛋白,利用PCR技术扩增得到pir1p(847 bp),连接至重组质粒pPICZαA-tres中,获得重组质粒pPICZαA-tres-pir1p。将重组质粒电击转入毕赤酵母GS115中,利用α-factor信号肽将蛋白引导分泌至细胞壁展示于毕赤酵母表面。通过Zeocin抗性筛选,挑选出阳性克隆子并摇瓶发酵。发酵产物经离心、破碎并使用昆布多糖酶水解,洗脱,结果显示,SDS-聚丙烯酰胺凝胶电泳分析可见明显融合蛋白条带,表明海藻糖合酶已成功地锚定在毕赤酵母。将重组毕赤酵母使用p H 7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在30℃~60℃水浴条件下作用2 h,反应产物利用HPLC检测,能够检测到酶学活性。在优化后的条件p H 7.5,50℃,表面展示海藻糖合酶酶活达到300.65 U/g。40℃~50℃酶活较稳定,保温60 min,残留酶活相对活力达75%以上;最适反应p H值为7.5,并在碱性环境下稳定。

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, 2 064 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. Fir series protein Pirlp which is covalently linked cell wall of Saccharomyces cerevisiae, was used as the anchoring protein, and the pirlp （ 847 bp） was amplified by PCR technique, then linked to recombinant plasmid pPICZαA-tres, and the recombinant plasmid pPICZA-tres-pirlp 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-polyac^clamide 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 rain,the residual activity was more than 75%. The optimum pH was 7.5, and in alkaline environment enzyme activities remained stable.