Persson M, Bülow L, Mosbach K
Pure and Applied Biochemistry, Chemical Center, University of Lund, Sweden.
FEBS Lett. 1990 Sep 17;270(1-2):41-4. doi: 10.1016/0014-5793(90)81230-l.
The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an 'affinity tag' to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.
将来自枯草芽孢杆菌的编码葡萄糖脱氢酶（EC 1.1.1.47）的基因插入到位于乳糖启动子下游1.0 kb处的质粒中，当在大肠杆菌中表达时，该酶的产量提高了70倍。在第44位含有半胱氨酸残基的葡萄糖脱氢酶突变体也能以同样高的水平表达。这个单一的半胱氨酸残基被用作“亲和标签”，以简化纯化过程，并用于将葡萄糖脱氢酶位点特异性固定在硫丙基琼脂糖上。该酶被纯化至同质，最终回收率为65%，比活性为240 U/mg。定向固定导致热稳定性增加。
