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.

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.

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.

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.

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.

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.

Optimized chromogenic dyes-based identification and quantitative evaluation of bacterial l-asparaginase with low/no glutaminase activity bioprospected from pristine niches in Indian trans-Himalaya.

Here, we report on the isolation of bacterial isolates from Himalayan niches, which produced extracellular l-asparaginase with low/no glutaminase activity. From the 235 isolates, 85 asparaginase positive bacterial isolates were identified by qualitative screening using optimized chromogenic dyes assay. Optimized concentration of different dyes revealed maximum color visualization in phenol red (0.003%). The diversity analysis of asparaginase positive isolates revealed that Proteobacteria (83%) are the most dominant, followed by Actinobacteria (12%), Firmicutes (3%), and Bacteriodetes (2%). Eleven isolates, which represented seven Pseudomonas species, one species each of the genus Arthrobacter, Janthinobacterium, Lelliottia, and Rahnella, were selected for further studies based on highest zone ratio and novel aspects for l-asparaginase production. Of these, five isolates, namely, Pseudomonas sp. PCH133, Pseudomonas sp. PCH146, Pseudomonas sp. PCH182, Rahnella sp. PCH162, and Arthrobacter sp. PCH138, produced l-asparaginase without glutaminase activity after 55 h of growth with the former isolate showing the highest l-asparaginase activity (1.67 U/ml). Interestingly, this is the first report of l-asparaginase production by members of the genera Janthinobacterium, Rahnella, and Lelliottia.

Antimicrobial Homoisoflavonoids from the Rhizomes of Polygonatum verticillatum.

Three homoisoflavonoids, including a new compound, 5,7-dihydroxy-3-(4-methoxybenzyl)-8-methyl chroman-4-one (1), together with two known compounds, 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-8-methylchroman-4-one (2) and 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-chroman-4-one (3), were isolated from the rhizomes of Polygonatum verticillatum (L.) All. (P. verticillatum). Isolated compounds were characterized on the basis of UV, FT-IR, ESI-MS, and 1D-, 2D-NMR data. Further, different extract fractions and pure compounds from Polygonatum verticillatum were screened for their antimicrobial potential. Among three pure compounds, compound 2 was found most potent with good zone of microbial growth inhibition as compared to the standards.