环糊精葡萄糖基转移酶的酿酒酵母表面展示及其生产2-O-α-D-吡喃葡萄糖基抗坏血酸

Anchorage of cyclodextrin glycosyltransferase on outer membrane of Saccharomyces cerevisiae to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid

摘要:

摘要:【目的】将环状芽孢杆菌251(Bacillus circulans 251)来源的环糊精葡萄糖基转移酶(Cyclodextrin Glycosyltransferase，CGTase)展示在酿酒酵母( Saccharomyces cerevisiae)细胞表面，构建全细胞催化剂生产2-O-α-D-吡喃葡萄糖基抗坏血酸(2-O-α-D-glucopyranosyl-L-ascorbic acid，AA-2G)，以提高AA-2G 的产量。【方法】将CGTase编码基因cgt连接到载体质粒pYD1中的a凝集素(a-agglutinin)Aga2p亚基基因的下游构建表面展示重组质粒pYD1-cgt，转化酿酒酵母EBY100获得重组菌EBY100-pYD1-cgt，对发酵条件(培养基、诱导温度和诱导剂半乳糖浓度)进行优化;同时先后对重组菌的发酵产酶以及表面展示CGTase的酶促合成AA-2G的条件进行了优化;进一步又比较了表面展示的CGTase与E.coli BL21发酵所得的游离CGTase在酶促制备AA-2G过程中副产物的积累情况。【结果】展示CGTase的酿酒酵母重组菌株以YPG培养基作为发酵培养基，诱导剂半乳糖初始添加浓度为20 g/L，经25 ℃诱导48 h后，表面展示CGTase最大酶产量为0.5 U/mL;表面展示CGTase 40 ℃条件下的温度稳定性比游离酶有所提高，pH稳定范围变宽。对表面展示的CGTase制备AA-2G转化条件的优化发现，其最适温度最适pH分别为30 ℃和4.5，转化48 h达到平衡，表面展示的CGTase制备AA-2G的产量较游离酶提高了37%。【结论】对于CGTase，a凝集素系统是一个有效的展示系统，构建的酿酒酵母全细胞催化剂用于酶促制备AA-2G时，产生的副产物葡萄糖可能被酵母细胞利用，从而降低了葡萄糖与VC的竞争作用使AA-2G的产量增加，该全细胞催化剂具有良好的应用前景。

关键词:关键词:环糊精葡萄糖基转移酶，酿酒酵母，表面展示，2-O-α-D-吡喃葡萄糖基抗坏血酸

Abstract:

Abstract:［Objective］ Displaying cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans 251 on the cell surface of Saccharomyces cerevisiae to improve 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) production.［Methods］CGTase encoding gene cgt was inserted into the 3' terminal of Aga2p of vector pYD1 and the obtained recombinant plasmid pYD1-cgt was then transformed into S.cerevisiae EBY100 to produce surface displayed CGTase and culture conditions (culture medium，inductive temperature and concentration of inducer galactose) were optimized． Moreover，resulted CGTase displayed on the yeast cell surface was used for the AA-2G biosynthesis under the optimized condition.［Results］ CGTase activity on the cell surface of recombinant yeast，S.cerevisiae EBY100-pYD1-cgt，reached 0.5 U/ml in 48 h fermentation using Yeast Peptone Galactose culture medium with 20% galactose as sole carbon source and inducer at 25 ℃．The displayed CGTase exhibited better thermostability and pH stability than that of free CGTase．The concentration of AA-2G produced by the surface displayed CGTase was 37% higher than that produced by free CGTase at its optimal transformation conditions of 30 ℃ and pH4.5．［Conclusion］The cell surface display system based on a-agglutinin is an effective system for displaying CGTase． During AA-2G production by surface displayed CGTase，the by-product glucose might be consumed by yeast cell and thus facilitated AA-2G production． The whole cell EBY100-pYD1-cgt will have better prospects for applications．

Key words:Keywords: cyclodextrin glycosyltransferase， Saccharomyces cerevisiae， surface display，2-O-α-D-glucopyranosyl-Lascorbic acid