Enzymatic characterization of a recombinant carbonyl reductase from Acetobacter sp. CCTCC M209061.

BACKGROUND: Acetobacter sp. CCTCC M209061 could catalyze carbonyl compounds to chiral alcohols following anti-Prelog rule with excellent enantioselectivity. Therefore, the enzymatic characterization of carbonyl reductase (CR) from Acetobacter sp. CCTCC M209061 needs to be investigated. RESULTS: A CR from Acetobacter sp. CCTCC M209061 (AcCR) was cloned and expressed in E. coli. AcCR was purified and characterized, finding that AcCR as a dual coenzyme-dependent short-chain dehydrogenase/reductase (SDR) was more preferred to NADH for biocatalytic reactions. The AcCR was activated and stable when the temperature was under 35 °C and the pH range was from 6.0 to 8.0 for the reduction of 4'-chloroacetophenone with NADH as coenzyme, and the optimal temperature and pH were 45 °C and 8.5, respectively, for the oxidation reaction of isopropanol with NAD+. The enzyme showed moderate thermostability with half-lives of 25.75 h at 35 °C and 13.93 h at 45 °C, respectively. Moreover, the AcCR has broad substrate specificity to a range of ketones and ketoesters, and could catalyze to produce chiral alcohol with e.e. >99% for the majority of tested substrates following the anti-Prelog rule. CONCLUSIONS: The recombinant AcCR exhibited excellent enantioselectivity, broad substrate spectrum, and highly stereoselective anti-Prelog reduction of prochiral ketones. These results suggest that AcCR is a powerful catalyst for the production of anti-Prelog alcohols.Graphical abstractThe biocatalytic reactions conducted with the recombinant AcCR.

Hairpin DNA as a biobarcode modified on gold nanoparticles for electrochemical DNA detection.

Hairpin DNA (hpDNA) as a novel biobarcode was conjugated with gold nanoparticles (AuNPs) and a reporter DNA (rpDNA) to form hpDNA/AuNP/rpDNA nanoparticles for the detection of an oligonucleotide sequence associated with Helicobacter pylori as a model target. The rpDNA is complementary to about a half-portion of the target DNA sequence (tDNA). A capture DNA probe (cpDNA), complementary to the other half of the tDNA, was immobilized on the surface of a gold electrode. In the presence of tDNA, a sandwich structure of (hpDNA/AuNP/rpDNA)/tDNA/cpDNA was formed on the electrode surface. The differential pulse voltammetry (DPV) detection was based on [Ru(NH3)5(3-(2-phenanthren-9-yl-vinyl)-pyridine)](2+), an electroactive complex that binds to the sandwich structure by its intercalation with the hpDNA and the double-stranded DNA (dsDNA) of the sandwich structure. The several factors--high density of biobarcode hpDNA on the surface of AuNPs, multiple electroactive complex molecules intercalated with each hpDNA and dsDNA molecule, and the intercalation binding mode of the electroactive complex with the DNA sandwich structure--contribute to the DNA sensor with highly selective and sensitive sensing properties. The DNA sensor exhibited a detection limit of 1 × 10(-15) M (i.e., 1 fM), the DNA levels in physiological samples, with linearity down to 2 × 10(-15) M. It can differentiate even one single mismatched DNA from the complementary tDNA. This novel biobarcode-based DNA sensing approach should provide a general platform for development of direct, simple, repetitive, sensitive, and selective DNA sensors for various important applications in analytical, environmental, and clinical chemistry.

Enhancement of dopamine sensing by layer-by-layer assembly of PVI-dmeOs and Nafion on carbon nanotubes.

In this study, carbon nanotubes (CNTs) were modified to further improve their performance in electrochemical sensing of dopamine (DA) levels. After a redox polymer, poly(vinylimidazole) complexed with Os(4, 4'-dimethyl- 2, 2-bipyridine)(2)Cl (termed PVI-dmeOs) was electrodeposited on multi-wall CNTs (MWCNTs), Nafion and PVI-dmeOs films were successfully layer-by-layer (LBL) assembled on the hydrophilic surface of the as-prepared PVI-dmeOs/CNTs nanocomposites through electrostatic interactions. The LBL assembly was proved by scanning electron microscopy (SEM), electrochemistry and UV-vis spectroscopy measurements. LBL assembly of Nafion/PVI-dmeOs films on CNTs significantly enhanced their linear sweep voltammetry (LSV) response sensitivity to DA, with a maximum enhancement for three Nafion/PVI-dmeOs film-modified MWCNTs. The LSV peak current density of (Nafion/PV I-dmeOs)(3)/CNT electrodes in response to 10 and 50 microM DA solutions was about 7.3 and 3.9 times those for bare CNTs. At the (Nafion/PV I-dmeOs)(3)/CNT electrodes, the limit of detection (LOD) (signal-to-noise ratio: 3) was 0.05 microM DA, the linear range was 0.1-10 microM DA (with a linear regression coefficient of 0.97) and the DA-sensing sensitivity was 8.15 microA cm( - 2) microM( - 1). The newly fabricated (Nafion/PV I-dmeOs)(3)/CNT electrodes may be developed as an ideal biosensor for direct and in situ measurement of DA levels.