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Pursuing new probiotic targets has surged, driven by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising candidate for probiotics. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Predictive analyses of genomic elements like prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes were conducted to assess safety. Comparative genomic analysis was performed using 18 published Geobacillus genomes and a few Lactobacillus and Bifidobacterium genomes as controls. Genes associated with probiotic traits, such as adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones, were uniformly detected in Geobacillus . Mobile genetic elements (such as plasmids, prophages, and insertion sequences), virulence factors, toxins, and antibiotic resistance genes were absent, while CRISPR-Cas systems and CAZymes were present. The pan-genome comprised 25,284 protein-coding genes. Comparative genomic analysis revealed an open pan-genome for Geobacillus . Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to confirm its probiotic potential.

With the advent of green chemistry, the use of enzymes in industrial processes serves as an alternative to the conventional chemical catalysts. A high demand for sustainable processes for catalysis has brought a significant attention to hunt for novel enzymes. Among various hydrolases, the α-amylase has a gamut of biotechnological applications owing to its pivotal role in starch-hydrolysis. Industrial demand requires enzymes with thermostability and to ameliorate this crucial property, various methods such as protein engineering, directed evolution and enzyme immobilisation strategies are devised. Besides the traditional culture-dependent approach, metagenome from uncultured bacteria serves as a bountiful resource for novel genes/biocatalysts. Exploring the extreme-niches metagenome, advancements in protein engineering and biotechnology tools encourage the mining of novel α-amylase and its stable variants to tap its robust biotechnological and industrial potential. This review outlines α-amylase and its genetics, its catalytic domain architecture and mechanism of action, and various molecular methods to ameliorate its production. It aims to impart understanding on mechanisms involved in thermostability of α-amylase, cover strategies to screen novel genes from futile habitats and some molecular methods to ameliorate its properties.

Here we present the construction and characterization of metagenome assembled genomes (MAGs) from two hot springs residing in the vicinity of Indian Himalayan Geothermal Belt (IHGB). A total of 78 and 7 taxonomic bins were obtained for Old Yume Samdong (OYS) and New Yume Samdong (NYS) hot springs respectively. After passing all the criteria only 21 and 4 MAGs were further studied based on the successful prediction of their 16 S rRNA. Various databases were used such as GTDB, Kaiju, EzTaxon, BLAST XY Plot and NCBI BLAST to get the taxonomic classification of various 16 S rRNA predicted MAGs. The bacterial genomes found were from both thermophilic and mesophilic bacteria among which Proteobacteria, Chloroflexi, Bacteroidetes and Firmicutes were the abundant phyla. However, in case of OYS, two genomes belonged to archaeal Methanobacterium and Methanocaldococcus. Functional characterization revealed the richness of CAZymes such as Glycosyl Transferase (GT) (56.7%), Glycoside Hydrolase (GH) (37.4%), Carbohydrate Esterase family (CE) (8.2%), and Polysaccharide Lyase (PL) (1.9%). There were negligible antibiotic resistance genes in the MAGs however, a significant heavy metal tolerance gene was found in the MAGs. Thus, it may be assumed that there is no coexistence of antibiotic and heavy metal resistance genes in these hot spring microbiomes. Since the selected hot springs possess good sulfur content thus, we also checked the presence of genes for sulfur and nitrogen metabolism. It was found that MAGs from both the hot springs possess significant number of genes related to sulfur and nitrogen metabolism.

The current study deals with cloning and expressing a maltogenic α-amylase gene from thermophilic Bacillus stercoris YSP18 ( AmyYSP ) in Escherichia coli BL21 (DE3). AmyYSP belongs to the GH13_20 subfamily of Glycoside Hydrolases and entails five conserved regions found in maltogenic α-amylases. As a monomer of 67 kDa, AmyYSP exhibits maximal activity at 80°C, pH 5.0 and retains > 75% residual activity at 70–100°C and pH 5.0–8.0. The kinetic and thermodynamic studies displayed that it has a high affinity for soluble starch ( K m = 1.54 ± 0.236 mgmL − 1 ), exhibits a longer half-life (38.5 h at 80°C and 8.88 h at 100°C), and a higher E a D value of 3824 ± 1.03 kJ mol − 1 . It was characterised as a Ca 2+ -independent α-amylase, resistant to various denaturing additives. It hydrolyses soluble starch and raw corn starch efficiently, liberating glucose, maltose, maltotriose and maltotetraose as the main products. The thermostable and acid-stable, maltooligosaccharide forming AmyYSP is a versatile enzyme with prerequisites for successful application in starch-saccharification industries.

Cloning and expression of the α-amylase gene ( AmyK2) of thermophilic Bacillus subtilis k2cm originated from Yume Samdong hot spring, North Sikkim, India was done in Escherichia coli BL-21(DE3). The 55.0 kDa purified recombinant enzyme exhibited optimum activity at 70 °C and pH 7.0 with significant stability in temperature and pH ranges from 30 to 90 °C and 6.0–8.0, respectively. The α-amylase is Ca + 2 independent, and can act with reasonably high efficiency in absence of any metal. Moreover, its activity increased in the presence of Fe + 2 ion and was inhibited by Hg + 2 ion and EDTA. The recombinant enzyme showed a half-life of 53 min at 70 °C and its V max and K m values were 22.22 U/mg and 5.06 mg/ml, respectively. Immobilization of the purified enzyme on low-cost coconut coir with high immobilization yield (98.27% specific activity), increased half-life (71 min), and higher thermostability with successive use up to 8 cycles with high efficacy validates the techno-economic merit of use of the immobilized biocatalyst. As a whole, cloning the α-amylase gene of thermostable bacteria into the mesophilic organism and subsequent immobilization of the enzyme will unravel its secrets within the confines of the laboratory which could expedite its commercial exploitation in future. Graphical abstract Highlights of the Study • The recombinant α-amylase AmyK2 is highly thermotolerant, stable in a wide range of pH and Ca + 2 independent enzyme. • The enzyme having low Km for starch; therefore, it exhibits high affinity for any starchy substrate. • The enzyme was successfully immobilized at its native state with coconut coir and effective after successive reuse.

The pursuit of new probiotic targets has seen a surge, aided by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising target for uncovering its potential as a probiotic. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Additionally, the research undertook predictive analyses of genomic elements such as prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes. These evaluations aimed to assess the safety aspects associated with the genus Geobacillus. A comparative genomic analysis was also carried out using 18 validly published genomes of the genus Geobacillus and a few other genomes of Lactobacillus and Bifidobacterium were taken as control. Genes associated with probiotic traits like adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones were uniformly detected in the Geobacillus genus. Notably, mobile genetic elements such as plasmids, prophages, and insertion sequences were absent, as were virulence factors, toxins, and Antibiotic resistance genes. Additionally, CRISPR-Cas systems and CAZymes were present. The pan-genome encompassed 25,284 protein-coding genes with translation. Comparative genomic analysis revealed an open pan-genome for Geobacillus. Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. In conclusion, Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to add to the knowledge of the probiotic potential of Geobacillus species. This comprehensive study highlights the significant probiotic potential and genetic makeup of the Geobacillus genus, shedding light on its unique attributes in adapting to extreme environmental conditions. Understanding the probiotic properties of Geobacillus is crucial amidst growing concerns over antibiotic resistance, offering promising alternatives for combating pathogenic microbes. Additionally, exploring the genetic diversity and adaptive mechanisms of Geobacillus through genomic and metagenomic approaches provides valuable insights into its biotechnological applications and evolutionary history. By employing in-silico methods and comparative analyses with established probiotic genera, this study contributes to elucidating the probiotic characteristics of Geobacillus, paving the way for further research in harnessing its beneficial traits for various applications in health, biotechnology, and environmental remediation.

E-learning market in India is growing at phenomenal speed. One such start-up Xbrain was started in 2013 with the objective of trying to change the way people learn. It is a Delhi based education marketplace incepted by two young entrepreneurs Rohit Kapoor and Aman Mehta with the vision of providing marketplace for self-paced online courses on professional skills, exam preparation and hobbies. In spite of increasing demand for online education the company had a negative net margin because of high cost of funds, depreciation expenses etc. The organization was no-where near break-even. Besides, the organization faced the challenge that students were not open to online education and therefore, winning their trust was very important to penetrate the market.