Biochemical characterization of glutaminase-free L-asparaginases from Himalayan Pseudomonas and Rahnella spp. for acrylamide mitigation.

L-asparaginase having low glutaminase activity is important in clinical and food applications. Herein, glutaminase-free L-asparaginase (type I) coding genes from Pseudomonas sp. PCH182 (Ps-ASNase I) and Rahnella sp. PCH162 (Rs-ASNase I) was amplified using gene-specific primers, cloned into a pET-47b(+) vector, and plasmids were transformed into Escherichia coli (E. coli). Further, affinity chromatography purified recombinant proteins to homogeneity with monomer sizes of ~37.0 kDa. Purified Ps-ASNase I and Rs-ASNase I were active at wide pHs and temperatures with optimum activity at 50 °C (492 ± 5 U/mg) and 37 °C (308 ± 4 U/mg), respectively. Kinetic constant Km and Vmax for L-asparagine (Asn) were 2.7 ± 0.06 mM and 526.31 ± 4.0 U/mg for Ps-ASNase I, and 4.43 ± 1.06 mM and 434.78 ± 4.0 U/mg for Rs-ASNase I. Circular dichroism study revealed 29.3 % and 24.12 % α-helix structures in Ps-ASNase I and Rs-ASNase I, respectively. Upon their evaluation to mitigate acrylamide formation, 43 % and 34 % acrylamide (AA) reduction were achieved after pre-treatment of raw potato slices, consistent with 65 % and 59 % Asn reduction for Ps-ASNase I and Rs-ASNase I, respectively. Current findings suggested the potential of less explored intracellular L-asparaginase in AA mitigation for food safety.

Apoptosis and cell cycle arrest of leukemic cells by a robust and stable L-asparaginase from Pseudomonas sp. PCH199.

Biomolecules obtained from microorganisms living in extreme environments possess properties that have pharmacokinetic advantages. Enzyme assay revealed recombinant L-ASNase, an extremozyme from Pseudomonas sp. PCH199 is to be highly stable with 90 % activity (200 h) at 37 °C. The stability of the enzyme in human serum (50 % activity maintained in 63 h) reveals high therapeutic potential with less dosage. The enzyme exhibited cytotoxicity to K562 blood cancer cell lines with IC50 of 0.37 U/mL without affecting the IEC-6 normal epithelial cell line. Due to the depletion of L-asparagine, K562 cells experience nutritional stress that results in the abruption of metabolic processes and eventually leads to apoptosis. Comparative studies on MCF-7 cells also revealed the same fate. Due to nutritional stress induced by L-ASNase treatment, mitochondrial membrane potential was lost, and reactive oxygen species were increased to 48 % (K562) and 21 % (MCF-7) as indicated by flow cytometric analysis. DAPI staining with prominent nuclear morphological changes visualized under the fluorescent microscope confirmed apoptosis in both cancer cells. Treatment increases pro-apoptotic Bax protein, and eventually, the cell cycle is arrested at the G2/M phase in both cell lines. Therefore, the current study paves the way for PCH199 L-ASNase to be considered a potential chemotherapeutic agent for treating acute lymphoblastic leukemia.

Genomics assisted characterization of plant growth-promoting and metabolite producing psychrotolerant Himalayan Chryseobacterium cucumeris PCH239.

Here, we report the first complete genome of a psychrotolerant and yellow-pigmented rhizobacteria Chryseobacterium cucumeris PCH239. It was obtained from the rhizospheric soil of the Himalayan plant Bergenia ciliata. The genome consists of a single contig (5.098 Mb), 36.3% G + C content, and 4899 genes. The cold adaptation, stress response, and DNA repair genes promote survivability in a high-altitude environment. PCH239 grows in temperature (10-37 °C), pH (6.0-8.0), and NaCl (2.0%). The genome derived plant growth-promoting activities of siderophore production (siderophore units 53 ± 0.6), phosphate metabolism (PSI 5.0 ± 0.8), protease, indole acetic acid production (17.3 ± 0.5 µg/ml), and ammonia (2.89 ± 0.4 µmoles) were experimentally validated. Interestingly, PCH239 treatment of Arabidopsis seeds significantly enhances germination, primary, and hairy root growth. In contrast, Vigna radiata and Cicer arietinum seeds had healthy radicle and plumule elongation, suggesting varied plant growth-promotion effects. Our findings suggested the potential of PCH239 as a bio-fertilizer and biocontrol agent in the challenging conditions of cold and hilly regions.

Depolymerization of lignin using laccase from Bacillus sp. PCH94 for production of valuable chemicals: A sustainable approach for lignin valorization.

Lignin is the most abundant aromatic polymer in nature, and its depolymerization offers excellent opportunities to develop renewable aromatic chemicals. In the present study, Bacillus sp. PCH94 was investigated for laccase production and lignin depolymerization. Maximum production of laccase enzyme was achieved within 6.0 h at 50 °C on a natural lignocellulosic substrate. Furthermore, Bacillus sp. PCH94 was used to bioconvert lignin dimeric and polymeric substrates, validated using FT-IR, NMR (1H, 13C), and LCMS. Genome mining of Bacillus sp. PCH94 revealed laccase gene (lacBl) as multicopper oxidase (spore coat CotA). Further, lacBl from Bacillus sp. PCH94 was cloned, expressed, and kinetically characterized. LacBl enzyme showed activity for substrates ABTS (40.64 IU/mg), guaiacol (5.43 IU/mg), and DMP (11.93 IU/mg). The LacBl was active in higher temperatures (10 to 100 °C) and showed a half-life of 36 and 27 h at 50 and 60 °C, respectively. The purified LacBl was able to depolymerize kraft lignin into valuable products (ferulic acid and acetovanillone), which have applications in the pharmaceutical and food industries. Overall, the current study demonstrated the role of bacterial laccase in the depolymerization of lignin and opened a promising prospect for the green production of valuable compounds from recalcitrant lignin.

Biochemical characterization of extremozyme L-asparaginase from Pseudomonas sp. PCH199 for therapeutics.

L-asparaginase (L-ASNase) from microbial sources is a commercially vital enzyme to treat acute lymphoblastic leukemia. However, the side effects associated with the commercial formulations of L-ASNases intrigued to explore for efficient and desired pharmacological enzymatic features. Here, we report the biochemical and cytotoxic evaluation of periplasmic L-ASNase of Pseudomonas sp. PCH199 isolated from the soil of Betula utilis, the Himalayan birch. L-ASNase production from wild-type PCH199 was enhanced by 2.2-fold using the Response Surface Methodology (RSM). Increased production of periplasmic L-ASNase was obtained using an optimized osmotic shock method followed by its purification. The purified L-ASNase was a monomer of 37.0 kDa with optimum activity at pH 8.5 and 60 ℃. It also showed thermostability retaining 100.0% (200 min) and 90.0% (70 min) of the activity at 37 and 50 ℃, respectively. The Km and Vmax values of the purified enzyme were 0.164 ± 0.009 mM and 54.78 ± 0.4 U/mg, respectively. L-ASNase was cytotoxic to the K562 blood cancer cell line (IC50 value 0.309 U/mL) within 24 h resulting in apoptotic nuclear morphological changes as examined by DAPI staining. Therefore, the dynamic functionality in a wide range of pH and temperature and stability of PCH199 L-ASNase at 37 ℃ with cytotoxic potential proves to be pharmaceutically important for therapeutic application.

In Vitro Characterisation Revealed Himalayan Dairy Kluyveromyces marxianus PCH397 as Potential Probiotic with Therapeutic Properties.

Recently, probiotics have gained much attention for their roles against various clinical conditions. Obesity is a worldwide health problem that triggers various other major complications like type 2 diabetes (T2D) and cancers, including colorectal cancer (CRC). Earlier, Kluyveromyces marxianus PCH397 isolated from yak (Bos grunniens) milk has been characterised by us for its efficient ß-galactosidase-producing ability, an important probiotic property. In the present study, yeast PCH397 has been evaluated for various parameters for its probiotic use. PCH397 exhibited tolerance to GI tract conditions (low pH, pancreatin, pepsin, and bile salts) with 78 to 99% survivability, possessed around 81% cell surface hydrophobicity, and 96% autoaggregation ability. The cell-free extract (CFE) and cell-free supernatant (CFS) from PCH397 improved insulin sensitisation by enhancing 2-NBDG (a glucose analogue) uptake in 3T3-L1 adipocytes, an approach useful in T2D treatment. They also exhibited lower intracellular lipid accumulation, triglyceride storage, and reactive oxygen species in differentiated adipocytes, indicating their anti-adipogenic ability. Also, CFE and intact cells (ICs) exhibited 73.33 ± 1.11% and 34.88 ± 2.80% DPPH radical scavenging activity, respectively. Furthermore, CFS showed a cytotoxic effect on SW-480 colorectal cancer (CRC) cells and induced the cell cycle phase arrest after 24 h of treatment. In conclusion, these results demonstrate that K. marxianus PCH397 could be used as a potential probiotic yeast and presents a therapeutic potential against obesity, T2D, and colon cancer.

Acrylamide mitigation in foods using recombinant L-asparaginase: An extremozyme from Himalayan Pseudomonas sp. PCH182.

Acrylamide has received worldwide attention due to its existence in commonly consumed foods. L-asparaginase reduces acrylamide formation in foods by hydrolyzing available L-asparagine. Herein, L-asparaginase (Ps-ASNase II) of Pseudomonas sp. PCH182 was expressed in Escherichia coli (E. coli), purified, and evaluated for acrylamide reduction in food samples. The monomeric 37 kDa Ps-ASNase II protein was purified to homogeneity with a 70 % yield. The enzyme was active at a wide pH range (5.0-11.0) and temperature (10-80 °C) with optimum activity at 45 °C in 50 mM Tris-HCl (pH 8.5) after 10 min. The Km and Vmax for L-asparagine were 0.52 ± 0.06 mM and 42.55 ± 4.0 U/mg, respectively. Also, the half-life and Kd value of the enzyme at 37 °C was 458 min and 1.51 × 10-3/min, suggesting its higher stability. Consistently, the enzyme retained 62 % residual activity after 60 days of storage at 4 °C. The Ps-ASNase II enzyme (5 U/mL) treatment of raw potato chips resulted in 90 % asparagine hydrolysis exhibiting high efficiency. Ps-ASNase II (5 U/mL) treated potato chips significantly reduced acrylamide content by 73 % at 37 °C within 24 min compared to untreated controls. Collectively, these findings verified Ps-ASNase's effectiveness and capability to lower acrylamide formation in fried potato chips without altering the food product's nutritional profile.

A thermo-alkali stable and detergent compatible processive ß-1,4-glucanase from Himalayan Bacillus sp. PCH94.

Present study reports a novel and robust GH9 processive endoglucanase ß-1,4-glucanase from Bacillus sp. PCH94 (EGaseBL) with thermo-alkali stable properties. The EGaseBL gene was cloned in pET-28b(+) and expressed in Escherichia coli BL21(DE3) cells. The recombinant protein was purified 94-fold with a yield of 67.8%. The biochemical characterization revealed an active enzyme at a wide pH (4.0-10.0) and temperature (4-100°C). It showed a Km and Vmax of 1.10 mg/ml and 208.24 IU/mg, respectively, using ß-glucan as a substrate. The EGaseBL showed dual activities for endoglucanase (134.17 IU/mg) and exoglucanase (28.76 IU/mg), assayed using substrates ß-glucan and Avicel, respectively. The enzyme is highly stable in neutral and alkaline pH and showed a half-life of 11.29 h, and 8.31 h in pH 7.0 and 9.0, respectively. The enzyme is also compatible with commercial detergents (Tide, Surf, Ghadi, Raj, and Healing tree) of the Indian market and retained > 85% enzyme activity. Concisely, robustness, extreme functionality, and detergent compatibility endorse EGaseBL as a potential bioresource for the detergent industry, in addition to its implications for the bioethanol industry. Highlights - Cloning, expression, and purification of putative novel GH9 family ß-1,4-glucanase.- Processive endoglucanase with CBM3 domain and bi-functional (endo/exo) activity.- Broad pH-temperature active and stable enzyme.- Compatible with commercial detergent powders.

Rahnella sikkimica sp. nov., a novel cold-tolerant bacterium isolated from the glacier of Sikkim Himalaya with plant growth-promoting properties.

The current study describes a novel species with the strain name ERMR1:05T isolated from the forefield soil of East Rathong Glacier in West Sikkim Himalaya (India). The isolate was facultatively anaerobic, gram-stain negative, non-spore-forming, rod-shaped, and oxidase negative. Whole-genome-based bacterial core gene phylogenetic analysis placed the strain in the genus Rahnella, well separated from Rouxiella spp. The digital DNA-DNA hybridisation and average nucleotide identity values between strain ERMR1:05T and other members of genus Rahnella were below the proposed thresholds for the species delineation. Based on these results, a new species, Rahnella sikkimica sp. nov., is proposed with strain ERMR1:05T (CIP 111636T, MTCC 12598T) as the type strain. The bacterium showed upregulation of cold-stress genes in cold conditions. Additionally, the genome analysis of the bacterium showed the presence of plant growth-promotion factors suggesting its role in crop improvement in cold hilly regions.

Microbial pigments: Learning from Himalayan perspective to industrial applications.

Pigments are an essential part of life on earth, ranging from microbes to plants and humans. The physiological and environmental cues induce microbes to produce a broad spectrum of pigments, giving them adaptation and survival advantages. Microbial pigments are of great interest due to their natural origin, diverse biological activities, and wide applications in the food, pharmaceutical, cosmetics, and textile industries. Despite noticeable research on pigment-producing microbes, commercial successes are scarce, primarily from higher, remote, and inaccessible Himalayan niches. Therefore, substantial bioprospection integrated with advanced biotechnological strategies is required to commercialize microbial pigments successfully. The current review elaborates on pigment-producing microbes from a Himalayan perspective, offering tremendous opportunities for industrial applications. Additionally, it illustrates the ecological significance of microbial pigments and emphasizes the current status and prospects of microbial pigments production above the test tube scale.

Multiple Adaptive Strategies of Himalayan Iodobacter sp. PCH194 to High-Altitude Stresses.

Bacterial adaption to the multiple stressed environments of high-altitude niches in the Himalayas is intriguing and is of considerable interest to biotechnologists. Previously, we studied the culturable and unculturable metagenome microbial diversity from glacial and kettle lakes in the Western Himalayas. In this study, we explored the adaptive strategies of a unique Himalayan eurypsychrophile Iodobacter sp. PCH194, which can synthesize polyhydroxybutyrate (PHB) and violacein pigment. Whole-genome sequencing and analysis of Iodobacter sp. PCH194 (4.58 Mb chromosome and three plasmids) revealed genetic traits associated with adaptive strategies for cold/freeze, nutritional fluctuation, defense against UV, acidic pH, and the kettle lake's competitive environment. Differential proteome analysis suggested the adaptive role of chaperones, ribonucleases, secretion systems, and antifreeze proteins under cold stress. Antifreeze activity inhibiting the ice recrystallization at -9°C demonstrated the bacterium's survival at subzero temperature. The bacterium stores carbon in the form of PHB under stress conditions responding to nutritional fluctuations. However, violacein pigment protects the cells from UV radiation. Concisely, genomic, proteomic, and physiological studies revealed the multiple adaptive strategies of Himalayan Iodobacter to survive the high-altitude stresses.

Molecular cloning, characterization, and in-silico analysis of l-asparaginase from Himalayan Pseudomonas sp. PCH44.

l-Asparaginase (l-ASNase) is a key enzyme used to treat acute lymphoblastic leukemia, a childhood blood cancer. Here, we report on the characterization of a recombinant l-ASNase (Ps44-asn II) from Pseudomonas sp. PCH44. The gene was identified from its genome, cloned, and overexpressed in the host Escherichia coli (E. coli). The recombinant l-ASNase (Ps44-ASNase II) was purified with a monomer size of 37.0 kDa and a homotetrameric size of 148.0 kDa. The purified Ps44-ASNase II exhibited optimum activity of 40.84 U/mg in Tris-HCl buffer (50 mM, pH 8.5) at 45 °C for 15 min. It retained 76.53% of enzyme activity at 45 °C after 120 min of incubation. The half-life and K d values were 600 min and 1.10 × 10-3 min-1, respectively, at 45 °C. The kinetic constants values K m and V max were 0.56, 0.728 mM, and 29.41, 50.12 U/mg for l-asparagine and l-glutamine, respectively. However, k cat for l-glutamine is more (30.91 s-1) than l-asparagine (18.06 s-1), suggesting that enzymes act more efficiently on l-glutamine than l-asparagine. The docking analysis of l-asparagine and l-glutamine with active site residues of the enzyme revealed a molecular basis for high l-glutaminase (L-GLNase) activity and provided insights into the role of key amino acid residues in the preferential enzymatic activities. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03224-0.

Polyphenolic Profiling, Antioxidant, and Antimicrobial Activities Revealed the Quality and Adaptive Behavior of Viola Species, a Dietary Spice in the Himalayas.

BACKGROUND: Himalayan Viola species (Banksha) are traditionally important herbs with versatile therapeutic benefits such as antitussive, analgesic, antipyretic, antimalarial, anti-inflammatory, and anticancerous ones. The current investigation was focused on exploring polyphenolic profiles, antioxidant, and antimicrobial potentials of wild viola species at 15 gradient locations (375-1829 m). METHODS: Morphological, physiochemical, and proximate analyses were carried out as per WHO guidelines for plant drug standardization. Total polyphenolic and flavonoid content were carried out using gallic acid and rutin equivalent. UPLC-DAD was used to profile the targeted polyphenols (gallic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, rutin, quercetin, luteolin, caffeic acid, and epicatechin). Similarly, all samples were screened for antioxidant and antimicrobial activity. Statistical analysis was used to correlate polyphenolic and targeted activities to assess Viola species adaptation behavior patterns. RESULTS: Viola canescens (V. canescens) and Viola pilosa (V. pilosa) were found abundantly at their respective sites. Among flowers and leaves, flowers of V. canescens and V. pilosa showed higher total polyphenolic and flavonoid content (51.4 ± 1.13 mg GAE/g and 65.05 ± 0.85 mg RE/g, and 33.26 ± 0.62 mg GAE/g and 36.10 ± 1.41 mg RE/g, respectively). Furthermore, UPLC-DAD showed the uppermost content of p-coumaric acid in flowers and ferulic acid in leaves, while rutin was significant in both the tissues. CONCLUSIONS: The adaptive behavior of Viola species showed variability in morphological characters with the altitudes, while targeted polyphenols and activities were significant at mid-altitudes. This research helps in the selection of right chemotype for agrotechnological interventions and the development of nutraceutical products.

Green bioprocess for degradation of synthetic dyes mixture using consortium of laccase-producing bacteria from Himalayan niches.

Microbial remediation of synthetic dyes from industrial effluents offers a sustainable and eco-friendly alternative. Herein, laccase-producing bacteria were isolated from decaying wood niches in the Himalayan region. A bacterial consortium (BC-I) was developed to decolorize synthetic dyes cocktail of three major groups (azo, anthraquinone, and triphenylmethane). BC-I consisted of Klebsiella sp. PCH427, Enterobacter sp. PCH428, and Pseudomonas sp. PCH429 can decolorize 77% of 240 mg/L dyes cocktail in 44 h at 37 °C. BC-I works under wide pH (4.0-10.0), a high salt concentration (NaCl, 10%), and low nutrients. Further, FT-IR and LC-MS validated the dyes cocktail degradation and identified the degraded products. Additionally, phytotoxicity analysis of BC-I treated dyes cocktail significantly reduced the toxicity to Vigna radiata and Cicer arietinum compared to untreated dyes cocktail. The present study has simulated environmental challenges of acidic, alkaline, and saline industrial dyes effluents, which are significant to bioremediation.

Metagenomic insights into Himalayan glacial and kettle lake sediments revealed microbial community structure, function, and stress adaptation strategies.

Glacial and kettle lakes in the high-altitude Himalayas are unique habitats with significant scope for microbial ecology. The present study provides insights into bacterial community structure and function of the sediments of two high-altitude lakes using 16S amplicon and whole-genome shotgun (WGS) metagenomics. Microbial communities in the sediments of Parvati kund (glacial lake) and Bhoot ground (kettle lake) majorly consist of bacteria and a small fraction of archaea and eukaryota. The bacterial population has an abundance of phyla Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Firmicutes, and Verrucomicrobia. Despite the common phyla, the sediments from each lake have a distinct distribution of bacterial and archaeal taxa. The analysis of the WGS metagenomes at the functional level provides a broad picture of microbial community metabolism of key elements and suggested chemotrophs as the major primary producers. In addition, the findings also revealed that polyhydroxyalkanoates (PHA) are a crucial stress adaptation molecule. The abundance of PHA metabolism in Alpha- and Betaproteobacteria and less representation in other bacterial and archaeal classes in both metagenomes was disclosed. The metagenomic insights provided an incisive view of the microbiome from Himalayan lake's sediments. It has also opened the scope for further bioprospection from virgin Himalayan niches.

The first draft genome of Picrorhiza kurrooa, an endangered medicinal herb from Himalayas.

Picrorhiza kurrooa is an endangered medicinal herb which is distributed across the Himalayan region at an altitude between 3000-5000 m above mean sea level. The medicinal properties of P. kurrooa are attributed to monoterpenoid picrosides present in leaf, rhizome and root of the plant. However, no genomic information is currently available for P. kurrooa, which limits our understanding about its molecular systems and associated responses. The present study brings the first assembled draft genome of P. kurrooa by using 227 Gb of raw data generated by Illumina and PacBio RS II sequencing platforms. The assembled genome has a size of n = ~ 1.7 Gb with 12,924 scaffolds. Four pronged assembly quality validations studies, including experimentally reported ESTs mapping and directed sequencing of the assembled contigs, confirmed high reliability of the assembly. About 76% of the genome is covered by complex repeats alone. Annotation revealed 24,798 protein coding and 9789 non-coding genes. Using the assembled genome, a total of 710 miRNAs were discovered, many of which were found responsible for molecular response against temperature changes. The miRNAs and targets were validated experimentally. The availability of draft genome sequence will aid in genetic improvement and conservation of P. kurrooa. Also, this study provided an efficient approach for assembling complex genomes while dealing with repeats when regular assemblers failed to progress due to repeats.

A New Extracellular ß-Galactosidase Producing Kluyveromyces sp. PCH397 from Yak Milk and Its Applications for Lactose Hydrolysis and Prebiotics Synthesis.

ß-Galactosidase is a crucial glycoside hydrolase enzyme with potential applications in the dairy, food, and pharmaceutical industries. The enzyme is produced in the intracellular environment by bacteria and yeast. The present study reports yeast Kluyveromyces sp. PCH397 isolated from yak milk, which has displayed extracellular ß-galactosidase activity in cell-free supernatant through the growth phase. To investigate further, cell counting and methylene blue staining of culture collected at different growth stages were performed and suggested for possible autolysis or cell lysis, thereby releasing enzymes into the extracellular medium. The maximum enzyme production (9.94 ± 2.53U/ml) was achieved at 37 °C in a modified deMan, Rogosa, and Sharpe (MRS) medium supplemented with lactose (1.5%) as a carbon source. The enzyme showed activity at a wide temperature range (4-50 °C), maximum at 50 °C in neutral pH (7.0). In addition to the hydrolysis of lactose (5.0%), crude ß-galactosidase also synthesized vital prebiotics (i.e., lactulose and galacto-oligosaccharides (GOS)). Additionally, ß-fructofuranosidase (FFase) activity in the culture supernatant ensued the synthesis of a significant prebiotic, fructo-oligosaccharides (FOS). Hence, the unique features such as extracellular enzymes production, efficient lactose hydrolysis, and broad temperature functionality by yeast isolate PCH397 are of industrial relevance. In conclusion, the present study unrevealed for the first time, extracellular production of ß-galactosidase from a new yeast source and its applications in milk lactose hydrolysis and synthesis of valuable prebiotics of industrial importance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00955-1.

A thermostable Fe/Mn SOD of Geobacillus sp. PCH100 isolated from glacial soil of Indian trans-Himalaya exhibits activity in the presence of common inhibitors.

Superoxide dismutases are the enzymes involved in dismutation of superoxide radicals into oxygen and hydrogen peroxide. The present work reports a thermostable Fe/Mn SOD of Geobacillus sp. strain PCH100 (GsSOD) isolated from glacial soil. Purified recombinant GsSOD is a dimeric protein of ~57 kDa that exhibited highest activity at a temperature of 10 °C and pH of 7.8. Maximum enzyme velocity and Michaelis constant of the GsSOD were 1098.90 units/mg and 0.62 µM, respectively. At 80 °C, thermal inactivation rate constant and half-life of GsSOD were 3.33 × 10-3 min-1 and 208 min, respectively. Interestingly, GsSOD tolerated a temperature of 100 °C and 130 °C up to 15 min and 5 min, respectively. Circular dichroism and differential scanning calorimetry confirmed thermostable nature of GsSOD. Apoenzyme of GsSOD regained enzymatic activity in the presence of Fe2+ and Mn2+ as metal ion cofactors. GsSOD was stable under varying concentrations of chemicals, namely ethylenediaminetetraacetic acid, potassium cyanide, hydrogen peroxide, chloroform-ethanol, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, Tween-20, Triton X-100, urea, and guanidine hydrochloride. The enzyme exhibited >70% activity in presence of 10 mM metal ions. Owing to its thermostable nature and resistance to chemical inhibitors, GsSOD is a potential enzyme for industrial applications.

Bioprocess for co-production of polyhydroxybutyrate and violacein using Himalayan bacterium Iodobacter sp. PCH194.

The co-production of industrially relevant biopolymers/biomolecules from microbes is of biotechnological importance. Herein, a unique bacterium, Iodobacter sp. PCH 194 from the kettle lake at Sach Pass in western Indian Himalaya was identified. It co-produces biopolymer polyhydroxyalkanoates (PHA) and biomolecule (violacein pigment). Statistical optimization yielded dual products in the medium augmented with glucose (4.0% w/v) and tryptone (0.5% w/v) as carbon and nitrogen sources, respectively. The purified PHA was polyhydroxybutyrate (PHB), and pigment constitutes of violacein (50-60%) and deoxyviolacein (40-50%). A bench-scale bioprocess in 22.0 L fermentor with 20% dissolved O2 supply produced PHB (11.0 ± 1.0 g/L, 58% of dry cell mass) and violacein pigment (1.5 ± 0.08 g/L). PHB obtained was used for the preparation of bioplastic film. Violacein pigment experimentally validated for anticancerous and antimicrobial activities. In summary, a commercially implied bioprocess developed for the co-production of PHB and violacein pigment using the Himalayan bacterium.

Biodegradation of organophosphorus pesticide chlorpyrifos by Sphingobacterium sp. C1B, a psychrotolerant bacterium isolated from apple orchard in Himachal Pradesh of India.

A psychrotolerant Sphingobacterium sp. was isolated from the apple orchard situated in the Kufri region of Shimla, Himachal Pradesh, India using an enrichment culture technique having chlorpyrifos (CP) as the sole source of carbon and energy. Based on biochemical characterization and 16S rRNA analysis, the strain was identified as Sphingobacterium sp. C1B. The bacterium C1B was able to degrade chlorpyrifos ≥ 42 ppm and ≥ 36 ppm within 14 days at 20 °C and 15 °C, respectively. The strain was also able to degrade chlorpyrifos ≤ 35 ppm at 28 °C within 14 days. The enzyme organophosphorus hydrolase might be responsible for the initial degradation of CP by the strain C1B. Based on the HPLC and GCMS analysis, a probable degradation pathway has been proposed, which followed the path from chlorpyrifos to 3,5,6-trichloro-2-pyridinol to benzene, 1,3-bis (1,1-dimethylethyl) and then entered into the TCA cycle. Our current study revealed that the bacterium C1B was found to be a useful strain for the degradation of pesticide chlorpyrifos in the cold climatic environment.