A novel thermotolerant L-rhamnose isomerase variant for biocatalytic conversion of D-allulose to D-allose.

A novel L-rhamnose isomerase was identified and cloned from an extreme-temperature aquatic habitat metagenome. The deduced amino acid sequence homology suggested the possible source of this metagenomic sequence to be Chloroflexus islandicus. The gene expression was performed in a heterologous host, Escherichia coli, and the recombinant protein L-rhamnose isomerase (L-RIM) was extracted and purified. The catalytic function of L-RIM was characterized for D-allulose to D-allose bioconversion. D-Allose is a sweet, rare sugar molecule with anti-tumour, anti-hypertensive, cryoprotective, and antioxidative properties. The characterization experiments showed L-RIM to be a Co++- or Mn++-dependent metalloenzyme. L-RIM was remarkably active (~ 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges. Optimal L-RIM activity with D-allulose as the substrate occurred at pH 7.0 and 75 °C. The enzyme was found to be excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively. L-RIM catalysis conducted at slightly acidic pH of 6.0 and 70 °C achieved biosynthesis of about 30 g L-1 from 100 g L-1 D-allulose in 3 h. KEY POINTS: • The present study explored an extreme temperature metagenome to identify a novel gene that encodes a thermostable l-rhamnose isomerase (L-RIM) • L-RIM exhibits substantial (80% or more) activity in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges • L-RIM is excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively.

Lactic acid bacteria in the functional food industry: biotechnological properties and potential applications.

With the growing demand for functional foods having better nutraceutical properties, lactic acid bacteria (LAB) has become an important industrial microorganism. LAB play a significant role in the functional food industry by exhibiting probiotic properties and has the ability to produce various biologically active metabolites such as γ-aminobutyric acid (GABA), exopolysaccharides (EPSs), conjugated linoleic acid (CLA), bacteriocins, reuterin and reutericyclin, which provides enhanced nutraceutical properties to the final food products. LAB are also known to produce several specific enzymes essential for producing substrate-derived bioactive compounds, such as polyphenols, bioactive peptides, inulin-type fructans and ß-glucans, fatty acids, and polyols. These compounds exhibit many health benefits, including better mineral absorption, oxidative stress protection, blood glucose and cholesterol-lowering properties, prevention of gastrointestinal tract infections and improved cardiovascular function. Further, metabolically engineered LAB have been widely used for the nutritive enhancement of different food products and the application of CRISPR-Cas9 holds tremendous potential for the engineering of food cultures. This review provides an overview of the use of LAB as probiotics, its application in producing fermented foods and nutraceutical products, and its health benefits on the host.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus.

BACKGROUND: Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. METHODS AND RESULTS: The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CONCLUSIONS: CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Metagenomic views on taxonomic and functional profiles of the Himalayan Tsomgo cold lake and unveiling its deterzome potential.

Cold habitat is considered a potential source for detergent industry enzymes. This study aims at the metagenomic investigation of Tsomgo lake for taxonomic and functional annotation, unveiling the deterzome potential of the residing microbiota at this site. The present investigation revealed molecular profiling of microbial community structure and functional potential of the high-altitude Tsomgo lake samples of two different temperatures, harvested during March and August. Bacteria were found to be the most dominant phyla, with traces of genomic pieces of evidence belonging to archaea, viruses, and eukaryotes. Proteobacteria and Actinobacteria were noted to be the most abundant bacterial phyla in the cold lake. In-depth metagenomic investigation of the cold aquatic habitat revealed novel genes encoding detergent enzymes, amylase, protease, and lipase. Further, metagenome-assembled genomes (MAGs) belonging to the psychrophilic bacterium, Arthrobacter alpinus, were constructed from the metagenomic data. The annotation depicted the presence of detergent enzymes and genes for low-temperature adaptation in Arthrobacter alpinus. Psychrophilic microbial isolates were screened for lipase, protease, and amylase activities to further strengthen the metagenomic findings. A novel strain of Acinetobacter sp. was identified with the dual enzymatic activity of protease and amylase. The bacterial isolates exhibited hydrolyzing activity at low temperatures. This metagenomic study divulged novel genomic resources for detergent industry enzymes, and the bacterial isolates secreting cold-active amylase, lipase, and protease enzymes. The findings manifest that Tsomgo lake is a potential bioresource of cold-active enzymes, vital for various industrial applications.

Cold-adaptive traits identified by comparative genomic analysis of a lipase-producing Pseudomonas sp. HS6 isolated from snow-covered soil of Sikkim Himalaya and molecular simulation of lipase for wide substrate specificity.

The genomic analysis of industrially important bacteria can help in understanding their capability to withstand extreme environments and shed light on their metabolic capabilities. The whole genome of a previously reported broad temperature active lipase-producing Pseudomonas sp. HS6, isolated from snow-covered soil of the Sikkim Himalayan Region, was analyzed to understand the capability of the bacterium to withstand cold temperatures and study its lipolytic nature. Pseudomonas sp. HS6 was found to be psychrotolerant with an optimal growth temperature ranging between 25 and 30 °C, with the ability to grow at 5 °C. The genome harbours various cold-adaptation genes, such as cold-shock proteins, fatty acid alteration, and cold stress-tolerance genes, supporting the psychrotolerant nature of the organism. The comparative analysis of Pseudomonas sp. HS6 genome showed the presence of amino acid substitutions in genes that favor efficient functioning and flexibility at cold temperatures. Genome mining revealed the presence of four triacylglycerol lipases, among which the putative lipase 3 was highly similar to the broad temperature-active lipase purified and characterized in our previous study. In silico studies of putative lipase 3 revealed broad substrate specificity with partial and no inhibition of the enzyme activity in the presence of PMSF and orlistat. The presence of genes associated with cold adaptations and true lipases with activity at broad temperature and substrate specificity in the genome of Pseudomonas sp. HS6 makes this bacterium a suitable candidate for industrial applications.

An acid-tolerant and cold-active ß-galactosidase potentially suitable to process milk and whey samples.

A novel ß-galactosidase gene (galM) was cloned from an aquatic habitat metagenome. The analysis of its translated sequence (GalM) revealed its phylogenetic closeness towards Verrucomicrobia sp. The sequence comparison and homology structure analysis designated it a member of GH42 family. The three-dimensional homology model of GalM depicted a typical (ß/α)8 TIM-barrel containing the catalytic core. The gene (galM) was expressed in a heterologous host, Escherichia coli, and the purified protein (GalM) was subjected to biochemical characterization. It displayed ß-galactosidase activity in a wide range of pH (2.0 to 9.0) and temperature (4 to 60 °C). The heat exposed protein showed considerable stability at 40 and 50 °C, with the half-life of about 100 h and 35 h, respectively. The presence of Na, Mg, K, Ca, and Mn metals was favorable to the catalytic efficiency of GalM, which is a desirable catalytic feature, as these metals exist in milk. It showed remarkable tolerance of glucose and galactose in the reaction. Furthermore, GalM discerned transglycosylation activity that is useful in galacto-oligosaccharides' production. These biochemical properties specify the suitability of this biocatalyst for milk and whey processing applications. KEY POINTS: • A novel ß-galactosidase gene was identified and characterized from an aquatic habitat. • It was active in extreme acidic to mild alkaline pH and at cold to moderate temperatures. • The ß-galactosidase was capable to hydrolyze lactose in milk and whey.

Molecular dissemination of emerging antibiotic, biocide, and metal co-resistomes in the Himalayan hot springs.

Growing resistance among microbial communities against antimicrobial compounds, especially antibiotics, is a significant threat to living beings. With increasing antibiotic resistance in human pathogens, it is necessary to examine the habitats having community interests. In the present study, a metagenomic approach has been employed to understand the causes, dissemination, and effects of antibiotic, metal, and biocide resistomes on the microbial ecology of three hot springs, Borong, Lingdem, and Yumthang, located at different altitudes of the Sikkim Himalaya. The taxonomic assessment of these hot springs depicted the predominance of mesophilic organisms, mainly belonging to the phylum Proteobacteria. The enriched microbial metabolism assosiated with energy, cellular processes, adaptation to diverse environments, and defence were deciphered in the metagenomes. The genes representing resistance to semisynthetic antibiotics, e.g., aminoglycosides, fluoroquinolones, fosfomycin, vancomycin, trimethoprim, tetracycline, streptomycin, beta-lactams, multidrug resistance, and biocides such as triclosan, hydrogen peroxide, acriflavin, were abundantly present. Various genes attributing resistance to copper, arsenic, iron, and mercury in metal resistome were detected. Relative abundance, correlation, and genome mapping of metagenome-assembled genomes indicated the co-evolution of antibiotic and metal resistance in predicted novel species belonging to Vogesella, Thiobacillus, and Tepidimona genera. The metagenomic findings were further validated with isolation of microbial cultures, exhibiting resistance against antibiotics and heavy metals, from the hot spring water samples. The study furthers our understanding about the molecular basis of co-resistomes in the ceological niches and their possible impact on the environment.

Biochemical characterization of a novel thermo-halo-tolerant GH5 endoglucanase from a thermal spring metagenome.

A novel endoglucanase gene, celM , was cloned from a thermal spring metagenome. The gene was expressed in Escherichia coli, and the protein was extracted and purified. The protein catalyzed the hydrolysis of amorphous cellulose in a wide range of temperatures, 30-95°C, with optimal activity at 80°C. It was able to tolerate high temperature (80°C) with a half-life of 8 h. Its activity was eminent in a wide pH range of 3.0-11.0, with the highest activity at pH 6.0. The enzyme was tested for halostability. Any significant loss was not recorded in the activity of CelM after the exposure to salinity (3 M NaCl) for 30 days. Furthermore, CelM displayed a substantial resistance toward metal ions, denaturant, reducing agent, organic solvent, and non-ionic surfactants. The amorphous cellulose, treated with CelM , was randomly cleaved, generating cello-oligosaccharides of 2-5 degree of polymerization. Furthermore, CelM was demonstrated to catalyze the hydrolysis of cellulose fraction in the delignified biomass samples, for example, sweet sorghum bagasse, rice straw, and corncob, into cello-oligosaccharides. Given that CelM is a thermo-halo-tolerant GH5 endoglucanase, with resistance to detergents and organic solvent, the biocatalyst could be of potential usefulness for a variety of industrial applications.

D-Allulose 3-epimerase of Bacillus sp. origin manifests profuse heat-stability and noteworthy potential of D-fructose epimerization.

BACKGROUND: D-Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. D-Allulose 3-epimerase family enzymes catalyze biosynthesis of D-allulose via epimerization of D-fructose. RESULTS: A novel D-allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0-11.0. DaeB was able to catalyze D-fructose to D-allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number (kcat 367 s- 1). The coupling of DaeB treatment and yeast fermentation of 700 g L- 1 D-fructose solution yielded approximately 200 g L- 1 D-allulose, and 214 g L- 1 ethanol. CONCLUSIONS: The novel D-allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of D-allulose.

Comparative genome analysis provides shreds of molecular evidence for reclassification of Leuconostoc mesenteroides MTCC 10508 as a strain of Leu. suionicum.

This study presents the whole-genome comparative analysis of a Leuconostoc sp. strain, previously documented as Leu. mesenteroides MTCC 10508. The ANI, dDDH, dot plot, and MAUVE analyses suggested its reclassification as a strain of Leu. suionicum. Functional annotation identified a total of 1971 genes, out of which, 265 genes were mapped to CAZymes, evincing its carbohydrate transforming capability. The genome comparison with 59 Leu. mesenteroides and Leu. suionicum strains generated the core and pan-genome profiles, divulging the unique genes in Leuconostoc sp. MTCC 10508. For the first time, this study reports the genes encoding alpha-xylosidase and copper oxidase in a strain of Leu. suionicum. The genetic information for any possible allergenic molecule could not be detected in the genome, advocating the safety of the strain. The present investigation provides the genomic evidence for reclassification of the Leuconostoc sp. strain and also promulgates the molecular insights into its metabolic potential.

Biotechnological approaches for the production of designer cheese with improved functionality.

Cheese is a product of ancient biotechnological practices, which has been revolutionized as a functional food product in many parts of the world. Bioactive compounds, such as peptides, polysaccharides, and fatty acids, have been identified in traditional cheese products, which demonstrate functional properties such as antihypertensive, antioxidant, immunomodulation, antidiabetic, and anticancer activities. Besides, cheese-making probiotic lactic acid bacteria (LAB) exert a positive impact on gut health, aiding in digestion, and improved nutrient absorption. Advancement in biotechnological research revealed the potential of metabolite production with prebiotics and bioactive functions in several strains of LAB, yeast, and filamentous fungi. The application of specific biocatalyst producing microbial strains enhances nutraceutical value, resulting in designer cheese products with multifarious health beneficial effects. This review summarizes the biotechnological approaches applied in designing cheese products with improved functional properties.

A Novel d-Allulose 3-Epimerase Gene from the Metagenome of a Thermal Aquatic Habitat and d-Allulose Production by Bacillus subtilis Whole-Cell Catalysis.

A novel d-allulose 3-epimerase gene (daeM) has been identified from the metagenomic resource of a hot-water reservoir. The enzyme epimerizes d-fructose into d-allulose, a functional sugar of rare abundance in nature. The metagenomic DNA fragment was cloned and expressed in Escherichia coli The purified recombinant protein (DaeM) was found to be metal dependent (Co2+ or Mn2+). It displayed the maximal levels of catalytic activity in a pH range of 6 to 11 and a temperature range of 75°C to 80°C. The enzyme exhibited remarkably high thermal stability at 60°C and 70°C, with half-life values of 9,900 and 3,240 min, respectively. To the best of our knowledge, this is the highest thermal stability demonstrated by a d-allulose 3-epimerase that has been characterized to date. The enzymatic treatment of 700 mg·ml-1 d-fructose yielded about 217 mg·ml-1 d-allulose, under optimal condition. The catalytic product was purified, and its nuclear magnetic resonance (NMR) spectra were found to be indistinguishable from those of standard d-allulose. For biomolecule production, the whole-cell catalysis procedure avoids the tedious process of extraction and purification of enzyme and also offers better biocatalyst stability. Further, it is desirable to employ safe-grade microorganisms for the biosynthesis of a product. The daeM gene was expressed intracellularly in Bacillus subtilis A whole-cell catalysis reaction performed with a reaction volume of 1 liter at 60°C yielded approximately 196 g·liter-1 d-allulose from 700 g·liter-1 d-fructose. Further, the whole recombinant cells were able to biosynthesize d-allulose in apple juice, mixed fruit juice, and honey.IMPORTANCE d-Allulose is a noncaloric sugar substitute with antidiabetes and antiobesity potential. With several characteristics of physiological significance, d-allulose has wide-ranging applications in the food and pharmacology industries. The development of a thermostable biocatalyst is an objective of mainstream research aimed at achieving industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered a potential metagenomic resource of heat-tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of the Tattapani geothermal region, Chhattisgarh, India, discovering a novel d-allulose 3-epimerase gene, daeM, encoding an enzyme of high-level heat stability. The daeM gene was expressed in the microbial cells of a nonpathogenic and safe-grade species, B. subtilis, which was found to be capable of performing d-fructose to d-allulose interconversion via a whole-cell catalysis reaction. The results indicate that DaeM is a potential biocatalyst for commercial production of the rare sugar d-allulose. The study established that extreme environmental niches represent a genomic resource of functional sugar-related biocatalysts.

Metagenomics revealing molecular profiling of community structure and metabolic pathways in natural hot springs of the Sikkim Himalaya.

BACKGROUND: Himalaya is an ecologically pristine environment. The geo-tectonic activities have shaped various environmental niches with diverse microbial populations throughout the Himalayan biosphere region. Albeit, limited information is available in terms of molecular insights into the microbiome, including the uncultured microbes, of the Himalayan habitat. Hence, a vast majority of genomic resources are still under-explored from this region. Metagenome analysis has simplified the extensive in-depth exploration of diverse habitats. In the present study, the culture-independent whole metagenome sequencing methodology was employed for microbial diversity exploration and identification of genes involved in various metabolic pathways in two geothermal springs located at different altitudes in the Sikkim Himalaya. RESULTS: The two hot springs, Polok and Reshi, have distinct abiotic conditions. The average temperature of Polok and Reshi was recorded to be 62 °C and 43 °C, respectively. Both the aquatic habitats have alkaline geochemistry with pH in the range of 7-8. Community profile analysis revealed genomic evidence of plentiful bacteria, with a minute fraction of the archaeal population in hot water reservoirs of Polok and Reshi hot spring. Mesophilic microbes belonging to Proteobacteria and Firmicutes phyla were predominant at both the sites. Polok exhibited an extravagant representation of Chloroflexi, Deinococcus-Thermus, Aquificae, and Thermotogae. Metabolic potential analysis depicted orthologous genes associated with sulfur, nitrogen, and methane metabolism, contributed by the microflora in the hydrothermal system. The genomic information of many novel carbohydrate-transforming enzymes was deciphered in the metagenomic description. Further, the genomic capacity of antimicrobial biomolecules and antibiotic resistance were discerned. CONCLUSION: The study provided comprehensive molecular information about the microbial treasury as well as the metabolic features of the two geothermal sites. The thermal aquatic niches were found a potential bioresource of biocatalyst systems for biomass-processing. Overall, this study provides the whole metagenome based insights into the taxonomic and functional profiles of Polok and Reshi hot springs of the Sikkim Himalaya. The study generated a wealth of genomic data that can be explored for the discovery and characterization of novel genes encoding proteins of industrial importance.

Characterization of a novel xylanase from an extreme temperature hot spring metagenome for xylooligosaccharide production.

In this study, the metagenomic resource generated from an aquatic habitat of extreme temperature was screened for the identification of a novel xylanase, XynM1. Gene sequence analysis designated it as a member of glycoside hydrolase (GH) family 10. The metagenomic DNA fragment was cloned, expressed in Escherichia coli, and the purified protein was biochemically characterized. The optimum temperature and pH for the XynM1 xylanase were found to be at 80 °C and 7, respectively. It exhibited worthwhile pH stability by retaining about 70% activity in the range of pH 6 to 9 after the exposure for 12 h at 25 °C. Thermostability analysis established considerable heat tolerance in XynM1 protein at elevated temperatures, displaying about 50% residual activity after the exposure of 40 °C, 50 °C, 60 °C, and 70 °C for 20 h, 12 h, 6 h, and 1.5 h, respectively. The effects of additives such as metals, surfactants, and organic solvents were evaluated on the activity of XynM1. It was able to retain about 50% of its initial activity in the presence of NaCl concentration of 1 to 5 M. The novel xylanase was capable of hydrolyzing the hemicellulosic polymer, derived from diverse biomass sources, e.g., beechwood xylan, wheat arabinoxylan, corncob xylan, and sweet sorghum xylan. The XynM1-treated beechwood xylan manifested catalytic release of xylooligosaccharides (XOS) of 2-6 DP. The novel GH10 xylanase is a promising biocatalyst that could be ascribed for biomass conversion and production of prebiotic XOS biomolecules.

Coping strategies of women survivors of domestic violence residing with an abusive partner after registered complaint with the family counseling center at Alwar, India.

Coping is one of the most important and widely studied aspects of domestic violence (DV). Women adopt a wide variety of coping strategies to manage abuse, ranging from individual estrangement to institutional entitlements. The condition of women becomes complex when they seek institutional support while living with an abusive husband and his family under the common hearth. The present study aims to analyze the coping strategy of mediation between informal and formal justice while residing with an abusive husband and his family. Further, the study explores the coping strategy as an outcome of the contextual factor and associated psychological distress. It is a prospective intervention study with a 4-month span building awareness, counseling (individual, couple, and family counseling), and case-specific advice (safety plan, choice-making, and problem-solving). A baseline and endline assessment with SRQ-20 and in-depth interviews were carried out on 299 married women who had registered a complaint with a family counseling center (FCC); Mahila Suraksha Evam Salah Kendra (MSSK) in Alwar district, India. To understand the context and the coping strategy adopted by women, in-depth interviews were carried out. The results show that there is a differential impact of DV, psychological distress, and coping strategy based on contextual factor; women having an informal support system have a better result in coping; and intervention at the formal system resulted in improving coping strategy and simultaneously reducing psychological distress. As the mediation period is interminable and traumatic, the institutional support to women survivors of DV is an important policy alternative for improving survivors' well-being, especially in an unsupportive informal context.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium.

Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.

An integrated bio-process for production of functional biomolecules utilizing raw and by-products from dairy and sugarcane industries.

The study investigated an integrated bioprocessing of raw and by-products from sugarcane and dairy industries for production of non-digestible prebiotic and functional ingredients. The low-priced feedstock, whey, molasses, table sugar, jaggery, etc., were subjected to transglucosylation reactions catalyzed by dextransucrase from Leuconostoc mesenteroides MTCC 10508. HPLC analysis approximated production of about 11-14 g L-1 trisaccharide i.e. 2-α-D-glucopyranosyl-lactose (4-galactosyl-kojibiose) from the feedstock prepared from table sugar, jaggery, cane molasses and liquid whey, containing about 30 g L-1 sucrose and lactose each. The trisaccharide was hydrolysed into the prebiotic disaccharide, kojibiose, by employing recombinant ß-galactosidase from Escherichia coli. The enzyme ß-galactosidase achieved about 90% conversion of 2-α-D-glucopyranosyl-lactose into kojibiose. The D-fructose generated by catalytic reactions of dextransucrase was targeted for catalytic transformation into rare sugar, D-allulose (or D-psicose), by treating the samples with Smt3-D-psicose 3-epimerase. The catalytic reactions resulted in the conversion of ~ 25% D-fructose to D-allulose. These bioactive compounds are known to exert a plethora of benefits to human health, and therefore, are preferred ingredients for making functional foods.

De novo transcriptome analysis of rose-scented geranium provides insights into the metabolic specificity of terpene and tartaric acid biosynthesis.

BACKGROUND: Rose-scented geranium (Pelargonium sp.) is a perennial herb that produces a high value essential oil of fragrant significance due to the characteristic compositional blend of rose-oxide and acyclic monoterpenoids in foliage. Recently, the plant has also been shown to produce tartaric acid in leaf tissues. Rose-scented geranium represents top-tier cash crop in terms of economic returns and significance of the plant and plant products. However, there has hardly been any study on its metabolism and functional genomics, nor any genomic expression dataset resource is available in public domain. Therefore, to begin the gains in molecular understanding of specialized metabolic pathways of the plant, de novo sequencing of rose-scented geranium leaf transcriptome, transcript assembly, annotation, expression profiling as well as their validation were carried out. RESULTS: De novo transcriptome analysis resulted a total of 78,943 unique contigs (average length: 623 bp, and N50 length: 752 bp) from 15.44 million high quality raw reads. In silico functional annotation led to the identification of several putative genes representing terpene, ascorbic acid and tartaric acid biosynthetic pathways, hormone metabolism, and transcription factors. Additionally, a total of 6,040 simple sequence repeat (SSR) motifs were identified in 6.8% of the expressed transcripts. The highest frequency of SSR was of tri-nucleotides (50%). Further, transcriptome assembly was validated for randomly selected putative genes by standard PCR-based approach. In silico expression profile of assembled contigs were validated by real-time PCR analysis of selected transcripts. CONCLUSION: Being the first report on transcriptome analysis of rose-scented geranium the data sets and the leads and directions reflected in this investigation will serve as a foundation for pursuing and understanding molecular aspects of its biology, and specialized metabolic pathways, metabolic engineering, genetic diversity as well as molecular breeding.

Advanced Absorption Correction for 3D Elemental Images Applied to the Analysis of Pearl Millet Seeds Obtained with a Laboratory Confocal Micro X-ray Fluorescence Spectrometer.

We present a novel absorption correction approach for elemental distribution images obtained with a laboratory confocal micro X-ray fluorescence spectrometer. The procedure is suited especially for biological samples, as a constant dark matrix with a varying minor or trace element distribution is assumed. The constant absorption in the sample is extracted from depth dependent measurements. By using the concept of an effective excitation energy, depth-dependent, and element-specific excitation energy values are calculated. For each voxel of a full 3D measurement, a correction is performed taking into account the actual number of voxels in the excitation and detection path. As proof of concept, the embryonic region of pearl millet seeds is investigated. Data are measured from the top and bottom side, resulting in a good agreement after the application of the absorption correction procedure. The distribution of elemental micronutrients is compared in seeds of two pearl millet genotypes. The corrected images illustrate different localization patterns of the micronutrient elements in pearl millet seed tissues.