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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 Al

Enhancing virus multiplication could assist in the rapid production of vaccines against viral diseases. Cold atmospheric plasma (CAP), a physical approach relying on reactive oxygen species to achieve the desirable cellular outcome, was shown to be effective in enhancing virus propagation, where bovine rhinotrachieitis virus and Madin-Darby Bovine Kidney cells were used as the modeling virus and cell line, respectively. CAP was shown to create synergies with virus infection in arresting host cells at the G2/M stage, decreasing cell membrane potential, increasing intracellular calcium level, and inducing selective autophagy. In addition, CAP was demonstrated to suppress virus-triggered immunogenic signaling as evaluated by IRF7 expression. We presented evidences on CAP-triggered maximization of host resources toward virus multiplication that is advantageous for viral vaccine production, and opened a novel regime for applying CAP in the sector of medical care and health.

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.

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.

Bacillus amyloliquefaciens YP6, a plant growth promoting rhizobacteria, is capable of efficiently degrading a wide range of organophosphorus pesticides (OPs). Here, we report the complete genome sequence of this bacterium with a genome size of 4 009 619 bp, 4 210 protein-coding genes and an average GC content of 45.9%. Based on the genome sequence, several genes previously described as being involved in solubilizing-phosphorus, OPs-degradation, indole-3-acetic acid (IAA) and siderophores synthesis. Interestingly, compared with the genomes of B. amyloliquefaciens species, strain YP6 had larger genome size and the most protein-coding genes. Moreover, the four categories of “cell envelope biogenesis, outer membrane (M),” “translation, ribosomal structure and biogenesis (J),” “transcription (K),” and “signal transduction mechanisms (T)” were fewer. These differences may be related to extensive environmental adaptability of the genus B. amyloliquefaciens. These results expand the application potential of strain YP6 for environmental bioremediation, provide gene resources involved in OPs degradation for biotechnology and gene engineering, and contribute to provide insights into the relationship between microorganism and living environment.

In the work Pichia pastoris system was selected to achieve secretory expression of a D501 G/L386 W/G417 L/G57 F mutant of CotA-laccase from Bacillus pumilus (GWLF) with highly improved catalytic efficiency and soluble expression level. The molecular mass of recombinant GWLF was estimated to be 75 kDa, and the active GWLF is a dimeric glycoprotein. A maximal activity of 17,080 ± 515.30 U/L in the culture supernatant was obtained by high cell density fermentation in a 5 L bioreactor. Biological toxicity test showed that GWLF can efficiently detoxify Evans blue. The mechanism for Evans blue decolorization and detoxification by GWLF was analyzed through liquid chromatography–mass spectrometry; the azo bond (-N N-) was transformed into N2 instead of toxic aniline compounds, in which water was the only by-product in the degradation process. This study is the first to clarify the mechanism of laccase-catalyzed degradation of Evans blue.

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.

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02321-2) contains supplementary material, which is available to authorized users.

Textile wastewater is characterized by high salinity and high temperature, and azo dye decolorization by mixed cultures under extreme salinity and thermophilic environments has received little attention. High salinity and temperature inhibit the biodecolorization efficiency in textile wastewater. In the present study, a halo-thermophilic bacterial consortium (HT1) that can decolorize azo dye at 10% salinity and 50 °C was enriched. Bacillus was the dominant genus, and this genus may play a key role in the decolorization process. HT1 can decolorize metanil yellow G (MYG) at a wide range of pH values (6–8), temperatures (40–60 °C), dye concentrations (100–200 mg/L) and salinities (1–15%). Laccase, manganese peroxidase, lignin peroxidase and azoreductase are involved in the decolorization process of MYG. In addition, the decolorization pathway of MYG was proposed based on GC-MS and FTIR results. The toxicity of MYG decreased after decolorization by HT1. A metagenomic sequencing approach was applied to identify the functional genes involved in degradation. Overall, this halo-thermophilic bacterial consortium could be a promising candidate for the treatment of textile wastewater under elevated temperature and salinity conditions.

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.

Multicopper oxidases (MCOs) are a pervasive family of enzymes that oxidize a wide range of phenolic and nonphenolic aromatic substrates, concomitantly with the reduction of dioxygen to water. MCOs are usually divided into two functional classes: metalloxidases and laccases. Given their broad substrate specificity and eco-friendliness (molecular oxygen from air as is used as the final electron acceptor and they only release water as byproduct), laccases are regarded as promising biological green tools for an array of applications. Among these laccases, those of bacterial origin have attracted research attention because of their notable advantages, including broad substrate spectrum, wide pH range, high thermostability, and tolerance to alkaline environments. This review aims to summarize the significant research efforts on the properties, mechanisms and structures, laccase-mediator systems, genetic engineering, immobilization, and biotechnological applications of the bacteria-source laccases and laccase-like enzymes, which principally include Bacillus laccases, actinomycetic laccases and some other species of bacterial laccases. In addition, these enzymes may offer tremendous potential for environmental and industrial applications.

Given that spore laccase from the Bacillus genus is heat- and alkali-resistant, it is more suitable for industrial applications than fungal laccase. To determine the optimal culture conditions for spore laccase production, the effects of Cu2+ concentration, oxygen content, and culture time on spore laccase production from Bacillus pumilus W3 were investigated. The optimal production parameters were 0.2 mM of Cu2+, 200 rpm shaking speed, 100 mL liquid loading, and 5 days of cultivation. Spore laccase was efficiently immobilized on amino-functionalized celite. When used in dye decolorization, the immobilized spore laccase removed 84.15% of methyl green and 69.70% of acid red 1 after 48 h of treatment. Moreover, the immobilized spore laccase retained 87.04% of its initial decolorization activity after six cycles in the decolorization of acid red 1. These insights into the culture conditions and immobilization of spore laccases should be useful in the development of spore laccase as a biocatalyst in the treatment of textile wastewater.

Eyestalk ablation is commonly used to promote ovarian development and maturation in captive economic crustaceans. Understanding the molecular mechanism of eyestalk ablation in Procambarus clarkii is essential in finding alternative measures to trigger ovarian maturation on this species without undesirable effects. The cell division cycle 2 (Cdc2) is a catalytic subunit of the maturation-promoting factor, which is a central factor for inducing oocyte meiotic maturation. This study analyzed the Cdc2 gene expression patterns and the progesterone level in the P. clarkii ovary during its eyestalk ablation-induced maturation course with or without synthetic pcGnRH injection. The RNA interference technique was used to elucidate the role of Cdc2 in the eyestalk ablation mechanism. A combination of the present and previous results revealed that the progesterone-mediated oocyte maturation pathway could exist in the ovary. Furthermore, this pathway could play a crucial role in the eyestalk ablation-induced ovarian maturation in P. clarkii . Statement of relevance The results of this study enable the utilization of the Cdc2 function to control oocyte maturation in Procambarus clarkii.

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&ndash, 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 &deg, C, pH 7.20, and an inoculum size of 4.17% (v/v). The phoxim metabolites, O,O-diethylthiophosphoric ester, phoxom, and &alpha, cyanobenzylideneaminooxy phosphonic acid, were confirmed by liquid chromatography&ndash, 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.

Increasing evidences suggest that intestinal microbiota balance closely correlated with host’s health status could affected by external environment. Integrated crayfish-rice cultivation model is a highly efficient artificial ecosystem widely practiced in subtropical China. Less information is available to estimate the influence response to the micro-ecology of crayfish intestine and so as to influence the biological processes. Thus, 16S rRNA high-throughput sequencing approach was employed to investigate the composition diversity and functions of bacterial community in the intestines of Procambarus clarkii farmed within this model. Results exhibited the highly diversity of microflora with dominant phyla Actinobacteria, Proteobacteria, Tenericutes, Firmicutes and Bacteroidetes. The genera of Candidatus Bacilloplasma and Ornithinibacter were presented as predominant population much exceeds in richness comparing to that of other genus. Despite the highly diversity in the bacterial community, the predicted functions indicated relative consistent in biological processing pathway. Collectively, significant richness of genes was observed involved in amino acid and carbohydrate metabolism and membrane transport processing. This study would contribute to the understanding of the impact of growth conditions on host–microbiota relation especially in aquatic animals.

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.

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.

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.

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 Mg

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 (

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.

A new α-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified α-glucosidase were 4.5 and 60 °C, respectively, using p-nitrophenyl-α-glucopyranoside (α-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe2+, Cu2+, and Ag+ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K m, V max, and k cat/K m of the α-glucosidase were 0.52 mM, 3.76 U mg−1, and 1.3 × 104 L s−1 mol−1, respectively. K m with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The α-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The α-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 α-glucosidase is an ascomycetes α-glucosidase. This is the first report of α-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and α-pNPG, transglycosylation and conversion activity of maltose into trehalose.

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, 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. 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.

Catechol siderophore plays an important role in microbial ecology, agriculture, and medicine, but its research is often limited by the difficulty in acquisition of it in large quantities. Based on evidence from the coordination chemistry and chemical biology, catechol siderophore could chelate Fe(3+) with high affinity. Therefore, Fe(III)-based immobilized metal–affinity chromatography (IMAC) was applied to capture siderophore from the culture filtrate of Bacillus tequilensis CD36. The ethanol-precipitated sample and the separated sample from Fe(III)-based IMAC were analyzed by liquid chromatography–mass spectrometry. According to the result, the pure siderophore DHB-Gly-Thr could be extracted from the ethanol-precipitated sample. Compared with other purifications, Fe(III)-based IMAC was convenient and had fewer steps. In addition, it also reduced the use of toxic chemical solvents in some traditional extraction process, such as extraction and ion exchange chromatography. Fe(III)-based IMAC was successfully used in separation of the catechol siderophore from B. tequilensis CD36. The results revealed that Fe(III)-based IMAC was an efficient and environmentally friendly method for the separation and purification of catechol siderophore.

&beta, 1,3-Glucanase is considered as a useful enzymatic tool for &beta, 1,3-glucan degradation to produce (1&rarr, 3)-linked &beta, glucan oligosaccharides with pharmacological activity properties. To validly isolate &beta, 1,3-glucanase-producing microorganisms, the soil of Wolfiporia extensa, considered an environment rich in &beta, 1,3-glucan-degrading microorganisms, was subjected to high throughput sequencing. The results demonstrated that the genera Streptomyces (1.90%) and Arthrobacter (0.78%) belonging to the order Actinomycetales (8.64%) in the phylum Actinobacteria (18.64%) were observed in soil for P. cocos cultivation (FTL1). Actinomycetes were considered as the candidates for isolation of glucan-degrading microorganisms. Out of 58 isolates, only 11 exhibited &beta, 1,3-glucan-degrading activity. The isolate SYBCQL belonging to the genus Kitasatospora with &beta, 1,3-glucan-degrading activity was found and reported for the first time and the isolate SYBC17 displayed the highest yield (1.02 U/mg) among the isolates. To check the &beta, 1,3-glucanase contribution to &beta, 1,3-glucan-degrading activity, two genes, 17-W and 17-Q, encoding &beta, 1,3-glucanase in SYBC17 and one gene QLK1 in SYBCQL were cloned and expressed for verification at the molecular level. Our findings collectively showed that the isolates able to secrete &beta, 1,3-glucanase could be obtained with the assistance of high-throughput sequencing and genes expression analysis. These methods provided technical support for isolating &beta, 1,3-glucanase-producing microorganisms.

Light is a very important signal for fungi since it influences many different physiological responses. The effects of dark or light at different wavelengths on growth, reproduction and hypocrellins of Shiraia sp. SUPER-H168 were studied: dark, white, red, yellow, green, blue and purple. All incubations under different light conditions had significant stimulating effects on aerial hyphae and suppressing effects on hypocrellin biosynthesis compared with dark incubation. Under blue and purple light especially blue light, the colonies with profuse growth of aerial mycelium were formed. Hypocrellin production reached 13.73 mg per dish under dark condition, and decreased to 4.01 mg and 2.83 mg per dish under white and blue light, respectively. Light condition not only influenced hypocrellin production but also influenced the composition of hypocrellins. Four types of hyphae, namely surface, aerial, biofilm and penetrative hyphae, were observed by light microscopy and SEM. This study found that biofilm hyphae was so closely connected with production of secondary metabolites, and hypocrellins were only produced by biofilm hyphae. Light promoted sexual development and inhibited asexual reproduction, especially blue light strongly inhibited asexual development.

Shiraia bambusicola is an important and valuable macrofungus and hypocrellins are its main secondary metabolites which have been widely applied in many medical fields. However, during SSF process of this filamentous fungus, use ratio of corn substrate and dissolved oxygen supply are two main limiting factors, which influence production cost, yield and product quality. To solve these problems, overexpressions of amy365-1 and vgb in S. bambusicola were investigated and three overexpression transformants were constructed. Results demonstrated that expressions and coexpression of AMY365-1 and VHb not only increased the productions of biomass, amylase, hypocrellin, but also up-regulated relative expression levels of four central carbon metabolism genes (pdc, ald, acs, acc) and seven hypocrellin biosynthesis genes (fad, mono, zftf, omef, msf, pks, mco). Furthermore, expression of VHb decreased SSF period. When amy365-1 and vgb were coexpressed, relative expression levels of zftf and pks reached their highest levels at 72 h under liquid fermentation, hypocrellin production reached the highest level 75.85 mg/gds which was 2.99-fold compared with wild type strain within 11 days under SSF, and residual starch of solid substrates was decreased from 35.47 to 14.57%. Electronic supplementary material The online version of this article (10.1186/s13568-018-0597-0) contains supplementary material, which is available to authorized users.

In recent decades, many microorganisms have been selected and bred for industrial production of alkali-stable catalases. Given the diversity and excellent properties of Bacillus catalases, the aim of this work was to screen potent acid-stable catalases from Bacillus strain(s). A strain with higher production of catalase activity was identified and designated as Bacillus altitudinis SYBC hb4 based on phenotypic properties, 16S rRNA and gyrB gene analyses. Its four catalase genes were cloned successfully and designated as katX, katB, katN1 and katN2, respectively. Three distinct catalases were detected by isozyme zymography; however, only one (KatB) was successfully identified by MALDI-TOF. KatB had an estimated molecular mass of 228 kDa, and consisted of four identical subunits of 57 kDa. KatB displayed optimal activity under conditions of pH 5.0, 30 °C and 25 mM H2O2. The apparent K m and V max were calculated to be 62 mM and 33.4 mol min−1 mg−1, respectively. KatB can maintain at least 60 % relative activity after 6 h under conditions of pH 5.0 and 30 °C. Its activity and stability were higher compared to bovine liver catalase under slightly acid conditions (pH 5.0). Thus, B. altitudinis SYBC hb4 KatB might represent a potential acid-stable catalase used in acidic conditions.

Cottonseed meal (CSM), a common agricultural by-product, was used as a nutrient source for the production of eicosapentaenoic acid (EPA) by Pythium irregulare. CSM can support good cell growth performance, as can yeast extract (YE). In terms of the maximum EPA content and EPA yield, CSM is superior to YE. Low concentrations of CSM are beneficial to lipid synthesis, and high concentrations favor the EPA content. Utilizing response surface methodology (RSM) analysis, the optimum contents of glucose and CSM in the fermentation medium were determined to be 40.2 and 16.1 g/l, respectively. After 6 days of fermentation at 25 °C and optimal conditions, the EPA yield and productivity were 245.3 and 40.9 mg/l day, respectively. Particle size of CSM was found to affect the EPA production, and a finely ground CSM (100 mesh) was determined to be best for EPA production. The variation in the fatty acid content of total fatty acid (TFA) indicates that EPA was synthesized through the n-6 route in P. irregulare and Δ12 desaturase was the key enzyme for EPA biosynthesis. Sodium carbonate was determined to be notably good at removing free gossypol attached to biomass. After fungal biomass from each flask had been harvested from Na2CO3-supplemented medium, 1 % (w/v) Na2CO3 solution was used to wash the mycelia three times; free gossypol (FG) was not detected (detection limit 0.0018 %). This work provides a new approach using cottonseed meal to produce EPA through fungal fermentation.

In this study, we established a Cre/loxP mutant recombination system (Cre/lox71-66 system) for markerless gene deletion to facilitate our follow-up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.

Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications.

Arachidonic acid (ARA) is one of the three essential fatty acids, and it is important for human body to keep healthy and is widely used. At present, expensive materials such as glucose and yeast extract are generally reported to be optimal for ARA production. A new cost-effective fermentation process including cheaper material for ARA production is of great significance.Feasibility of using corn meal and powdered soybean for fungal growth and lipid accumulation was evaluated by means of single factor test. N-hexadecane concentration was optimized, and the effect of temperature on biomass and ARA content was examined.Mortierella alpina made better use of the aforementioned material as carbon and nitrogen sources for both hyphae growth and ARA production compared with glucose and yeast extract. Maximal levels of 10.9 g/L ARA and 26.1 g/L total lipids were obtained when 66 g/L corn meal, 54 g/L soybean meal and 6% (v/v) n-hexadecane were supplemented. A temperature-shift strategy involved three steps, namely, 30°C (3 days) - 25°C (4 days) - 20°C (4 days), which further improved ARA production by 24.7%.Several factors such as carbon and nitrogen sources, temperature and dissolved oxygen had great influence on biomass and microbial oil production. Mortierella alpina preferred corn and soybean meal compared with glucose and yeast extract, which would surely alleviate the high cost of ARA production. Based on this study, the new process is both low cost and practicable.

In order to obtain a more stable and reusable immobilized spore laccase for dye decolorization, the spores from Bacillus pumilus W3 were immobilized on diethylaminoethyl cellulose (DEAE-cellulose). Free and immobilized spore laccase retained 34.38% and 46.11% of their initial activity, respectively, after 10 days incubation at pH 9.0. Their residual activity remained at 27.36% and 31.84% after 10 h incubation at 70 °C. Immobilized spore laccase was more stable than free spore laccase in the presence of most organic solvents, metal ions and inhibitors. The tested dyes, including methyl green, methyl red and acid red 1, were removed 86.82%, 78.14% and 88.60%, respectively, by immobilized spore laccase after 24 h at 37 °C, and 74.34% of initial decolorization activity after 7 cycles was retained when it decolorized acid red 1. These properties indicated that immobilized spore laccase may be useful in textile effluent treatment. Keywords : Spore laccase; Bacillus pumilus ; DEAE-cellulose; Decolorization; Immobilization.

Incubation with different concentrations of different carbon, nitrogen and metal ion sources was found to significantly influence Shiraia sp. SUPER-H168 laccase production. Based on the magnitude of the range, the factors were ranked as follows: starch > Cu2+ > yeast powder for laccase production. The optimal combination of factors was 20 g/L starch, 4 g/L yeast powder and 0.6 mM Cu2+. Other fermentation media and conditions were also studied. The maximal laccase yield of Shiraia sp. SUPER-H168 varied from 5,058 to 1.105 × 105 U/L. Shiraia sp. SUPER-H168 laccase DNA and its corresponding full-length cDNA were cloned and characterized; furthermore, the Shiraia laccase gene sequence is reported for the first time. The coding region of Shiraia sp. SUPER-H168 laccase consisted of a 1,848 bp open reading frame encoding a protein of 615 aa with a 21 aa signal peptide sequence. Three introns of 50, 49 and 51 bp in length were located at upstream of the N terminus. The molecular mass (66.7 kDa) and isoelectric point (7.63) were predicted. Additionally, the protein contained six potential N-glycosylation sites (Asn-X-Ser/Thr) and four conserved copper-binding domains. The neighbor joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 laccase is an ascomycetes laccase closely related to Phoma sp. UHH 5-1-03 laccase.

Fungal laccases are typically unstable at high temperatures and alkaline conditions, thereby limiting their practical application. In this study, the novel laccase-producing Bacillus pumilus strain W3 was isolated from raw gallnut honey samples. The CotA–laccase gene was cloned from W3 and efficiently expressed by recombinant Escherichia coli in its biologically active form. The purified recombinant laccase had an extensive pH range for substrate catalysis. The enzyme was highly stable in alkaline pH and high temperatures, with considerable tolerance to NaCl and organic solvents. Laccase activity remained constant after 10 d of incubation at pH 9.0, whereas approximately 45% of the initial activity was detected after 10 h incubation at 80 °C. Two azo dyes and two anthraquinonic dyes could be efficiently decolorized by purified laccase in the presence of a mediator under alkaline condition. More than 90% decolorization was observed at pH 9.0 after incubation for 5 h. These unusual properties indicated a high potential of the novel CotA–laccase for industrial application on decolorisation of textile dyeing effluent.

Soil bacteria are important drivers of biogeochemical cycles and participate in many nutrient transformations in the soil. Meanwhile, bacterial diversity and community composition are related to soil physic-chemical properties and vegetation factors. However, how the soil and vegetation factors affect the diversity and community composition of bacteria is poorly understood, especially for bacteria associated with evergreen and deciduous trees in subtropical forest ecosystems. In the present paper, the microbial communities of rhizospheric soils associated with different types of trees were analyzed by Illumina MiSeq sequencing the V3-V4 region of the 16S rRNA gene. A total of 121,219 effective 16S rRNA gene sequences were obtained, which were classified into 29 bacterial phyla and 2 archaeal phyla. The dominant phyla across all samples (>5% of good-quality sequences in each sample) were Proteobacteria, Acidobacteria, Firmicutes and Bacteroidetes. The bacterial community composition and diversity were largely affected by both soil pH and tree species. The soil pH was the key factor influencing bacterial diversity, with lower pH associated with less diverse communities. Meanwhile, the contents of NO3- were higher in evergreen tree soils than those associated with deciduous trees, while less NH4+ than those associated with deciduous trees, leading to a lower pH and indirectly influencing the diversity and composition of the bacteria. The co-occurrence patterns were assessed by network analysis. A total of 415 pairs of significant and robust correlations (co-occurrence and negative) were identified from 89 genera. Sixteen hubs of co-occurrence patterns, mainly under the phyla Acidobacteria, Proteobacteria, Firmicutes and Bacteroidetes, may play important roles in sustaining the stability of the rhizospheric microbial communities. In general, our results suggested that local environmental conditions and soil pH were important in shaping the bacterial community of the Taihu Lake zone in east China.

The gummosis disease is caused by Botryosphaeria dothidea (Moug. ex. Fr) Ces. et de Not., and it is one of the most important diseases of stone fruits worldwide. The use of biocontrol as an alternative approach to synthetic chemical fungicides has aroused general concern about how to control plant diseases that are caused by phytopathogens. The aim of this study is to isolate Bacillus strains from raw honeys with the capacity to inhibit B. dothidea and to explore the mechanisms by which they could be used in the biocontrol of peach gummosis. Bacillus amyloliquefaciens SYBC H47 was isolated and identified on the basis of its physiological and biochemical characteristics and its 16S rRNA and gyrB gene sequences. The cell suspension and the cell-free supernatant of its culture showed significant antifungal activity against Aspergillus niger, Mucor racemosus, Fusarium oxysporum, Penicillium citrinum, and Candida albicans by agar-diffusion assays. The primary antifungal substances were bacillomycin L, fengycin, and surfactin, which were analyzed by HPLC LC/ESI-MS/MS. Bacillomycin L showed the best inhibitory effect against conidial germination of B. dothidea, followed by fengycin and surfactin. Surfactin had limited effects on mycelial growth, contrary to those of bacillomycin L and fengycin. However, a mixture of the three lipopeptides had a synergistic effect that disrupted the structure of the conidia and mycelia. In order to reduce the production cost, the use of waste frying peanut oil and soy oil as the sole carbon source increased the lipopeptide yield levels by approximately 17% (2.42 g/L) and 110% (4.35 g/L), respectively. In a field trial, the decreases in the infected gummosis rate (IGR) and the disease severity index (DSI) through cell suspension treatments were 20% and 57.5% (in 2014), respectively, and 40% and 57.5% (in 2015), respectively, in comparison with the control. In conclusion, B. amyloliquefaciens SYBC H47 could inhibit the germination of conidia and the growth of mycelia from B. dothidea; therefore, this strain behaves as a potential biocontrol agent against the gummosis disease.

Encoded by seven genes, angiopoietin-like (ANGPTL) family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here we reports for the first time the identification and expression analysis of all the seven members of bovine ANGPTL gene family, which were designated bANGPTL1 to bANGPTL7 in order. The seven bANGPTL genes consist of 4-9 exons, span 3800-43000 bp and are located on different chromosomes. The deduced amino acid sequences of the members all possess an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, both characteristics of angiopoietins. Phylogenetic analysis showed that the 32 identified ANGPTL homologs from 9 species could be classified into two major groups. Real-time quantitative PCR (Q-PCR) analysis revealed that the bANGPTL family members have different expression patterns. This study will be helpful for investigation on the biological role of the bANGPTL family in this economically important species. Furthermore, it provides an insight into the molecular evolution of the emerging ANGPTL family.

Relative expression levels of twenty-four amylase genes in Shiraia sp. SUPER-H168 were investigated by real-time quantitative PCR when various carbohydrates, including glucose, sucrose, maltose, amylose, amylopectin and corn flour, were used as carbon source. Genes, including an α-glucosidase gene Amy33 (2997 bp), an α-amylase gene Amy365-1 (1749 bp) and a glycogen debranching enzyme gene Amy130 (2487 bp), were overexpressed, and four overexpression transformants were constructed, respectively. When Amy365-1 and Amy130 were co-overexpressed, relative expression levels of seven hypocrellin biosynthesis genes and four related genes in central carbon catabolism were all increased. Expression of Amy33 was also increased along with increase of Amy365-1 and Amy130. Under liquid state fermentation, biomasses and hypocrellin productions were both gradually increased in four overexpression strains than those of wild type strain. Under SSF, hypocrellin production of Amy365-1 and Amy130 co-expression strain reached 71.85 mg/gds, which was 2.83 fold than that of wild type strain, and residual sugar was decreased from 35.47% to 16.68%. These results can provide a practical approach for other secondary metabolites by filamentous fungi under SSF when raw starch material is used as carbon source.

Here we report the full genome sequence of Bacillus pumilus W3, which was isolated from raw gallnut honey in Nandan County, Guangxi Province of China, showing high CotA-laccase activity. The W3 strain contains 3,745,123bp with GC content of 41.39%, and contains 3695 protein-coding genes, 21 rRNAs and 70 tRNAs.