AzoR is a homodimeric, flavin mononucleotide (FMN)-containing, NADH-dependent azoreductase from Escherichia coli. In this paper, we investigated the effect of the concentration of both AzoR and R59G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra. Two cross peaks (530, 490) and (580, 530) were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR, while only one cross peak appeared at (600, 530) for R59G mutant. This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates. The specific activity of both AzoR and R59G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L, which suggested that no significant dissociation of FMN occurred at low concentrations. Additionally, in order to explore the origin of these intermediates, we carried out a 2D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy. The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm, respectively. Taken together, these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.Dilution-induced two-dimensional synchronic and asynchronic fluorescence emission spectral changes of wt AzoR (A and D), R59G mutant (B and E) and free FMN (C and F) in 20 mmol/L Tris–HCl (pH 7.4) at room temperature upon excitation at 450 nm.

Gibberellic acid stimulated transcriptional protein from Gymnadenia conopsea (GcGAST) is a novel member of GA-induced cysteine-rich protein family, which shared 12 highly conserved cysteine residues with other members in C-terminal domain. In the present paper, the recombinant plasmid, as well as two mutants Serine–Proline–Cysteine (SPC) and Cysteine–Proline–Serine (CPS), were constructed to investigate for the first time the effects of the cysteines in Cysteine–Proline–Cysteine (CPC) sequence on the antioxidant activity of GcGAST protein. It was found that E.coli expressing wt GcGAST exhibited significant resistance against exogenous H2O2. Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to H2O2. Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate H2O2, whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of H2O2-eliminating ability. Fourier transform Infrared spectroscopy analysis indicated that the IB of CPS mutant contained more β-sheet secondary structure than wt and SPC mutant. Non-reducing SDS-PAGE combined western-blotting analysis revealed that the disulfide bonds were important for the formation of IBs of wt GcGAST and SPC mutant, whereas non-reducing SDS-PAGE of resolubilized IBs showed that hydrophobic interaction favored the aggregation of IBs in CPS mutant. Taken together, these results suggested that GcGAST possessed antioxidant activity in the level of IB, which made some contribution to cellular resistance to H2O2. More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.

In this work steady-state absorption spectroscopy, circular dichroism spectroscopy and sub-microsecond time-resolved absorption spectroscopy were used to investigate the effect of pH on the structures and functions of LH2 complex for Rhodopseudomonas palustris. The results revealed that: (1) B800 Bchla was gradually transformed to free pigments absorbing around 760 nm on the minutes timescale upon the induction of strong acidic pH, and subsequently there disappeared the CD signal for Qy band of B800 in the absence of B800. In addition, Carotenoids changed with the similar tendency to B850 BChl. (2) The introduction of strong basic pH gave rise to no significant changes for B800 Bchla, while B850 BChla experienced remarkable spectral blue-shift from 852 to 837 nm. Similar phenomenon was seen for the CD signal for Qy band of B850. Carotenoids displayed strong and pH-independent CD signals in the visible range. (3) In the case of both physiological and basic pH, broad and asymmetrical positive Tn ← T1 transient absorption appeared following the pulsed photo-excitation of Car at 532 nm. By contrast, the featureless and weak positive signal was observed on the sub-microsecond timescale in the acidic pH environment. The aforementioned experimental results indicated that acidic pH-induced removal of B800 Bchla prevented the generation of the carotenoid triplet state (3Car*), which is known to be essential for the photo-protection function. Nevertheless, carotenoids can still perform this important physiological role under the basic pH condition, where the spectral blue shift of B850 exerts little effect on the overall structure of the cyclic aggregate, therefore favoring the formation of carotenoid triplet state.

•An MDC-Analyzer-facilitated combinatorial strategy was developed for enzyme evolution.•The mutant DG9 with 5.9-fold higher kcat value than the wild-type enzyme was obtained.•The mutant ZB8 with 2.3-fold higher half-life than DG9 was evolved after screening 384 clones.Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) displays a broad substrate range with high regio- and enantioselectivity of both ring-closure and ring-opening reactions, making the enzyme a useful catalyst for the production of optically pure epoxides and β-substituted alcohols. In this study, we report a novel method using an MDC-Analyzer-facilitated combinatorial strategy to improve the activity and stability of HheC by simultaneously randomizing multiple contiguous residues. Six contiguous active-site residues, which are the hotspots for improving the activity of HheC, were simultaneously selected and randomized using the MDC-Analyzer-facilitated combinatorial strategy, resulting in a high-quality mutagenesis library. After screening a total of 1152 clones, three positive mutants were obtained, which exhibited approximately 3.5–5.9-fold higher kcat values than the wild-type HheC toward 1,3-dichloro-2-propanol (1,3-DCP). However, the inactivation half-life of the best mutant (DG9) at 55 °C decreased 9-fold compared with that of the wild-type HheC. To improve the stability of mutant DG9, seven contiguous potential surface amino acids were revealed by using the B-FITTER tool. Two charged amino acids, Glu and Lys, which are more abundant in thermophilic proteins than in their mesophilic counterparts, were selected to substitute those seven amino acids and were combined together via an MDC-Analyzer-facilitated combinatorial strategy. Two mutants displaying 1.6- and 2.3-fold higher half-life τ1/2 (55 °C) values than their DG9 template were obtained after screening only 384 clones. The results indicated that an MDC-Analyzer-facilitated combinatorial strategy represents an efficient tool for the directed evolution of functional enzymes with multiple contiguous targeting sites.