Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

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.

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.

Preparation and evaluation of bilayer-core osmotic pump tablets contained topiramate.

Topiramate (TPM) was an antiepileptic agent commonly used in clinical. Studies showed that an oral preparation of TPM with extended-release manner could bring some benefits for epileptics. In this paper, controlled release push-pull osmotic pump (PPOP) tablets of sparingly water-soluble TPM were successfully prepared. This bi-layer tablet core mainly consisted of sodium chloride as osmotic promoting agent and polyethylene oxide as suspending and pushing agents. The influences of osmotic agents, pushing agents and the compositions of coating membrane on TPM release profiles were evaluated. An optimal formulation of TPM-PPOP was obtained through single-factor experiments. In vitro release tests showed that the optimum formulation could release TPM at an approximate zero-order rate up to 8 h. Pharmacokinetic behaviors of TPM-PPOP tablets were evaluated and compared with the immediate release capsules after an oral single dose in beagle dogs. Pharmacokinetics results demonstrated that the TPM-PPOP tablet was able to provide a prolonged release of TPM with longer tmax and mean residence time. Lower fluctuations of drug plasma levels could also be achieved with TPM-PPOP tablets. These results suggested that sparely water-soluble drugs as TPM can be designed to PPOP for efficacy and safety use.

Improving the decolorization activity of Bacillus pumilus W3 CotA-laccase to Congo Red by rational modification.

Congo Red (CR) is a typical azo dye with highly toxic and carcinogenic properties. This study aimed to improve the decolorization activity of Bacillus pumilus W3 CotA-laccase for azo dye CR. This work analyzed the interaction between CotA-laccase and CR based on homology modeling and molecular docking. The three amino acids (Gly323, Thr377, Thr418) in the substrate-binding pocket were rationally modified through saturation mutation. Finally, the obtained multi-site mutants T377I/T418G and G323S/T377I/T418G decolorized 76.59% and 59.37% of CR within 24 h at pH 8.0 without a mediator, which were 3.15- and 2.44-fold higher than the wild-type CotA. The catalytic efficiency of the multi-site mutants T377I/T418G and G323S/T377I/T418G to CR were increased by 2.21- and 2.01-fold compared with the wild-type CotA, respectively. The mechanism of activity enhancement of mutants was proposed by structural analysis. This evidence suggests that the mutants T377I/T418G and G323S/T377I/T418G could be used as novel bioremediation tools.

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.

Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: • Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. • Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. • The mechanisms of awarding enhanced activity to the mutants were supposed.

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.

Recombinant Horseradish Peroxidase C1A Immobilized on Hydrogel Matrix for Dye Decolorization and Its Mechanism on Acid Blue 129 Decolorization.

In this study, horseradish peroxidase C1A (HRP C1A) from Armoracia rusticana was expressed in Escherichia coli as an inclusion body. Subsequently, an active recombinant HRP C1A was obtained by refolding gradually using dilution-ultrafiltration. The recombinant HRP C1A was immobilized on agarose-chitosan hydrogel at 86.9 ± 2.5% of immobilization efficiency. After immobilization of the recombinant HRP C1A, the pH and temperature stability were improved and the reusability of the recombinant HPR C1A was achieved. The immobilized HRP C1A activity was retained above 80% after 6 cycles. The immobilized recombinant HRP C1A was used for the decolorization of four various dyes, including acid blue 129 (AB129), methyl blue (MB), methyl red (MR), and trypan blue (TB). The decolorization rates are all more than 70%, among which the decolorization effect of AB129 was the most significant (the decolorization rate was 76.3 ± 1.6%). Furthermore, a plausible decolorization pathway for AB129 was proposed based on the identified intermediates by ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS). This is the first report of the putative mechanism on the decolorization of AB129 by HRP.

Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green.

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

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.

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.

Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis.

CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7-11. Mutant S208G/F227A showed optimal temperature at 80°C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis.

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.

The effects of phytoremediation on soil bacterial communities in an abandoned mine site of rare earth elements.

Overexploitation of rare earth elements (REEs) has caused serious desertification and environmental pollution. The ecological restoration of mining areas has attracted increasing attention in China. Soil microbiota is important for successful ecological remediation of abandoned mine land. In this study, soil samples were collected from a restored REE mine site, and the bacterial community composition and diversity were assessed by Illumina high-throughput sequencing targeting the V3-V4 region of the 16S rRNA gene. Microbiota significantly developed in the remediated land. A total of 663,781 effective 16S rRNA gene sequences were obtained, which were classified into 28 bacterial phyla and 3 archaeal phyla. The dominant phyla across all samples (>5% of total effective sequences) were Proteobacteria, Acidobacteria and Firmicutes. Bacterial diversity indices (OTU number, Shannon index and Chao1 index) of the restored soils were higher than those of the tailings and even surpassed those in the unmined site. Redundancy analysis indicated that soil nutrients (soil organic carbon, available phosphorus and total nitrogen) were the dominant factors, followed by soil pH, affecting bacterial community structure. In general, these results suggested that soil amendment and phytoremediation effectively improved the soil environment of the abandoned mine site, which also increased our understanding of the correlation between microbial variation and soil properties in restored REE mine soils.

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.