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Carbon nanotube (CNT) has been proved to be a kind of novel support for enzyme immobilization. In this study, we tried to find the relationship between conformation and catalytic performance of immobilized enzyme. Two C C bond hydrolases BphD and MfphA were immobilized on CNTs (SWCNT and MWCNT) via physical adsorption and covalent attachment. Among the conjugates, the immobilized BphD on chemically functionalized SWCNT (BphD-CSWCNT) retained the highest catalytic efficiency ( k cat / K m value) compared to free BphD (92.9%). On the other hand, when MfphA bound to pristine SWCNT (MfphA-SWCNT), it was completely inactive. Time-resolved fluorescence spectrum indicated the formation of static ground complexes during the immobilization processes. Circular dichroism (CD) showed that the secondary structures of immobilized enzymes changed in varying degrees. In order to investigate the inhibition mechanism of MfphA by SWCNT, molecular dynamics simulation was employed to analyze the adsorption process, binding sites and time evolution of substrate tunnels. The results showed that the preferred binding sites (Trp201 and Met81) of MfphA for SWCNT blocked the main substrate access tunnel, thus making the enzyme inactive. The “tunnel-block” should be a novel possible inhibition mechanism for enzyme-nanotube conjugate.

The introduction of hydroxyl groups into indole molecule by different mono- and dioxygenases leads to the production of indigo. As a well-known biocatalyst, biphenyl dioxygenase possessed the ability to transform indole to indigo. However, there has been little information about this enzymatic transformation process. In this study, the genes encoding biphenyl dioxygenase (BphA) and biphenyl-2,3-dihydrodiol 2,3-dehydrogenase (BphB) were cloned from Dyella ginsengisoli LA-4 and heterologously expressed in Escherichia coli (DE3) (designated as AB _IND ). The feasibility of indole transformation to indigo by strain BphA _LA-4 was predicted by molecular docking studies. The biotransformation ratios of indole (100 mg/L) reached the maximum (95%) when cells were induced at 15 °C with 0.25 mM IPTG in M9 medium. In addition, 44 mg/L indigo was produced from 200 mg/L indole when supplied with 0.28 g/L of biomass and 0.2% (w/v) glucose. HPLC–MS was used to identify the products, which showed that indigo was the major product. Meanwhile, indirubin and isatin were also identified during the transformation process. Furthermore, the pathway of indole transformation by strain AB _IND was also proposed.

meta-Cleavage product (MCP) hydrolases can catalyze relatively low reactive carbon-carbon bond hydrolysis of products, which are derived from the meta-cleavage of catechols. The strict substrate selectivity of MCP hydrolases attracts an interest to understand the determinants of substrate specificity. Compared with conventional site-directed mutagenesis, domain swapping is an effective strategy to explore substrate specificity due to the large-scale reorganization of three-dimensional structure. In the present study, the hybrid MCP hydrolases BphDLidA and MfphALidD were constructed by exchanging the lid domain of two parental enzymes MfphA and BphD. The residues Gly130/Ala196 (MfphA) and Gly136/Ala211 (BphD) were selected as crossover points according to structural disruption score analysis and molecular dynamics simulations. It was shown that the hybrid enzymes exhibited similar substrate selectivity with the parent enzyme providing the lid domain. Docking studies suggested that the lid domain may play a key role in determining substrate specificity by reshaping the active pocket and modulating the orientation of the substrate.

Indirubin, a red isomer of indigo, can be used for the treatment of various chronic diseases. However, the microbial production of indirubin did not receive much attention probably due to its low yield compared with indigo. In this study, the recombinant Escherichia coli containing the naphthalene dioxygenase (NDO) genes from Comamonas sp. MQ was used to produce indirubin from tryptophan. To enhance the production of indirubin, the induction conditions for NDO expression were optimized. The optimal induction conditions were carried out with 0.5 mM isopropyl-β-d-thiogalactopyranoside at 30 °C when cells were grown to OD600 ≈ 1.20. Subsequently, the effects of medium composition on indirubin production were investigated by response surface methodology, and 9.37 ± 1.01 mg/l indirubin was produced from 3.28 g/l tryptophan. Meanwhile, the indirubin production was further improved by adding 2-oxindole and isatin to the tryptophan medium after induction. About 57.98 ± 2.62 mg/l indirubin was obtained by the addition of 500 mg/l 2-oxindole after 1-h induction, which was approximately 6.2-fold to that without additional 2-oxindole. The present study provided a possible way to improve the production of indirubin and should lay the foundation for the application of microbial indirubin production.

meta-Cleavage product (MCP) hydrolases (EC 3.7.1.9) can catalyze a specific C–C bond fission during the microbial aerobic degradation of aromatics. The previous studies on structure–function relationship of MCP hydrolases mainly focus on the active site residues by site-directed mutagenesis. However, the information about the role of the non-active-site residues is still unclear. In this study, a non-active-site residue Met148 of MCP hydrolase BphD was selected as the mutagenesis site according to the sequence alignments, structure superimpose and the tunnel analysis, which underwent the saturation mutagenesis resulting 19 mutants. The catalytic efficiencies of the mutants on 6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were all decreased compared with the wild-type one except for the M148D mutant. Especially, the M148P mutant exhibited 290-fold lower k cat/K m than that of the wild-type BphD. Transient kinetic analyses of M148P showed the reciprocal relaxation time corresponded to C–C bond cleavage and product release steps (9.6 s−1) was 4.08-fold lower than BphD WT (39.2 s−1). Tunnel cluster analysis of BphD WT, M148P and M148W demonstrated that only the bulky Trp148 could block tunnel T2 in the BphD WT, but it exhibited slight effects on the catalytic efficiency (0.94-fold of BphD WT). Therefore, product release was not the main reason for the efficiency decrease of M148P. On the other hand, molecular dynamics simulations on the BphD WT and BphD M148P in complex with HOPDA indicated that the dramatic decrease of the catalytic efficiencies of BphD M148P should be due to the unproductive binding of HOPDA. The study demonstrated the catalytic efficiency of MCP hydrolase can be engineered by modification of non-active site residue.

A multistep conversion system of para-substituted phenols by recombinant phenol hydroxylase (PHIND) and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphCLA-4) was constructed in this study. Docking studies with different para-substituted phenols and corresponding catechols inside of the active site of PHIND and BphCLA-4 predicted that all the substrates should be transformed. High-performance liquid chromatography–mass spectrometry analysis showed that the products of multistep conversion were the corresponding para-substituted catechols and semialdehydes. For the first-step conversion, the formation rate of 4-fluorocatechol (0.39 μM/min/mg dry weight) by strain PHIND hydroxylation was 1.15, 6.50, 3.00, and 1.18-fold higher than the formation of 4-chlorocatechol, 4-bromocatechol, 4-nitrocatechol, and 4-methylcatechol, respectively. For the second-step conversion, the formation rates of semialdehydes by strain BphCLA-4 were as follows: 5-fluoro-HODA > 5-chloro-HODA > 2-hydroxy-5-nitro-ODA > 5-bromo-HODA > 2-hydroxy-5-methyl-ODA. The present study suggested that the multistep conversion by both ring hydroxylase and cleavage dioxygenase should be potential in the synthesis of industrial precursors and provide a novel avenue in the wastewater recycling treatment.

A C–C hydrolase gene (bphD LA-4 ) from strain Dyella ginsengisoli LA-4 was cloned and expressed in Escherichia coli BL21 (DE3). BphDLA-4 together with another hydrolase MfphALA-4, which derived from the same strain, possessed esterase activities. p-Nitrophenyl butyrate was the best substrate for both enzymes. BphDLA-4 had high catalytic efficiency to p-nitrophenyl benzoate, whereas MfphALA-4 had no activity. Homology modeling and docking studies demonstrated that the proper hydrogen bond interaction was important for the reactivity of specific substrate.

Aromatic oxygenases,which can catalyze the hydroxylation of aromatic ring,have been widely used in bioremediation and chemical synthesis.In this paper,the classification and application area of aromatic oxygenases are summarized.Moreover,their discovery strategies based on pure culture technology and metagenomic technology are discussed in detail and first evaluated.Abundant of aromatic oxygenases have been obtained using pure culture technology,but these enzymes exhibit with single type,high repetition rate and low novelty.Emerging metagenomic technology provides new ideas for discovering microbial resource from uncultured microbes.However,it is still in the initial stage of exploiting aromatic oxygenases by metagenomic methods,and many problems such as low screening efficiency and hard to express heterologously have not been resolved.This review evaluates the two strategies by analyzing the content of genetic information,screening throughout and efficiency,the time consume,cost,and expression feasibility of aromatic oxygenase.And the metagenomic methods suitable for screening different types of aromatic oxygenases are proposed.In the future,pure culture and metagenomic technology combined with other omics technologies,will contribute to promoting the theoretical study of aromatic oxygenases and rapid development of biocatalysis industry.