Parlak Mahmut, Ustek Duran, Tanriseven Aziz
Department of Biochemistry, Gebze Institute of Technology, Muallimkoy, Gebze, Kocaeli, Turkey.
Department of Genetics, Research Institute of Experimental Medicine, Istanbul University, Fatih, Istanbul, Turkey.
Carbohydr Res. 2014 Mar 11;386:41-7. doi: 10.1016/j.carres.2014.01.003. Epub 2014 Jan 14.
Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119 kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 5' end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature.
葡聚糖蔗糖酶由明串珠菌属、链球菌属和乳杆菌属的细菌产生。该酶能合成葡聚糖和一些受体产物，其中一些受体产物可作为益生元，越来越多地应用于食品、医药和化妆品等行业。嗜渗明串珠菌B-512F葡聚糖蔗糖酶（DSR-S）是葡聚糖和益生元商业化生产中的首选酶。在本研究中，设计了一种在益生元生产中高效的新型葡聚糖蔗糖酶。通过截短来自嗜渗明串珠菌B-512 FMC基因中的信号、可变区和葡聚糖结合区，并在5'端融合谷胱甘肽S-转移酶（GST）基因，在大肠杆菌中于最佳条件下生产该酶。这种新型酶（分子量：119 kDa）具有活性，能有效地进行葡聚糖生物合成和受体反应。新型葡聚糖蔗糖酶（fTDSR-S）是通过截短基因中的信号、可变区和葡聚糖结合区，并在5'端融合gst基因而产生的。对fTDSR-S进行了详细表征，并与DSR-S进行了比较。截短和融合使fTDSR-S在大肠杆菌BL21（DE3）中的生物合成增加了35倍。fTDSR-S能产生葡聚糖并增加受体反应。由于GST融合，可以成功地将fTDSR-S共价固定到Eupergit C上。还发现，葡聚糖蔗糖酶活性位点的大小比文献中先前报道的短49个氨基酸。
