The present study investigates the low temperature tolerance strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1, which grows optimally at 55 °C , by subjecting it to a temperature down-shift of 10 °C (45 °C) for 4 and 6 h followed by studying its growth, morphophysiological, molecular and proteomic responses. Results suggested that although TPH1 experienced increased growth inhibition, ROS production, protein oxidation and membrane disruption after 4 h of incubation at 45 °C yet maintained its DNA integrity and cellular structure through the increased expression of DNA damage repair and cell envelop synthesizing proteins and also progressively alleviated growth inhibition by 20% within two hours i.e., 6 h, by inducing the expression of antioxidative enzymes, production of unsaturated fatty acids, capsular and released exopolysaccharides and forming biofilm along with chemotaxis proteins. Conclusively, the adaptation of Anoxybacillus rupiensis TPH1 to lower temperature is mainly mediated by the synthesis of large numbers of defense proteins and exopolysaccharide rich biofilm formation.

In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg-1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg-1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg-1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5\'-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme\'s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL-1 and 1580.3 ± 183.7 μmol mg-1 protein min-1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.

Hyperthermostable enzymes are highly desirable biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme exhibited remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new β-sheets at 80 °C, making the core of the hydrolase fold more stable. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, which resided at the interface between the large and cap domains. To further investigate the structural adaptation in extreme conditions, the intramolecular interactions of the native structure were investigated. EstD9 revealed 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters, which is higher than the previously reported thermostable Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to both fundamental and applied research involving protein engineering of industrial thermostable enzymes.

Xylanases are the main enzymes to hydrolyze xylan, the major hemicellulose found in lignocellulose. Xylanases also have a wide range of industrial applications. Therefore, the discovery of new xylanases has the potential to enhance efficiency and sustainability in many industries. Here, we report a xylanase with thermophilic character and superior biochemical properties for industrial use. The new xylanase is discovered in Anoxybacillus ayderensis as an intracellular xylanase (AAyXYN329) and recombinantly produced. While AAyXYN329 shows significant activity over a wide pH and temperature range, optimum activity conditions were determined as pH 6.5 and 65 °C. The half-life of the enzyme was calculated as 72 h at 65 °C. The enzyme did not lose activity between pH 6.0-9.0 at +4 °C for 75 days. Km, kcat and kcat/Km values of AAyXYN329 were calculated as 4.09824 ± 0.2245 μg/μL, 96.75 1/sec, and 23.61/L/g.s -1, respectively. In conclusion, the xylanase of A. ayderensis has an excellent potential to be utilized in many industrial processes.

In this study, our aim was to elucidate the relationship between Anoxybacillus rupiensis DSM 17127T and Anoxybacillus geothermalis GSsed3T through whole-genome phylogenetic analysis. The obtained 16S rRNA gene sequence from the genome of A. rupiensis DSM 17127T exhibited a 99.8% similarity with A. geothermalis GSsed3T. In the phylogenetic trees constructed using whole-genome sequences and 16S rRNA gene sequences, A. rupiensis DSM 17127T and A. geothermalis GSsed3T were observed to form a clade, indicating a close relationship between them. Moreover, the average amino acid identity, average nucleotide identity, and digital DNA-DNA hybridization values calculated between A. rupiensis DSM 17127T and A. geothermalis GSsed3T exceeded the threshold values typically used for species demarcation. Furthermore, the phylogenomic analysis based on the core genome of the strains in question provided additional support for the formation of a monophyletic clade by A. rupiensis DSM 17127T and A. geothermalis GSsed3T. Most phenotypic and chemotaxonomic features between both strains were almost identical except for a few exceptions. These findings suggest that both strains should be classified as belonging to the same species, and we propose that A. geothermalis GSsed3T is a later heterotypic synonym of A. rupiensis DSM 17127T.

Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, Anoxybacillus flavithermus, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn2+ ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/β structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Thermophilic and alkaliphilic microorganisms are unique organisms that possess remarkable survival strategies, enabling them to thrive on a diverse range of substrates. Anoxybacillus, a genus of thermophilic and alkaliphilic bacteria, encompasses 24 species and 2 subspecies. In recent years, extensive research has unveiled the diverse array of thermostable enzymes within this relatively new genus, holding significant potential for industrial and environmental applications. The biomass of Anoxybacillus has demonstrated promising results in bioremediation techniques, while the recently discovered metabolites have exhibited potential in medicinal experiments. This review aims to provide an overview of the key experimental findings related to the biotechnological applications utilizing bacteria from the Anoxybacillus genus.

α-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60-80 °C and is active and stable over a wide pH range (4.0-9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry.