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.

An alkaline phosphatase from Bacillus amyloliquefaciens YP6 of new application in biodegradation of five broad-spectrum organophosphorus pesticides.

In recent decades, biodegradation has been considered a promising and eco-friendly way to eliminate organophosphorus pesticides (OPs) from the environment. To enrich current biodegrading-enzyme resources, an alkaline phosphatase (AP3) from Bacillus amyloliquefaciens YP6 was characterized and utilized to test the potential for new applications in the biodegradation of five broad-spectrum OPs. Characterization of AP3 demonstrated that activity was optimal at 40 °C and pH 10.3. The activity of AP3 was enhanced by Mg2+, Ca2+, and Cu2+, and strongly inhibited by Mn2+, EDTA, and L-Cys. Compared to disodium phenyl phosphate, p-nitrophenyl phosphate (pNPP) was more suitable to AP3, and the Vm, Km, kcat, kcat/Km values of AP3 for pNPP were 4,033 U mg-1, 12.2 mmol L-1, 3.3 × 106 s-1, and 2.7 × 108 s-1mol-1L, respectively. Degradation of the five OPs, which included chlorpyrifos, dichlorvos, dipterex, phoxim, and triazophos, was 18.7%, 53.0%, 5.5%, 68.3%, and 96.3%, respectively, after treatment with AP3 for 1 h. After treatment of the OP for 8 h, AP3 activities remained more than 80%, with the exception of phoxim. It can be postulated that AP3 may have a broad OP-degradation ability and could possibly provide excellent potential for biodegradation and bioremediation in polluted ecosystems.

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.

Bacterial diversity of stingless bee honey in Yunnan, China: isolation and genome sequencing of a novel acid-resistant Lactobacillus pentosus (SYBC-MI) with probiotic and L. tryptophan producing potential via millet fermentation.

Stingless bee (Hymenoptera, Apidae, and Trigona) honey is a remarkable "miracle liquid" with a wide range of medical benefits for conditions including gastroenteritis, cataracts, and wound healing. Our study aimed to isolate, identify, and characterize acid-resistant Lactobacillus spp. from sour honey distributed in Yunnan, China. To assess the safety of an entirely novel Lactobacillus pentosus strain, S4 (OM618128), based on probiotic property evaluation and whole-genome sequencing analysis. A 16S rRNA gene high-throughput sequencing analysis showed that Lactobacillus was abundant at the genus level in sour honey. Seven Lactobacillus strains (viz. S1-7) were isolated from sour honey using a multiple-anaerobic culture enrichment method. One potential acid-resistant isolate, Lactobacillus sp. S4, was obtained after screening the seven Lactobacillus isolates, and it had the highest lactic acid production (17.62 g/L), followed by Lactobacillus sp. S3 (17.07 g/L). Phylogenetic and comparative analyses of conserved sequence regions have shown that all seven strains are phylogenetically located in the Lactobacillus pentosus sub-cluster. In L. pentosus SYBC-MI, there is a circular chromosome (3288615 bps) and 11,466 bps plasmids. GC content is 44.03%. The number of predicted genes is 3,129, with 16 rRNAs and 74 tRNAs present. During the fermentation of foxtail millet by seven Lactobacillus pentosus (S1-7) strains isolated from sour honey, a potential tryptophan accumulating isolate, Lactobacillus pentosus S4, was obtained, which could reach a maximum tryptophan content of 238.43 mgL-1 that is 1.80 times the initial tryptophan content in the fermentation broth. This strain has strong acid tolerance, salt tolerance, and fermentation acid production abilities. This strain degrades nitrite at a rate of over 99%, and it has high probiotic potential as well. This project has established a solid foundation for further exploring the excellent lactic acid bacteria in sour honey. It is also investigating the key taxa and their role in the environment. According to the results of our studies, these LAB isolates provide a lot of potential for use in the future, as a source of probiotics for human, animals, and starter cultures for food applications.

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.

Mining of alkaline proteases from Bacillus altitudinis W3 for desensitization of milk proteins: Their heterologous expression, purification, and characterization.

In this study, three active alkaline proteases (AprEs) (BaApr1, BaApr2, and BaApr9) from Bacillus altitudinis W3 were obtained through bioinformatics analysis and verification. Multiple sequence alignment showed low identity of 64.60% and suggested that the three AprEs belonged to the S8A subfamily of serine proteases. They showed maximal activity with pH of 9.5 at 55 °C, 8.5 at 50 °C, and 10.5 at 45 °C, respectively. They were stable at alkaline condition and below 50 °C. In the presence of Ca2+, the optimal temperatures and thermostability of them were significantly improved. They were activated by Ca2+ and Mg2+ but inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Surfactants had little effect on them, but most organic solvents had some inhibitory effect except for n-hexane. They were effective in hydrolyzing natural proteins such as casein and NON-fat powdered milk. BaApr1 exhibited the highest catalytic efficiency towards casein and showed an excellent effect on the desensitization of milk proteins. The present study reveals some useful characteristics of the three AprEs, and indicates that AprEs have potential application values in the desensitization process of milk proteins.

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.

Poly[diaqua-µ(4)-oxalato-di-µ(6)-phosphato-tetra-cobalt(II)].

In the structure of the title compound, [Co(4)(C(2)O(4))(PO(4))(2)(H(2)O)(2)](n), there are layers composed of the phosphate anions and two independent Co(II) cations. These layers are parallel to (001) and are bridged by the oxalate anions that are situated in special positions on centres of symmetry. One independent Co atom has an octa-hedral coordination, while the second independent Co atom is coordinated in a trigonal-bipyramidal coordination that includes the water mol-ecule. The crystal packing is stabilized by O-H⋯O hydrogen bonds between the coordinated water mol-ecules and oxalate O atoms.