Bioprospecting potential of microbial communities in solid waste landfills for novel enzymes through metagenomic approach.

Landfills are repository for complex microbial diversity responsible for bio-degradation of solid waste. To elucidate this complexity, samples from three different landfill sites of North India (sample V: Bhalswa near Karnal byepass road, New Delhi, India; sample T: Chandigarh, India and sample S3: Una, H.P., India) were analyzed using metagenomic approach. Selected landfill sites had different geographical location, varied in waste composition, size of landfill and climate zone. For comparison, one sample from high altitude (sample J) having less human interference was taken in this study. The aim of this study was to explore microbial diversity of communities responsible for degradation of landfill. Samples were characterized by 16S rRNA gene sequencing. Data from three landfill sites showed abundance of phylum Proteobacteria while less contaminated sample from high altitude showed abundance of phylum Cholroflexi followed by phylum Proteobacteria. The most abundant genus was unknown suggesting that these landfills could be repository for various novel bacterial communities. Sample T was relatively more active in terms of microbial activity. It was relatively abundant in enzymes responsible for dioxin degradation, styrene degradation, steroid degradation, streptomycin biosynthesis, carbapenem biosynthesis, monobactam biosynthesis, furfural degradation pathways while sample J was predicted to be enriched in plant cell wall degrading enzymes. Co-occurrence analysis revealed presence of complex interaction networks between microbial assemblages responsible for bio-degradation of hydrocarbons. The data provides insights about synergetic interactions and functional interplay between bacterial communities in different landfill sites which could be further exploited to develop an effective bioremediation process.

Atmospheric cold plasma (ACP) treatment improved in-package shelf-life of strawberry fruit.

The aim of this study was to investigate the effect of atmospheric cold plasma (ACP) treatment on the microbial inactivation, physicochemical properties, and shelf-life of strawberry fruit with its extended in-package storage at room (25 °C) and refrigerated (4 °C) temperature. ACP treatment of 10, 15 and 30 min was studied on strawberry fruit using a dielectric barrier discharge (DBD) at 60 kV with an input voltage of 260 V at 50 Hz. The shelf-life of ACP treated strawberry was extended to 5 days at 25 °C and 9 days at 4 °C in sealed ACP package. However, non-treated packaged strawberry was degraded in 2 days. ACP treatment of 15 min resulted in 2 log reduction of microbial load and enhanced the concentration of chlorogenic acid, hyprin, phloretin, vanillin, gallic acid, 4-hydroxybenzaldehyde and rutin during in-package storage of 5 day (~ 120 h) at 25 °C with respect to control (p < 0.05). In addition, ACP treatment of 15 min at 60 kV was also found to increase the total phenolic content and antioxidant activity. However, total soluble solids, pH and moisture were not affected with ACP treatment (p > 0.05). Therefore, ACP treatment of 15 min with in-package storage of 5 days (~ 120 h) was found to be advantageous for increasing the shelf-life and functional quality of strawberry fruit.

Tomato processing byproduct valorization in bread and muffin: improvement in physicochemical properties and shelf life stability.

Tomato processing industry generates huge waste like tomato skin, seed, and pulp which creates environmental issues. Since tomato pomace contains bioactive compounds and pigments, present study was conducted to investigate the effect of tomato pomace addition on physicochemical characteristics and shelf-life stability of the developed bread and muffin. Refined flour was partially substituted with 35 and 40% tomato pomace in bread and muffin, respectively. Tomato pomace addition in bread and muffin was observed to significantly (p < 0.05) increase the dietary fiber, vitamin C, antioxidant activity and minerals (Na, K, Mg, Ca, Fe). The color parameters for bread and muffins were quantified in terms of L* (lightness), a* (redness/greenness) and b* (yellowness/blueness). There was an increase in a* and b*, while L* values decreased. Tomato based bread and muffin were found to possess softer texture as compared to control products. Microbial study has depicted the enhanced shelf-life of tomato based bread and muffin. Shelf life of preservative added tomato based bread was 8 days and muffins were 12 days. Tomato pomace could be a very useful commodity for incorporation into bread and muffin to have a complete nutritive food product.

Anthocyanins enriched purple tea exhibits antioxidant, immunostimulatory and anticancer activities.

Purple coloured tea shoot clones have gained interest due to high content of anthocyanins in addition to catechins. Transcript expression of genes encoding anthocyanidin reductase (ANR), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), flavonol synthase (FLS) and leucoantho cyanidin reductase (LAR) enzymes in three new purple shoot tea clones compared with normal tea clone showed higher expression of CsDFR, CsANR, CsANS and lower expression of CsFLS and CsLAR in purple shoot clones compared to normal clone. Expression pattern supported high content of anthocyanins in purple tea. Four anthocyanins (AN1-4) were isolated and characterized by UPLC-ESI-QToF-MS/MS from IHBT 269 clone which recorded highest total anthocyanins content. Cyanidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN2) showed highest in vitro antioxidant activity (IC50 DPPH = 25.27 ± 0.02 µg/mL and IC50 ABTS = 10.71 ± 0.01 µg/mL). Anticancer and immunostimulatory activities of cyanidin-3-glucoside (AN1), cyanidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN2), delphinidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN3), cyanidin-3-O-(2-O-ß-xylopyranosyl-6-O-acetyl)-ß-glucopyranoside (AN4) and crude anthocyanin extract (AN5) showed high therapeutic perspective. Anthocyanins AN1-4 and crude extract AN5 showed cytotoxicity on C-6 cancer cells and high relative fluorescence units (RFU) at 200 µg/mL suggesting promising apoptosis induction activity as well as influential immunostimulatory potential. Observations demonstrate potential of purple anthocyanins enriched tea clone for exploitation as a nutraceutical product.

Global De Novo Protein-Protein Interactome Elucidates Interactions of Drought-Responsive Proteins in Horse Gram (Macrotyloma uniflorum).

Inspired by the availability of de novo transcriptome of horse gram (Macrotyloma uniflorum) and recent developments in systems biology studies, the first ever global protein-protein interactome (PPI) map was constructed for this highly drought-tolerant legume. Large-scale studies of PPIs and the constructed database would provide rationale behind the interplay at cascading translational levels for drought stress-adaptive mechanisms in horse gram. Using a bidirectional approach (interolog and domain-based), a high-confidence interactome map and database for horse gram was constructed. Available transcriptomic information for shoot and root tissues of a sensitive (M-191; genotype 1) and a drought-tolerant (M-249; genotype 2) genotype of horse gram was utilized to draw comparative PPI subnetworks under drought stress. High-confidence 6804 interactions were predicted among 1812 proteins covering about one-fourth of the horse gram proteome. The highest number of interactions (33.86%) in horse gram interactome matched with Arabidopsis PPI data. The top five hub nodes mostly included ubiquitin and heat-shock-related proteins. Higher numbers of PPIs were found to be responsive in shoot tissue (416) and root tissue (2228) of genotype 2 compared with shoot tissue (136) and root tissue (579) of genotype 1. Characterization of PPIs using gene ontology analysis revealed that kinase and transferase activities involved in signal transduction, cellular processes, nucleocytoplasmic transport, protein ubiquitination, and localization of molecules were most responsive to drought stress. Hence, these could be framed in stress adaptive mechanisms of horse gram. Being the first legume global PPI map, it would provide new insights into gene and protein regulatory networks for drought stress tolerance mechanisms in horse gram. Information compiled in the form of database (MauPIR) will provide the much needed high-confidence systems biology information for horse gram genes, proteins, and involved processes. This information would ease the effort and increase the efficacy for similar studies on other legumes. Public access is available at http://14.139.59.221/MauPIR/ .

Overexpression of MuHSP70 gene from Macrotyloma uniflorum confers multiple abiotic stress tolerance in transgenic Arabidopsis thaliana.

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H2O2, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.

PLA nanovectors with encapsulated betulin: plant leaf extract-synthesized nanovectors are more efficacious than PVA-synthesized nanovectors.

OBJECTIVES: Betulin (BT) is an abundant triterpene found predominantly in the bark of Himalayan birch. It is difficult to deliver it in vivo because of its low aqueous solubility. We have therefore developed novel formulations of BT for improving its solubility, bioavailability and therapeutic efficacy. RESULTS: Poly-D,L-lactide nanovectors (PLA NVs) were synthesized using poly(vinyl alcohol) and Lonicera japonica leaf extract (LE) as a stabiliser and named as PLA-1 NVs and PLA-2 NVs. PLA-1 NVs and PLA-2 NVs were used for the encapsulation of betulin (BT) and named as BT-En-1 and BT-En-2 NVs. The encapsulation efficiency of BT-En-1 and BT-En-2 NVs were 99.3 and 100 % respectively. Prepared nanoformulations were physically stable. An in vitro study revealed 45 % BT was released over 24 h. BT had a prolonged release from BT-En-2 NVs as compared to BT-En-1 NVs. BT-En-2 NVs had better anticancerous activity against SiHa cells than BT-En-1 NVs. CONCLUSIONS: Developed BT-EN-2 NVs had better biocompatibility, excellent stability and enhanced release characteristics than BT-En-1 NVs.

Diversion of carbon flux from gibberellin to steviol biosynthesis by over-expressing SrKA13H induced dwarfism and abnormality in pollen germination and seed set behaviour of transgenic Arabidopsis.

This paper documents the engineering of Arabidopsis thaliana for the ectopic over-expression of SrKA13H (ent-kaurenoic acid-13 hydroxylase) cDNA from Stevia rebaudiana. HPLC analysis revealed the significant accumulation of steviol (1-3 µg g(-1) DW) in two independent transgenic Arabidopsis lines over-expressing SrKA13H compared with the control. Independent of the steviol concentrations detected, both transgenic lines showed similar reductions in endogenous bioactive gibberellins (GA1 and GA4). They possessed phenotypic similarity to gibberellin-deficient mutants. The reduction in endogenous gibberellin content was found to be responsible for dwarfism in the transgenics. The exogenous application of GA3 could rescue the transgenics from dwarfism. The hypocotyl, rosette area, and stem length were all considerably reduced in the transgenics. A noteworthy decrease in pollen viability was noticed and, similarly, a retardation of 60-80% in pollen germination rate was observed. The exogenous application of steviol (0.2, 0.5, and 1.0 µg ml(-1)) did not influence pollen germination efficiency. This has suggested that in planta formation of steviol was not responsible for the observed changes in transgenic Arabidopsis. Further, the seed yield of the transgenics was reduced by 24-48%. Hence, this study reports for the first time that over-expression of SrKA13H cDNA in Arabidopsis has diverted the gibberellin biosynthetic route towards steviol biosynthesis. The Arabidopsis transgenics showed a significant reduction in endogenous gibberellins that might be responsible for the dwarfism, and the abnormal behaviour of pollen germination and seed set.

AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco.

In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants.

Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Nanotechnology in agri-food sector.

The emergence of nanotechnology developments using nanodevices/nanomaterials opens up potential novel applications in agriculture and food sector. Smart delivery systems, biosensors, and nanoarrays are being designed to solve the problems faced in agriculture sector. Similarly, food sector is also benefited through the use of smart biosensors, packaging materials, and nanonutraceuticals. Despite the great potential of nanotechnology in agri-food sector, people are ambiguous about use in food applications because of suspected potential health risks and environmental concerns. Nanoparticles, due to their unique characteristics, including small size, shape, high surface area, charge, chemical properties, solubility and degree of agglomeration can cross cell boundaries or pass directly from the lungs into the blood stream and ultimately reach to all of the organs in the body. This is the reason why they may pose higher risk than the same mass and material of larger particles. In this paper, we have made an attempt to give an overview of nanotechnology developments in agri-food sector, risks associated with nanomaterials and toxicity regulations for policy framework.

Overexpression of a glycosyltransferase gene SrUGT74G1 from Stevia improved growth and yield of transgenic Arabidopsis by catechin accumulation.

Steviol glycoside and gibberellin biosynthetic routes are known as divergent branches of a common origin in Stevia. A UDP-glycosyltransferase encoded by SrUGT74G1 catalyses the conversion of steviolbioside into stevioside in Stevia rebaudiana leaves. In the present study, transgenic Arabidopsis thaliana overexpressing SrUGT74G1 cDNA from Stevia were developed to check the probability of stevioside biosynthesis in them. However, stevioside accumulation was not evident in transgenics. Also, the transgenic Arabidopsis showed no change in GA3 content on SrUGT74G1 overexpression. Surprisingly, significant accumulation of catechin was noticed in transgenics. The transgenics showed a considerable increase in shoot length, root length and rosette area. An increase in free radical scavenging activity of transgenics was noticed. Moreover, the seed yield of transgenics was also increased by 6-15% than control. Additionally, variation in trichome branching pattern on leaf surface of transgenics was observed. The trichome branching pattern was also validated by exogenous catechin exposure (10, 50, 100 ng ml(-1)) to control plants. Hence, present study reports the probable role of SrUGT74G1 from Stevia in catechin accumulation of transgenic Arabidopsis thaliana. Thus, detailed study in present perspective has revealed the role of Stevia SrUGT74G1 gene in trichome branching pattern, improved vegetative growth, scavenging potential and seed yield by catechin accumulation in transgenic Arabidopsis.

Gain of function mutation in tobacco MADS box promoter switch on the expression of flowering class B genes converting sepals to petals.

One mutant transgenic line displaying homeotic conversion of sepals to petals with other phenotypic aberrations was selected and characterized at molecular level. The increased transcript level of gene encoding anthocyanidin synthase and petal specific class B genes, GLOBOSA and DEFECIENS in sepals of mutant line may be responsible for its homeotic conversion to petaloid organs. While characterizing this mutant line for locus identification, T-DNA was found to be inserted in 3' untranslated region of promoter of class B MADS box gene, GLOBOSA. Here, CaMV 35S promoter of T-DNA might be deriving the expression of class B genes.

Encapsulation of podophyllotoxin and etoposide in biodegradable poly-D,L-lactide nanoparticles improved their anticancer activity.

To improve the efficacy podophyllotoxin (PODO) and etoposide (ETOPO) were encapsulated in poly-d,l-lactide nanoparticles (PLA NPs). The size of synthesised PODO-loaded PLA NPs and ETOPO-loaded PLA NPs was 100 ± 17 nm and 163 ± 20 nm and their encapsulation efficiency was 17 and 48%, respectively. In vitro release studies showed initial burst release followed by slow and sustained release. In vitro cytotoxicity of synthesised NPs was assessed using A549 and CHO-K1 cells. Blank PLA NPs did not show any toxicity. While PODO-loaded PLA NPs showed higher in vitro cytotoxicity in comparison to ETOPO-loaded PLA NPs against both cell lines. Also, the cytotoxicity of both PODO-loaded PLA NPs and ETOPO-loaded PLA NPs was higher compared to pure drugs. Hence, this study documents the improvement in efficacy of these molecules upon encapsulation in PLA NPs and could be an important strategy for better therapeutics.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Recent advances in 3D bioprinting for cancer research: From precision models to personalized therapies.

Cancer remains one of the most devastating diseases, necessitating innovative and precise therapeutic solutions. The emergence of 3D bioprinting has revolutionized the platform of cancer therapy by offering bespoke solutions for drug screening, tumor modeling, and personalized medicine. The utilization of 3D bioprinting enables the fabrication of complex tumor models that closely mimic the in vivo microenvironment, facilitating more accurate drug testing and personalized treatment strategies. Moreover, 3D bioprinting also provides a platform for the development of implantable scaffolds as a therapeutic solution to cancer. In this review, we highlight the application of 3D bioprinting for cancer therapy along with current advancements in cancer 3D model development with recent case studies.

Comprehensive transcriptomic study on horse gram (Macrotyloma uniflorum): De novo assembly, functional characterization and comparative analysis in relation to drought stress.

BACKGROUND: Drought tolerance is an attribute maintained in plants by cross-talk between multiple and cascading metabolic pathways. Without a sequenced genome available for horse gram, it is difficult to comprehend such complex networks and intercalated genes associated with drought tolerance of horse gram (Macrotyloma uniflorum). Therefore, de novo transcriptome discovery and associated analyses was done for this highly drought tolerant yet under exploited legume to decipher its genetic makeup. RESULTS: Eight samples comprising of shoot and root tissues of two horse gram genotypes (drought-sensitive; M-191 and drought-tolerant; M-249) were used for comparison under control and polyethylene glycol-induced drought stress conditions. Using Illumina sequencing technology, a total of 229,297,896 paired end read pairs were generated and utilized for de novo assembly of horse gram. Significant BLAST hits were obtained for 26,045 transcripts while, 3,558 transcripts had no hits but contained important conserved domains. A total of 21,887 unigenes were identified. SSRs containing sequences covered 16.25% of the transcriptome with predominant tri- and mono-nucleotides (43%). The total GC content of the transcriptome was found to be 43.44%. Under Gene Ontology response to stimulus, DNA binding and catalytic activity was highly expressed during drought stress conditions. Serine/threonine protein kinase was found to dominate in Enzyme Classification while pathways belonging to ribosome metabolism followed by plant pathogen interaction and plant hormone signal transduction were predominant in Kyoto Encyclopedia of Genes and Genomes analysis. Independent search on plant metabolic network pathways suggested valine degradation, gluconeogenesis and purine nucleotide degradation to be highly influenced under drought stress in horse gram. Transcription factors belonging to NAC, MYB-related, and WRKY families were found highly represented under drought stress. qRT-PCR validated the expression profile for 9 out of 10 genes analyzed in response to drought stress. CONCLUSIONS: De novo transcriptome discovery and analysis has generated enormous information over horse gram genomics. The genes and pathways identified suggest efficient regulation leading to active adaptation as a basal defense response against drought stress by horse gram. The knowledge generated can be further utilized for exploring other underexploited plants for stress responsive genes and improving plant tolerance.

Comparative analysis of DNA methylation polymorphism in drought sensitive (HPKC2) and tolerant (HPK4) genotypes of horse Gram (Macrotyloma uniflorum).

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.

Immobilization of L-ribose isomerase on the surface of activated mesoporous MCM41 and SBA15 for the synthesis of L-ribose.

A hexagonal mesoporous molecular sieve-like structure of MCM41 and SBA15 with a large surface area was used to immobilize protein L-ribose isomerase (L-RI) through covalent linkages. The amino group of APTES functionalized nanosilica support MCM41 and SBA15 interacted with glutaraldehyde to promote bidentate linkage and on other side with amino group of enzyme. The use of mesoporous silica matrix for immobilization was observed to conserve the distinctive properties of the protein. The various operational conditions optimized for covalent conjugation of protein with the silica support were found to be dependent on enzyme support ratio, immobilization temperature and time. The immobilization yield of L-RI on MCM41 and SBA15 was achieved to be 60 % (600 mg enzyme /g matrix) and 45 % (450 mg enzyme/g matrix), respectively under the optimized conditions. The immobilized biocatalyst was characterized by various analytical techniques like HR-TEM, EDS, FTIR, TGA and BET. Effects of different experimental conditions were optimized to study enzyme kinetics, pH, temperature, bioconversion, reusability, metal ion effect and storage stability. The biocatalytic efficiency (kcat/Km) was increased by 1.2 fold on immobilization with the catalytic activity of 39.64 IU. Increase in the catalytic efficiency after immobilization could be due to the suitable orientation of enzyme active site and improved accessibility for substrate binding. The immobilization of L-RI on mesoporous silica support could improve the biocatalytic activity, storage stability and reusability. The immobilized biocatalyst was found to be reusable for more than 4 cycles retaining more than 50 % of catalytic activity and promoting the synthesis of a rare sugar L-ribose from L-ribulose with a conversion yield of 22 % in 2 h time.