Detoxification of azo dye Direct Black G by thermophilic Anoxybacillus sp. PDR2 and its application potential in bioremediation.

Direct Black G (DBG) is a highly toxic synthetic azo dye which is difficult to degrade. Biological treatment seems to be a promising option for the treatment of azo dye containing effluent. A thermophilic bacterial strain (Anoxybacillus sp. PDR2) previously isolated from the soil can effectively remove DBG. However, the molecular underpinnings of DBG degradation and the microbial detoxification ability remains unknown. In the present study, the genetic background of PDR2 for the efficient degradation of DBG and its adaptation to azo dye-contaminated environments was revealed by bioinformatics. Moreover, the possible biodegradation pathways were speculated based on the UV-vis spectral analysis, FTIR, and intermediates identified by LC-MS. Additionally, phytotoxicity and the comet experiment studies clearly indicated that PDR2 converts toxic azo dye (DBG) into low toxicity metabolites. The combination of biodegradation pathways and detoxification analysis were utilized to explore the molecular degradation mechanism and bioremediation of azo dye for future applications. These findings will provide a valuable theoretical basis for the practical treatment of azo dye wastewater.

Genome and transcriptome analysis of a newly isolated azo dye degrading thermophilic strain Anoxybacillus sp.

Understanding azo dye degrading enzymes and the encoding of their functional genes is crucial for the elucidation of their molecular mechanisms. In this study, a thermophilic strain capable of degrading azo dye was isolated from the soil near a textile dye manufacturing factory. Based on its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis, the strain was identified as Anoxybacillus sp. PDR2. The decolorization ratios of 100-600 mg/L Direct Black G (DBG) by strain PDR2 reached 82.12-98.39% within 48 h of dyes. Genome analysis revealed that strain PDR2 contains a circular chromosome of 3791144 bp with a G + C content of 42.48%. The genetic basis of azo dye degradation by strain PDR2 and its capacity to adapt to harsh environments, were further elucidated through bioinformatics analysis. RNA-Seq and qRT-PCR technology confirmed that NAD(P)H-flavin reductase, 2Fe-2S ferredoxin and NAD(P)-dependent ethanol dehydrogenase genes expressed by strain PDR2, were the key genes involved in DBG degradation. The combination of genome and transcriptome analysis was utilized to explore the key genes of strain PDR2 involved in azo dye biodegradation, with these findings providing a valuable theoretical basis for the practical treatment of azo dye wastewater.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive.

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R2 = 0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4 IU/g at an optimum temperature of 57.5 °C, pH 8.31, moisture 50% and 1.2 mg of bio-surfactant. Experimental production of 30.3 IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.

Global transcriptomic response of Anoxybacillus sp. SK 3-4 to aluminum exposure.

Anoxybacillus sp. SK 3-4 is a Gram-positive, rod-shaped bacterium and a member of family Bacillaceae. We had previously reported that the strain is an aluminum resistant thermophilic bacterium. This is the first report to provide a detailed analysis of the global transcriptional response of Anoxybacillus when the cells were exposed to 600 mg L-1 of aluminum. The transcriptome was sequenced using Illumina MiSeq sequencer. Total of 708 genes were differentially expressed (fold change >2.00) with 316 genes were up-regulated while 347 genes were down-regulated, in comparing to control with no aluminum added in the culture. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the majority of genes encoding for cell metabolism such as glycolysis, sulfur metabolism, cysteine and methionine metabolism were up-regulated; while most of the gene associated with tricarboxylic acid cycle (TCA cycle) and valine, leucine and isoleucine metabolism were down-regulated. In addition, a significant number of the genes encoding ABC transporters, metal ions transporters, and some stress response proteins were also differentially expressed following aluminum exposure. The findings provide further insight and help us to understand on the resistance of Anoxybacillus sp. SK 3-4 toward aluminium.

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1.

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by ß-mercaptoethanol (ß-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.

Characterization of aluminum resistant Anoxybacillus sp. SK 3-4 isolated from a hot spring.

The Anoxybacillus sp. SK 3-4, previously isolated from a hot spring, was screened for its heavy metals resistance (Al(3+), Mn(2+), Cu(2+), Co(2+), Zn(2+), and Ni(2+)) and the strain was found to be most resistant to aluminum. Significant growth of the strain was observed when it was grown in medium containing aluminum (200 mg L(-1)-800 mg L(-1)) with relative growth rates ranging between 77% and 100%. A gene encoding the aluminum resistance protein (accession number: WP_021095658.1) was found in genome of strain SK 3-4, which revealed high sequence identity (>95%) to its homologues from Anoxybacillus species. Sequence comparisons with two functionally characterized aluminum resistance proteins, namely G2alt and ALU1-P, showed 97% and 81% of sequence identity, respectively. Four putative metal binding sites were detected in SK 3-4 aluminum resistance protein and G2alt at same amino acid residue positions of 186, 195, 198, and 201. Strain SK 3-4 was found to be able to remove aluminum from aqueous solution. This study demonstrated that Anoxybacillus sp. SK 3-4 could be applied in the treatment of aluminum contaminated wastewater.

Molecular response of Anoxybacillus sp. PDR2 under azo dye stress: An integrated analysis of proteomics and metabolomics.

Treating azo dye wastewater using thermophilic bacteria is considered a more efficient bioremediation strategy. In this study, a thermophilic bacterial strain, Anoxybacillus sp. PDR2, was regarded as the research target. This strain was characterized at different stages of azo dye degradation by using TMT quantitative proteomic and non-targeted metabolome technology. A total of 165 differentially expressed proteins (DEPs) and 439 differentially metabolites (DMs) were detected in comparisons between bacteria with and without azo dye. It was found that Anoxybacillus sp. PDR2 can degrade azo dye Direct Black G (DBG) through extracellular electron transfer with glucose serving as electron donors. Most proteins related to carbohydrate metabolism, including acetoacetate synthase, and malate synthase G, were overexpressed to provide energy. The bacterium can also self-synthesize riboflavin as a redox mediator of in vitro electron transport. These results lay a theoretical basis for industrial bioremediation of azo dye wastewater.

Improvement of the Thermostability and Activity of Pullulanase from Anoxybacillus sp. WB42.

Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (PulAC) derived from the pullulanase (PulA) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type PulA was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and kcat/Km value of the PulAC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type PulA. The optimum temperature of the mutant PulAC was 65 °C. The PulAC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type PulA. Due to its high optimum temperature, thermostability, and specific activity, the mutant PulAC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.

A novel ß-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharification.

An intracellular ß-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by ß-mercaptoethanol. In the presence of p-nitrophenyl-ß-d-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s-1, Kcat Km-1 of 2322 s-1 mM and was moderately influenced by xylose (Ki 21.25 mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.

Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp. LM18-11 for improved thermostability.

Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Enhancing the thermostability of Pullulanase is required for industrial application. In this study, we used two methods to improve the thermostability of the pullulanase from Anoxybacillus sp. LM18-11; these methods were the modified amino acid consensus method combined with the analyses of the residue water-exposed surface (ACC) and the deletion of flexible domains. Four mutants (Y477A, Y175C, L215C and R473E) were obtained via the modified consensus method exhibited varying degrees of improvements in terms of thermostability. One deletion mutant termed D3 (residues(686-688)) was obtained and exhibited enhanced thermostability due to deletion of the flexible region at the C-terminus. The combination of the two strategies yielded the mutant M18 (Y477A/D3/Y175C/L215P/R473E). It retained 66% of its initial activity after incubation at 60 °C for 72 hrs, whereas that of the wild-type enzyme was only 35%. After incubation at 65 °C for 4 h, M18 retained 50.6% of its initial activity, whereas that of the wild-type was only 16.8%, respectively. Additionally, kinetic studies revealed that the Km of M17 (Y477A/D3/Y175C/L215P) was decreased by 33.9% and that the Kcat/Km value of M17 increased by 50%, while M18 exhibited Km and Kcat/Km values that were similar to those of the wild-type enzyme. The attractive improved thermostability and the high catalytic efficiency made M17 and M18 more suitable for industrial application.