Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1-12 nmol/L) and high (10-500 nmol/L) concentration was observed, whose standard curve was y = 772.97× + 4041.1, R = 0.9884 and y = 1710× + 4.99 × 10 , R = 0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H-dependent flavin mononucleotide (FMN) reductases.

We established a method based on a luminescence system driven by NADH conversion to quantitatively detect ethanol in alcoholic beverages. NADH produced by an ADH-catalyzed enzymatic reaction was used as an indicator to directly quantify luminescence in vitro. A strong linear relationship (R 2 ¼ 0.9942) was observed between the luminescence intensity and ethanol concentration over a range of 0.002-0.1 mol L À1 , providing a detection limit of 1.43 Â 10 À4 mol L À1 for ethanol. This system has advantages such as rapid detection, high specificity, and repeatability in operation; therefore, it could be an advantageous alternative method for accurate quantification of ethanol content in beer, and may be suitable for other applications in the food industry.

LuxG from P. leiognathi YL was successfully purified and characterized. Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection, which showed higher sensitivity than existing bioluminescent systems.