Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30–70 °C) and pH (6.2–9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45C after 48h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100rpm. The enzyme was partially purified by 80 ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60C and pH 5. The cellulase enzyme was stable within the pH ranges of 35 and retained 67 of activity even at 55C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd²⁺, Pb²⁺, and Ba²⁺. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The K_m and V_max values of the enzyme were 1.8mg/ml and 10.92μg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.

This study is aimed at isolating and identifying a thermophilic cellulolytic bacterium from hot spring water and characterizing thermostable cellulase produced by the isolate. Enrichment and culture of water sample was used for isolation of bacterial strains and an isolate with highest cellulase activity was chosen for the production, partial purification, and biochemical characterization of the enzyme. Different staining techniques, enzymatic tests, and 16s ribosomal DNA (16s rDNA) gene sequencing were used for the identification of the isolate. The cellulase producing isolate was Gram positive, motile, and sporulated rod‐shaped bacterium growing optimally between 55°C and 65°C. Based on partial 16s rDNA sequence analysis, the isolate was identified as Geobacillus sp. and was designated as Geobacillus sp. KP43. The cellulase enzyme production condition was optimized, and the product was partially purified and biochemically characterized. Optimum cellulase production was observed in 1% carboxymethyl cellulose (CMC) at 55°C. The molecular weight of the enzyme was found to be approximately 66 kDa on 12% SDS‐PAGE analysis. Biochemical characterization of partially purified enzyme revealed the temperature optimum of 70°C and activity over a wide pH range. Further, cellulase activity was markedly stimulated by metal ion Fe^2+. Apart from cellulases, the isolate also depicted good xylanase, cellobiase, and amylase activities. Thermophilic growth with a variety of extracellular enzyme activities at elevated temperature as well as in a wide pH range showed that the isolated bacteria, Geobacillus sp. KP43, can withstand the harsh environmental condition, which makes this organism suitable for enzyme production for various biotechnological and industrial applications.

Visceral Leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes. VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020. This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients’ blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts. Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts (p = 0.001) while it increased in the non-program districts (p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients’ samples (n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex. All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex. We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal. The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram‐positive, straight or slightly curved, rod‐shaped, spore‐forming, catalase, and oxidase‐positive facultative anaerobes. It grows over a wide range of pH (5.0‐11) and temperature (37‐75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK‐KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole‐genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK‐KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate‐related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D‐xylose transport ATP‐binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase‐producing genes could be useful in lignocellulosic biomass‐utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

Mosquitoes (family: Culicidae) include several species that act as vectors of major human diseases by transmitting pathogens such as Plasmodium spp. (malaria), dengue virus (dengue), filarial worms (filariasis), and various other arboviruses. Species from the subfamilies Anophelinae and Culicinae are key transmitters of pathogens. This research aimed to assess genetic diversity and phylogenetic relationships among different species of mosquitoes collected from five districts of central Nepal. The mitochondrial cytochrome c oxidase I ( COI ) and Internal Transcribed Spacer 2 ( ITS2 ) genes were employed for genetic diversity. A total of 7223 mosquitoes were gathered, encompassing 18 species from eight genera across an altitudinal range from 62 to 3840 m above sea level. From the collected mosquitoes, 114 high‐quality sequences were obtained. Ae . aegypti exhibited lower diversity, with a haplotype diversity (Hd) of 0.46 ± 0.20 and a nucleotide diversity ( π ) of 0.006. The significantly negative Tajima's D value (−1.76, p < 0.05) suggests the possibility of a recent population expansion or the effect of purifying selection on this species. Phylogenetic analysis revealed distinct clades for most genera, while Aedes exhibited paraphyly. Intraspecific genetic divergence (K2P distances) was below 2.39%, while interspecific divergence exceeded 8%. The highest interspecific divergence was observed between Tx . splendens and An . subpictus (25.28%), and the lowest between Cx . pipiens and Cx . quinquefasciatus (0.43%). Haplotype network analysis revealed that Ae. aegypti exhibited a star‐like pattern with a widely shared central haplotype and low‐frequency variants, whereas Ae. albopictus showed a more structured network with two main clusters. These findings provide valuable information on the genetic diversity and evolutionary relationships among mosquito species in Nepal.