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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.

In this study, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) from Dyella ginsengisoli LA-4 was immobilized on the mesoporous silica SBA-15 in order to improve its stability with relatively high retaining activities. By Fourier transformed infrared spectroscopy (FTIR) and N 2 adsorption/desorption isotherms, BphC was confirmed to be successfully adsorbed and captured on SBA-15. Under the experimental conditions, the maximum loading amount could reach 124.6 mg protein g −1 support. The immobilized BphC could keep 16% of its initial activity after being stored at 4 °C for 5 days, whereas the free enzyme lost its activity rapidly within 12 h. Furthermore, the immobilized BphC possessed 90% of its initial activity after incubating at 40 °C for 4 h, indicating that the thermostability of BphC was significantly improved by immobilization. The biodegradation of catecholics compounds has been studied by immobilized BphC in batch system. The enhanced properties of immobilized BphC were further analyzed by the circular dichroism (CD), which supposed that the activity and stability alteration was due to the changes of secondary structure of BphC. This study would contribute to realizing the more effective biodegradation and bioremediation processes by immobilizing enzymes on the mesoporous silica materials.

Single-wall carbon nanotubes (SWCNTs) possess unique mechanical properties and extraordinary thermal conductivity, and were used as the matrix for immobilized biocatalysts. The C-C bond hydrolase BphD was immobilized on SWCNTs by physical adsorption and covalent bonding. The relative activity, stability, and reusability of the immobilized enzyme were investigated. BphD immobilized by physical adsorption retained 52.5% of the activity of free BphD, and BphD thermal stability and denaturant resistance were also improved. Covalently bound BphD exhibited the activity nearly the same as that of free BphD (99.7% of initial activity), but its stability showed no significant improvement over that of free BphD.

The naphthalene dioxygenase (NDO) genes were cloned from Comamonas sp. MQ and successfully expressed in Escherichia coli BL21 (DE3) (designated as ND_IND). The whole cells of recombinant strain ND_IND possessed relatively high transformation efficiency towards indole and most indole derivatives. According to the UV–vis and HPLC–MS analyses, the major products derived from the indoles could be indigo with different substituent groups. Furthermore, strain ND_IND was able to produce 205 mg/l indigo from 300 mg/l indole with a specific production rate of 8.4 mg/(g dry cell weight h). The effects of phenol, pyridine and quinoline on indigo production were determined, which indicated that phenol and pyridine had little inhibition on indigo production while quinoline would result in a 32% decrease in indigo yield. The present study proposed the potential application of recombinant strain ND_IND in indigoid pigments production, and offered the promise of applying strain ND_IND for the production of indigo using indole-containing wastewater as the raw materials.

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.

A molecular simulation strategy based on homology modeling and electrostatic potential calculations has been proposed to assist the immobilization process of a CC hydrolase MfphA onto mesoporous SBA-15. The size of the enzyme, pH-dependence of the immobilization process, and possible orientation of MfphA onto mesoporous materials (MPs) were predicted by molecular simulation. The adsorption of MfphA onto SBA-15 reached equilibrium in 1.5 hours at pH 7.0 and the maximum loading capacity was 34 mg g−1. Meanwhile at pH 9.0, no obvious adsorption was observed after 12 hours which corresponded to the molecular simulation prediction. It was also shown that, after immobilization, the catalytic activity of immobilized MfphA decreased to some extent, but the thermal stability was significantly improved. Guanidinium chloride (GdmCl) exhibited similar activity attenuation for both immobilized and free enzyme. By contrast, the immobilized MfphA was more resistant to urea at high concentration than was the free enzyme. Meanwhile, recycling experiments showed that the immobilized enzyme retained 30 % of its initial activity after ten reaction cycles.

Dyella ginsengisoli strain LA-4 can efficiently degrade environmental pollutants such as biphenyl and azo dyes. Here, we present a 4.55-Mb draft genome sequence of strain LA-4, which may provide further insights into the molecular mechanism in environmental pollution remediation.

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.