Simultaneous removal of tetracycline and sulfamethoxazole by laccase-mediated oxidation and ferrate(VI) oxidation: the impact of mediators and metal ions.

This study explores the impact of mediators and metal ions of laccase-mediated oxidation and ferrate(VI) oxidation for the simultaneous removal of tetracycline antibiotics (TCs) and sulfonamide antibiotics (SAs) and to effectively remove their antimicrobial activity. The results showed that the antimicrobial activity of tetracycline against Bacillus altitudinis and Escherichia coli was significantly reduced, and the antimicrobial activity of sulfamethoxazole against B. altitudinis disappeared completely after treatment with the laccase-ABTS system. The combination of 6.0 U/mL of laccase and 0.2 mmol/L of ABTS removed 100% of 20.0 mg/L of tetracycline after 1.0 min at pH 6.0 and 25.0 °C, whereas the removal ratio of 20.0 mg/L of sulfamethoxazole was only 6.7%. The Al3+ and Cu2+ ions promoted the oxidation, and the Mn2+ ion decelerated the oxidation of tetracycline and sulfamethoxazole by the laccase-mediator systems. In contrast, the antimicrobial activity of tetracycline against B. altitudinis and E. coli was shown to be significantly reduced, and the sulfamethoxazole still retained high antimicrobial activity against B. altitudinis after treatment with Fe(VI) oxidation. The removal ratio of 20.0 mg/L of tetracycline was 100% after 1.0 min of treatment with 982.0 mg/L of K2FeO4 at pH 6.0 and 25.0 °C, whereas the removal ratio of 20.0 mg/L of sulfamethoxazole was only 49.5%. The Al3+, Cu2+, and Mn2+ ions both decelerated the oxidation of tetracycline and sulfamethoxazole by Fe(VI) oxidation. In general, the combination of the laccase-ABTS system and Fe(VI) was proposed for the simultaneous treatment of TCs and SAs in wastewater and to effectively remove their antimicrobial activity.

Pigment production by a newly isolated strain Pycnoporus sanguineus SYBC-L7 in solid-state fermentation.

Natural pigments are playing important roles in our daily lives. They not only make products colorful but also provide various health benefits for humans. In addition, Pycnoporus genus, listed as food- and cosmetic-grade microorganism, is one of the promising organisms for developing natural pigments. In this study, a new fungal strain with high efficiency in producing intense orange pigments was isolated and identified as Pycnoporus sanguineus SYBC-L7. Different agro-industrial wastes were applied to evaluate the growth and pigment production of strain SYBC-L7. SYBC-L7 can grow rapidly and effectively produce pigments using wood chips as substrate in solid-state fermentation (SSF). Culture conditions were also optimized for value-added pigments production and the optimum production conditions were glucose as carbon source, ammonium tartrate as nitrogen source, initial pH 6.0, and relative humidity of 65%. Pigment components, cinnabarinic acid, tramesanguin, and 2-amino-9-formylphenoxazone-1-carbonic acid were confirmed by liquid chromatography-mass spectrometry. Meanwhile, an agar plate diffusion assay was performed to evaluate the antimicrobial activity of the pigment. These pigments showed more significant inhibition of Gram-positive than Gram-negative bacteria. The results showed that Pycnoporus sanguineus SYBC-L7 was able to cost-effectively produce intense natural orange pigments with antibacterial activity in SSF, which is the basis of their large-scale production and application.

Identification of a novel glycerophosphodiester phosphodiesterase from Bacillus altitudinis W3 and its application in degradation of diphenyl phosphate.

Diphenyl phosphate (DPHP) has been increasingly detected in environmental samples, posing a potential hazard to humans and other organisms and arousing concern regarding its adverse effects. Biological degradation of DPHP is considered a promising and environmentally friendly method for its removal. In this study, the bagdpd gene was mined from the Bacillus altitudinis W3 genome and identified as a glycerophosphodiester phosphodiesterase by bioinformatics analysis. The enzyme was expressed and its biochemical properties were studied. When using bis(4-nitrophenyl) phosphate as substrate, enzyme activity was optimal at 55 °C and a pH of 8.5. The enzyme remained stable in the pH range of 8.0 - 10.0. The rBaGDPD enzyme degraded DPHP and the reaction product was identified as phenyl phosphate by LC-MS. This is the first report of a glycerophosphodiester phosphodiesterase exhibiting hydrolytic activity against DPHP. This study demonstrated that rBaGDPD could have the potential for bioremediation and industrial applications.

Improving the catalytic thermostability of Bacillus altitudinis W3 ω-transaminase by proline substitutions.

As a green biocatalyst, transaminase with high thermostability can be better employed to synthesize many pharmaceutical intermediates in industry. To improve the thermostability of (R)-selective amine transaminase from Bacillus altitudinis W3, related mutation sites were determined by multiple amino acid sequence alignment between wild-type ω-transaminase and four potential thermophilic ω-transaminases, followed by replacement of the related amino acid residues with proline by site-directed mutagenesis. Three stabilized mutants (D192P, T237P, and D192P/T237P) showing the highest stability were obtained and used for further analysis. Comparison with the wild-type enzyme revealed that the double mutant D192P/T237P exhibited the largest shift in thermostability, with a 2.5-fold improvement of t 1/2 at 40 °C, and a 6.3 °C increase in T 50 15, and a 5 °C higher optimal catalytic temperature. Additionally, this mutant exhibited an increase in catalytic efficiency (k cat/K m) relative to the wild-type enzyme. Modeling analysis indicated that the improved thermostability of the mutants could be associated with newly formed hydrophobic interactions and hydrogen bonds. This study shown that proline substitutions guided by sequence alignment to improve the thermostability of (R)-selective amine transaminase was effective and this method can also be used to engineering other enzymes.

Characterization of a robust cold-adapted and thermostable laccase from Pycnoporus sp. SYBC-L10 with a strong ability for the degradation of tetracycline and oxytetracycline by laccase-mediated oxidation.

A native laccase (Lac-Q) with robust cold-adapted and thermostable characteristics from the white-rot fungus Pycnoporus sp. SYBC-L10 was purified, characterized, and used in antibiotic treatments. Degradation experiments revealed that Lac-Q at 10.0 U mL-1 coupled with 1.0 mmol L-1 ABTS could degrade 100% of the tetracycline or oxytetracycline (50 mg L-1) within 5 min with a static incubation at 0 °C (pH 6.0). The presence of the Mn2+ ion inhibited the removal rate of tetracycline and oxytetracycline by the Lac-Q-ABTS system, and the presence of Al3+, Cu2+, and Fe3+ accelerated the removal rate of tetracycline and oxytetracycline by the Lac-Q-ABTS system. Furthermore, the growth inhibition of Bacillus altitudinis SYBC hb4 and E. coli by tetracycline antibiotics revealed that the antimicrobial activity was significantly reduced after treatment with the Lac-Q-ABTS system. Finally, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC-MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. These results suggest that the Lac-Q-ABTS system shows a great potential for the treatment of antibiotic wastewater containing different metal ions at various temperatures.

Characterization of a novel carboxylesterase from Bacillus velezensis SYBC H47 and its application in degradation of phthalate esters.

Recently, residual plasticizer phthalate esters (PAEs) in the different environments pose a serious health threat to humans and mammals. Biodegradation has been considered a promising and eco-friendly way to eliminate PAEs. In this study, a gene (baces04) encoding the novel PAEs hydrolase, carboxylesterase (BaCEs04), was screened from the genome of Bacillus velezensis SYBC H47 via bioinformatics analysis. Then, baces04 was cloned and expressed in Escherichia coli BL21 (DE3). BaCEs04 belonged to the esterase family VI. It contained a conserved domain (Gly159-His160-Ser161-Leu162-Gly163) and a typical serine hydrolase catalytic site (Ser161-Asp204-His261). The characterization of BaCEs04 showed that the activity was optimal at 60°C and pH 7.5. This enzyme also displayed high resistance to metal ions, organic solvents, and detergents. After treatment with BaCEs04 for 5 h, the degradation ratio of four different 1 mM PAEs, including dimethyl phthalate, diethyl phthalate, dipropyl phthalate, and dibutyl phthalate, was 32.4%, 50.5%, 77.9%, and 86.8%, respectively. The degradation products of four PAEs were identified as their corresponding monoalkyl phthalates. This is the first report that family VI esterase displaying PAE-hydrolysis activity. This study also proved that BaCEs04 could be used as an ideal candidate for the application in bioremediation and industry.

Evaluation of the Strain Bacillus amyloliquefaciens YP6 in Phoxim Degradation via Transcriptomic Data and Product Analysis.

Phoxim, a type of organophosphorus pesticide (OP), is widely used in both agriculture and fisheries. The persistence of phoxim has caused serious environmental pollution problems. In this study, Bacillus amyloliquefaciens YP6 (YP6), which is capable of promoting plant growth and degrading broad-spectrum OPs, was used to study phoxim degradation. Different culture media were applied to evaluate the growth and phoxim degradation of YP6. YP6 can grow rapidly and degrade phoxim efficiently in Luria-Bertani broth (LB broth) medium. Furthermore, it can also utilize phoxim as the sole phosphorus source in a mineral salt medium. Response surface methodology was performed to optimize the degradation conditions of phoxim by YP6 in LB broth medium. The optimum biodegradation conditions were 40 °C, pH 7.20, and an inoculum size of 4.17% (v/v). The phoxim metabolites, O,O-diethylthiophosphoric ester, phoxom, and α-cyanobenzylideneaminooxy phosphonic acid, were confirmed by liquid chromatography-mass spectrometry. Meanwhile, transcriptome analysis and qRT-PCR were performed to give insight into the phoxim-stress response at the transcriptome level. The hydrolase-, oxidase-, and NADPH-cytochrome P450 reductase-encoding genes were significantly upregulated for phoxim hydrolysis, sulfoxidation, and o-dealkylation. Furthermore, the phoxim biodegradation pathways by YP6 were proposed, for the first time, based on transcriptomic data and product analysis.

Comparison of aminotransferases of three Bacillus strains Bacillus altitudinis W3, Bacillus velezensis SYBC H47, and Bacillus amyloliquefaciens YP6 via genome analysis and bioinformatics.

Aminotransferases have attracted considerable attention due to their extraordinary potential for the biosynthesis of chiral amines. Research on transaminase genes can facilitate their application to various fields. Herein, 89 putative aminotransferase genes potentially encoding useful biocatalysts were identified in three Bacillus strains genomes by genome annotation. Enzymes encoded by genes ota3, ota8, otae6, otae21, otaf1, otaf8, and otaf26 belong to pyridoxine 5'-phosphate-dependent enzyme class IV. These seven ω-aminotransferase genes are highly conserved according to phylogenetic tree and bioinformatics analyses, as are the putative lysine catalytic residues in the corresponding enzymes (ω-BPTA 1-7). The enzymes may possess similar activity to ω-aminotransferases from Arthrobacter sp. KNK 168. The potential application of these novel enzymes for the synthesis of medicinal amino compounds will be explored in future genetic engineering studies.

Effect of residue substitution via site-directed mutagenesis on activity and steroselectivity of transaminase BpTA from Bacillus pumilus W3 for sitafloxacin hydrate intermediate.

Aminotransferases are widely employed as biocatalysts for the asymmetric synthesis of biologically active pharmaceuticals. Transaminase BpTA from Bacillus pumilus W3 can accept a broad spectrum of sterically demanding substrates, but it does not process the key five-membered ring intermediate of sitafloxacin. In the present study, we rationally constructed numerous single-point mutants and six multi-point mutants by combining the structural characteristics of transaminase and its substrates. Biochemical characteristics of wild-type and mutant enzymes were initially analyzed, and mutants I215M, I215F, and Y32L displayed increased catalytic efficiency, K155A, I215V and T252A completely lost enzyme activity. Residues K155 and T252 had a particularly strong influence on catalytic activity. Four multi-point mutants (L212M/I215M, Y32L/S190A/L212M/I215M, Y32L/Y159F/T252A and Y32W/Y159F/I215M/T252A) possess potential for industrial production of the key five-membered ring intermediate of sitafloxacin. Furthermore, mutants Y32L/Y159F/T252A and Y32W/Y159F/I215M/T252A can catalyze conversion of (R)-α-phenethylamine, albeit at an extremely low rate (<8%). In summary, mutants L212M/I215M and Y32L/S190A/L212M/I215M are more suitable for industrial production of the antibiotic, sitafloxacin, via an enzymatic approach.

Mining of aminotransferase gene ota3 from Bacillus pumilus W3 via genome analysis, gene cloning and expressing for compound bioamination.

Aminotransferases are widely employed as biocatalysts to produce chiral amines and biologically active pharmaceuticals via asymmetric synthesis. In this study, transaminase genes in the Bacillus pumilus W3 genome were analysed, and gene ota3 encoding a putative (R)-selective transaminase was identified. The sequence of ota3 shares highest sequence identity (24.7%) with the first (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Amino acid sequence and conserved domains analyses indicated that ω-BPAT encoded by ota3 belonged to the pyridoxal 5'-phosphate-dependent class IV (PLPDE_IV) superfamily. Both native and codon-optimised ω-BPAT genes were recombinantly expressed, and the purified proteins had a molecular mass of ~33.4 kDa. Furthermore, enantioselectivity tests with (S)- and (R)-α-phenethylamine revealed its (R)-selectivity. The optimal conditions for catalytic reaction were 45 °C and pH 7.0, and ω-BPAT retained stability at 20 °C and pH 7.0. Thus, ω-BPAT is a novel (R)-selective aminotransferase with great potential as a universal biocatalyst.