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Despite the widespread use of platinum(Pt)-drugs for cancer-therapy, their development has stalled, as no new Pt-drugs have been approved in over a decade. Packaging small molecule drugs into nanoparticles is a way to enhance their therapeutic efficacy. However, an important consideration in designing Pt-nanoparticles, and the focus of this study, is the rational choice of oxidation-state of Pt. To date, there has been no direct comparison of relative merits of the choice of Pt oxidation-state in the same nanoparticle-system that would allow its optimal design. To address this lacuna, we designed an recombinant asymmetric triblock polypeptide (ATBP) that self-assembles into rod-shaped micelles and chelates Pt(II) or enable covalent conjugation of Pt(IV) with similar morphology and stability. Both ATBP-Pt(II) and ATBP-Pt(IV) nanoparticles enhanced the half-life of Pt by ~45-fold but ATBP-Pt(IV) had superior tumor regression efficacy compared to ATBP-Pt(II) and cisplatin. These results suggest loading Pt(IV) into genetically engineered nanoparticles may yield a new generation of more effective platinum-drug nanoformulations.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Pectin is a complex form of polysaccharide and is composed of several structural components that require the concerted action of several pectinases for its complete degradation. In this study, in s...

The structure and molecular dynamics of α-L-arabinofuranosidase (PsGH43_12) of family 43 glycoside hydrolase, subfamily 12 from Pseudopedobacter saltans were studied. The modeled PsGH43_12 structure displayed 5-bladed β-propeller fold at N-terminal and β–sandwich fold at C terminal. Ramachandran plot showed 95.7% residues in favored and 3.3% in the generously allowed region and only 1% residues in the disallowed region. The secondary structure analysis of PsGH43_12 by circular dichroism revealed 2.7% α-helices, 30.33% β-strands and 66.97% random coils. Protein melting study of PsGH43_12 showed complete unfolding at 65°C and did not require any metal ion for its stability. Molecular docking analysis confirmed the involvement of active site residues Asp71, Asp180 and Glu247 in the catalysis, which was also confirmed by the site-directed mutagenesis of these residues. SAXS analysis displayed that PsGH43_12 is monomeric and a fully folded state in solution form. Guinier analysis gave the radius of gyration (Rg) 2.8 ± 0.09 nm. The maximum dimension and Rg of PsGH43_12 estimated from P(R) plot were 9.7 nm and 2.81 nm, respectively. The ab initio derived dummy model of PsGH43_12 displayed a bell-like shape. The ab initio derived dummy model superposed well with its comparative modeled structure except the N-terminal His6-tag region.

Xylan extracted from neem sawdust gave 22.5%, (w/w) yield. The extracted xylan was composed of xylose and glucuronic acid at a molar ratio of 8:1 and with a molecular mass, ~66 kDa. FTIR and NMR analyses indicated a backbone of xylan substituted with 4-O-methyl glucuronic acid at the O-2 position. FESEM analysis showed a highly porous and granular surface structure of xylan. A thermogravimetric study of xylan showed thermal denaturation at 271 °C. The hydrolysis of xylan by recombinant endo-β-1,4-xylanase produced a mixture of xylooligosaccharides ranging from degree of polymerization 2–7. Xylooligosaccharides inhibited cell growth of human colorectal cancer (HT-29) cells but did not affect the mouse fibroblast cells confirming its biocompatibility. Western blotting, DNA laddering and flow cytometric analysis displayed inhibition of HT-29 cells by xylooligosaccharides. FLICA staining and mitochondrial membrane potential analyses confirmed the activation of the intrinsic pathway of apoptosis. The study amply indicated that the xylooligosaccharides produced from neem xylan could be potentially used as an antiproliferative agent.

Effect of bed roughness on flow field and turbulence characteristics due to spur dyke in rigid bed meandering channel is presented in this study. This is an extension of authors’ earlier work. In t...

Newcastle disease virus (NDV) has been explored to a great extent to understand the biology of negative-sense RNA viruses. Nucleoprotein (N) is the most abundant protein in the virus particles, and its primary function is to encapsidate the virus genome for its transcription, replication, and packaging. Here, we report the structural investigations of the N protein of NDV (NDV-N) in solution. The N gene of NDV was cloned and expressed in E. coli as a soluble protein of ~53 kDa in size. The FE-TEM imaging of the purified NDV-N displayed a nearly spherical shape with a diameter of 28 nm and the DLS analysis of the purified NDV-N displayed a monodispersed nature, with averaged hydrodynamic radius, 26.5 nm. The conformational behavior of the NDV-N in solution was studied by SAXS analysis, which suggested two ring structures of NDV-N formed by thirteen monomeric units each. Each ring interacts with RNA molecules and forms a large molecule with a size of ~1450 kDa and are stacked on each other in a spiral arrangement. More profound knowledge of the N protein structure will help us in deciphering the control of viral RNA synthesis at the early stage of NDV life-cycle.

Chimera (CtGH1-L1-CtGH5-F194A) developed by fusing β-glucosidase (CtGH1) at N-terminal and endoglucanase (CtGH5-F194A) at C-terminal was structurally characterized. Its secondary structure analysis by CD showed 38% α-helix, 9.3% β-sheets and 52.7% random coils corroborating with prediction. In-silico modeled structure of Chimera comprised two modules, CtGH1 and CtGH5-F194A displaying (α/β)8 fold. Ramachandran plot of Chimera showed 99.9% residues in allowed region. Binding interaction of Chimera with cello-oligosaccharides suggested active forms of CtGH1 and CtGH5-F194A and their involvement in catalysis. MD simulation of cellohexaose bound endoglucanase module of Chimera showed favourable flexibility in loops, LA with H-bond formation with Asn510 and in loop LC relocation of Tyr687 away from active site efficiently releasing the product after catalysis. Higher short range interaction energy of Chimera, −383 kJ/mol than the individual endoglucanase, 254 kJ/mol against cellohexaose suggested higher efficient catalysis by Chimera. β-Glucosidase module of Chimera showed fluctuations in outer loops suggesting conformational changes that might be contributing to improved hydrolysis. SAXS analysis of Chimera displayed monodispersed state. Guinier analysis of Chimera showed globular shape (Rg= 3.15 ± 0.10 nm). Kratky plot confirmed fully folded and flexible behaviour in solution. Gasbor modeled structure of Chimera displayed an elongated structure with two modules having shape similar to bean-bag contour.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: A cost-effective, high-throughput, adaptable pipeline capable of identifying effective humanized nanobodies against SARS-CoV-2.

Enzymes that participate in the hydrolysis of complex carbohydrates display a modular architecture, although the significance of enzyme modularity to flexibility and catalytic efficacy is not fully understood. α-L-arabinofuranosidase from Clostridium thermocellum (CtAraf43) catalyzes the release of α-1,2-, α-1,3-, or α-1,5- linked L-arabinose from arabinose decorated polysaccharides. CtAraf43 comprises an N-terminal catalytic domain (CtAbf43A) connected with two family 6 carbohydrate-binding modules (CBMs), termed as CtCBM6A and CtCBM6B, through flexible linker peptides. Here, we modeled the structure of CtAraf43 revealing that the module, CtAbf43A displays a 5-fold β-propeller fold and the CBMs the typical jellyroll type β-sandwich folds. Ramachandran plot showed 98.5% residues in the favored region and 1.5% residues in the disallowed region. Molecular dynamics simulation analysis of CtAraf43 revealed significant flexibility that is more expressive in the C-terminal CtCBM6B module in terms of structure and orientation. Small angle X-ray scattering analysis of CtAraf43 revealed its elongated structure. CtAraf43 at 1.2 mg/mL demonstrated the monomeric nature and a multi-modular shaped molecular envelope in solution with a Dmax of 12 nm. However, at 4.7 mg/mL, CtAraf43 displayed a dimeric structure and elongated molecular envelope. Kratky plot analysis revealed the folded state of CtAraf43 with limited flexibility at both concentrations. The data revealed higher flexibility at the C-terminal of CtAraf43 suggesting a coordinated action of the N-terminal catalytic module CtAbf43A and the internal CtCBM6A.AbbreviationCBMsCarbohydrate Binding ModulesCtAraf43α-L-arabinofuranosidaseGHsGlycoside HydrolasesMDMolecular DynamicsRMSDRoot Mean Square DeviationRMSFRoot Mean Square FluctuationSAXSSmall angle X-ray scatteringCommunicated by Ramaswamy H. Sarma.

Endo-β-1,4-mannanase named as RfGH5_7 from Ruminococcus flavefaciens cloned, expressed and purified earlier was structurally characterized in present study. The RaptorX modeled structure of RfGH5_7...

The structure of heparinase II/III belonging to family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was generated by homology modeling. Multiple sequence alignment showed conserved (Asn216, Tyr270 and His400) and semi-conserved active site amino acid residues. The modeled structure of PsPL12a displayed α/α toroid domain at N-terminal and antiparallel β sheets at C-terminal domain. The modeled structure was similar to those of heparinases from polysaccharide lyase 12 and 21 families. Validation of PsPL12a model by Ramachandran plot showed 94.6% of residues in the favored region, 5.2% of residues in the allowed region and only 0.2% of residues in the outlier region. The area and volume computed for PsPL12a displayed nearly a closed conformation of the active site, similar to HepIII from Bacteroides thetaiotaomicron. The charge calculation on the surface of the PsPL12a structure showed the higher distribution of positive charge in the active site cleft as compared with other homologous structures. Molecular docking study of MD-simulated PsPL12a structure with heparin oligosaccharide showed high binding affinity as compared with heparan sulfate oligosaccharides. Comparison of the active site of modeled PsPL12a with other homologous heparinases revealed putative catalytic triad involving the residues Asn216, His400 and Tyr270. Small-angle X-ray scattering analysis of PsPL12a displayed a fully folded and boxing glove-like envelop.Communicated by Ramaswamy H. Sarma.

Site-directed mutagenesis of β-1,4-endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a β-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and β-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both β-1,4-endoglucanase and β-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cello-oligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (Tm), 68 °C and of CtGH1, 79 °C, whereas, chimera showed 78 °C. The improved structural integrity, thermostability and enhanced bi-functional enzyme activities of chimera makes it potentially useful for industrial application in converting biomass to glucose and thus bioethanol.

Pectin is a complex form of polysaccharide and is composed of several structural components that require the concerted action of several pectinases for its complete degradation. In this study

Safe disposal of waste glass is one of the biggest environmental challenges due to its inertness property across the globe. Among the various types of glasses, disposal of cathode ray tube (CRT) and liquid crystal display (LCD) is more challenging due to emission of harmful contents. It is not easy and safe to dispose of these glass waste in the landfills. As an environmental friendly solution, researchers evolve the possibility of using CWG in the construction industry. It may be used both as coarse and fine aggregate in concrete production. This review paper offers an overview of previous work in which the CWG is used as fine aggregate. The optimum workability, dry and wet density, and compressive, tensile, and flexural strength results presented by various researchers are compiled for various compositions of concrete with CWG. It can be observed that the results are inconsistent and did not follow a trend. CWG will marginally increase the strengths when it is used in the lower percentage. After reaching an optimum value, the strengths again reduce.

A critical review about the challenges of riverfront projects in arid or semi-arid regions is present in the present study. Dravayawati Riverfront of Jaipur city is taken as a case study. Riverfronts are the new landscapes in the urban areas to utilize the undeveloped banks of the river reaches. Recently, Dravyavati, Gomti, Patna, Sabarmati and Yamuna riverfronts are some of the landscape projects developed in the various cities. As most of the rivers are either ephemeral (water is available only during monsoon period) or tapped for canal systems, the availability of freshwater is the major challenge. A 47 km long riverfront project was planned in Jaipur city on the banks of Dravayawati River. As Jaipur is located in the semi-arid part of the Rajasthan, the water is available for limited duration during the monsoon period. In this project, treated sewage water is the prime source of water. A concrete channel is constructed in the middle of river section and parks or some developments on both the banks. This ultimately reduces the possibility of groundwater recharge in a dark zone city. There are few more hydrologic challenges in the execution of the projects and discussed in the present study. The results of the present study may be useful for the policy makers, when such projects are planned in the arid and semi-arid part of the country.

The production of bioenergy from wastes attracts worldwide attention to overcome energy crisis and increasing pollution (Thakur et al., Microbial fermentation and enzyme technology, Taylor and Francis Group, Boca Raton, FL, 257–268, 2020). Lignocellulosic biomass can serve as an alternative source for bioenergy production. Thermostable enzymes can hydrolyze the lignocellulosic biomass and produce reducing sugars, which can be fermented to produce bioethanol by using fermenting microbes. Clostridium thermocellum is a gram-positive, anaerobic and rod-shaped, thermophilic microorganism having great potential applications. It can directly transform lignocellulosic biomass into valuable products such as acetate, ethanol, formate, and lactate. Clostridium thermocellum expresses a multi-enzyme complex bound to scaffoldin proteins called cellulosome that contains cellulolytic, hemicellulolytic, and other carbohydrate degrading enzymes. The thermophilic enzymes possess wide applications in several industries for producing sustainable green products. This chapter evaluates the production and properties of recombinant thermostable cellulases, hemicellulases, and pectinases from C. thermocellum, their structure, and applications in different industrial processes.

Xylan is the major hemicellulose present in sugarcane stem secondary cell walls. Xylan is composed of xylose backbone with a high degree of substitutions, which affects its properties. In the present study, the xylan from sugarcane tops (SCT) was extracted and characterized. Compositional analysis of xylan extracted from SCT (SCTx) displayed the presence of 74% of d -xylose residues, 16% of d -glucuronic acid residues and 10% of l -arabinose. High performance size exclusion chromatographic analysis of SCTx displayed a single peak corresponding to a molecular mass of ∼57 kDa. The Fourier transform infrared spectroscopic analysis of SCTx displayed the peaks corresponding to those obtained from commercial xylan. FESEM analysis of SCTx showed the granular and porous surface structure. Differential thermogravimetric analysis (DTG) of SCTx displayed two thermal degradation temperatures (Td) of 228°C, due to breakdown of the side chains of glucuronic acid and arabinose and 275°C, due to breakdown of xylan back bone. The presence of arabinose and glucuronic acid as a side chains was confirmed by the DTG and thermogravimetric analysis. The CHNS analysis of SCTx showed the presence of only carbon and hydrogen supporting its purity. The recombinant xylanase (CtXyn11A) from Clostridium thermocellum displayed a specific activity of 1394 ± 51 U/mg with SCTx, which was higher than those with commercial xylans. The thin layer chromatography and electrospray ionization mass spectroscopy analyses of CtXyn11A hydrolysed SCTx contained a series of linear xylo-oligosaccharides ranging from degree of polymerization 2-6 and no substituted xylo-oligosaccharides because of the endolytic activity of enzyme. The extracted xylan from SCT can be used as an alternative commercial substrate and for oligo-saccharide production.

α-l-Arabinofuranosidase (PsGH43_12) of family 43 glycoside hydrolase and subfamily 12 from Pseudopedobacter saltans was cloned, over-expressed and biochemically characterized. PsGH43_12 displayed molecular mass, ~ 65 kDa. It exhibited activity in pH (5–9) and temperature range (35–55 °C) with maxima at pH 6.5 and 50 °C. PsGH43_12 gave 88.7 U/mg specific activity against rye arabinoxylan and 78.9 U/mg against wheat arabinoxylan. PsGH43_12 displayed Km and Vmax, 3.02 mg/ml and 103 µmole/min/mg, respectively, against rye arabinoxylan and 2.17 mM and 100.7 µmole/min/mg, respectively, against pNP-α-l-arabinofuranoside. 10 mM Mg2+ or Ca2+ ions enhanced PsGH43_12 activity by 54% or 33%, respectively. PsGH43_12 hydrolyzed rye arabinoxylan and released only l-arabinosyl moiety as main product, confirming its specificity towards α-l-arabinofuranoside. The regioselective analysis by NMR showed that PsGH43_12 belongs to type III α-l-arabinofuranoside. The synergistic behavior of PsGH43_12 in saccharification of mild alkali pretreated finger miller stalk (FMS) along with xylanase (CtXyn11A) from Clostridium thermocellum and xylosidase (BoGH43) from Bacteroides ovatus gave twofold higher total reducing sugar (TRS) yield. TLC analysis of pretreated FMS hydrolysed by CtXyn11A and BoGH43 showed xylooligosaccharides and xylose. Addition of PsGH43_12 to above combination gave mostly xylose and arabinose confirming their synergistic behavior and displaying their applicability in hydrolysis of hemicellulosic biomass.

The cellulases of family 9 glycoside hydrolase with subtle difference in amino acid sequence have shown different types of catalytic activities such as endo-, exo- or processive endocellulase. However, the reason behind the different types of catalytic activities still unclear. In this study, the processive endocellulase, HtGH9 of family 9 GH from Hungateiclostridium thermocellum was modeled by homology modeling. The catalytic module (HtGH9t) of HtGH9 modeled structure displayed the (α/α)6 barrel topology and associated family 3 carbohydrate binding module (HtCBM3c) displayed β-sandwich fold. Ramachandran plot of HtGH9 modeled structure displayed all the amino acid residues in allowed region except Asn225 and Asp317. Secondary structure analysis of modeled HtGH9 showed the presence of 41.3% α-helices and 11.0% β-strands which was validated through circular dichroism analysis that showed the presence of 42.6% α-helices and 14.5% β-strands. Molecular Dynamic (MD) simulation of HtGH9 structure for 50 ns showed Root Mean Square Deviation (RMSD), 0.84 nm and radius of gyration (Rg) 3.1 nm. The Small-angle X-ray scattering of HtGH9 confirmed the monodisperse state. The radius of gyration for globular shape (Rg) was 5.50 ± 0.15 nm and for rod shape (Rc) by Guinier plot was 2.0 nm. The loop formed by amino acid residues, 264–276 towards one end of the catalytic site of HtGH9 forms a barrier, that blocks the non-reducing end of the cellulose chain causing the processive cleavage resulting in the release of cellotetraose. The position of the corresponding loop in cellulases of family 9 GH is responsible for different types of cleavage patterns.

The world is currently facing two major challenges, the resource scarcity and overproduction of waste. Due to the uneven distribution and excess consumption of resources, the demand for energy is high and increasing day by day. On the other hand, the mounting of waste needs strategic waste management. In the environment, microbes present utilize organic waste by releasing enzymes, but the rate of degradation is much slower than the production of waste. Organic waste majorly constitutes agricultural waste or municipal solid waste that can be converted to bioenergy (methane, methanol, bioethanol and biobutanol). Cellulose and hemicellulose portion of the solid wastes are hydrolyzed by cellulases and hemicellulases into fermentable sugars like glucose, xylose and arabinose. Fermenting microbes further utilize these sugars for the production of bioethanol, and residual biomass used as compost in the agricultural field. Ethanol produced from cellulose and hemicellulose is environmental friendly fuel as it emits lower carbon monoxide. An overview of breakdown of cellulosic and hemicellulosic polysaccharides present in agro-waste and production of bioethanol with focus on recent developments is described.

Aims The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards a global health emergency. Currently, no proper medicine or effective treatment strategies are available; therefore, repurposing of FDA approved drugs may play an important role in overcoming the situation. Materials and methods The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2′OMTase), which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2′OMTase of SARS-CoV-2 was analyzed, and its structure was modeled by a comparative modeling approach and validated. The library of 3000 drugs was screened against the active site of 2′OMTase followed by re-docking analysis. The apo and ligand-bound 2′OMTase were further validated and analyzed by using molecular dynamics simulation. Key findings The modeled structure displayed the conserved characteristic fold of class I MTase family. The quality assessment analysis by SAVES server reveals that the modeled structure follows protein folding rules and of excellent quality. The docking analysis displayed that the active site of 2′OMTase accommodates an array of drugs, which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids, and other drugs. The redocking and MD simulation analysis of the best 5 FDA approved drugs reveals that these drugs form a stable conformation with the 2′OMTase. Significance The results suggested that these drugs may be used as potential inhibitors for 2′OMTase for combating the SARS-CoV-2 infection.

Over the past two decades, birchwood and beechwood xylans have been used as a popular substrate for the characterization of xylanases. Recently, major companies have discontinued their commercial production. Therefore, there is a need to find an alternative to these substrates. Xylan extraction from Acacia sawdust resulted in 23.5% (w/w) yield. The extracted xylan is composed of xylose and glucuronic acid residues in a molar ratio of 6:1 with a molecular mass of ∼70 kDa. The specific optical rotation analysis of extracted xylan displayed that it is composed of the d-form of xylose and glucuronic acid monomeric sugars. The nuclear magnetic resonance analysis of extracted xylan revealed that the xylan backbone is substituted with 4-O-methyl glucuronic acid at the O2 position. Fourier transform infrared analysis confirmed the absence of lignin contamination in the extracted xylan. Xylanase from Clostridium thermocellum displayed the enzyme activity of 1761 U/mg against extracted xylan, and the corresponding activity against beechwood xylan was 1556 U/mg, which confirmed that the extracted xylan could be used as an alternative substrate for the characterization of xylanases.

This paper proposes the solution for electricity issues in agriculture sector of particular location in Rajasthan. The issues reported about the availability of ground water and availability of Electricity from electric Grid. The study proposes a centralized solar photovoltaic system for available nearby open wells, which are being operated by diesel engine pump-sets, which emits greenhouse gases and pollute the environment. The proposed centralized photovoltaic system will provide power for submersible pump sets. The additional available power can be stored in batteries, utilized for charging the batteries of electrical vehicles or sell back to electric grid. This paper shows the cost analysis for proposed systems that can be applied and effective centralized solar photovoltaic system in the area considered for study. Centralized solar photovoltaic generation supply to different consumers in different slots and may increase the conflict between consumers, demand side management provide suitable solution to consumers is proposed and analyse in future work.

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 A resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

This study compares different types of pretreatment methods, such as thermal pretreatment at 120 °C, autoclaving, microwaving and ultrasonication in the presence of water, dilute acid (1% H2SO4) or dilute alkali (1% NaOH) on Sorghum stalk with respect to the holocellulose and Acid Detergent/Insoluble Lignin content. Among all the methods, pretreatment with 1% NaOH along with autoclaving at 121 °C and 15 psi for 30 min was the most effective method for Sorghum stalk. Fourier Transform Infra-Red spectroscopy analysis of this pretreated biomass showed the removal of lignin and Field Emission Scanning Electron Microscope analysis displayed enhanced surface roughness. The enzymatic hydrolysis of raw and best pretreated Sorghum stalk using recombinant endo-β-1,4-glucanase (CtCel8A) and β-1,4-glucosidase (CtBgl1A) both from Clostridium thermocellum gave glucose yields, 22.4 mg/g raw biomass and 34 mg/g pretreated biomass, respectively, resulting in 1.5-fold increase of glucose yield after the pretreatment.

An endo-xylanase (PsGH10A) of family 10 glycoside hydrolase from Pseudopedobacter saltans was cloned, expressed and purified. Substrate specificity analysis of PsGH10A showed activity against β-1,4-xylans. It showed maximum activity against beechwood xylan (59.7 ± 1.1 U/mg) followed by xylan (Mw. 20,000–30,000) (57.1 ± 0.7 U/mg). PsGH10A displayed maximum activity at pH 6.0 and 40 °C. The enzyme was stable in the pH range, from 6.0 to 7.5 and showed thermostability up to 40 °C. The kinetic parameters of PsGH10A using beechwood xylan determined were Km 6.2 mg/mL and Vmax 72 U/mg. The activity of PsGH10A was 29 ± 2.4% enhanced by 2 mM Mn2+ ions and inhibited by less than 50 ± 1.6% by 2 mM Zn2+, Pb2+ or Cu2+ ions. The time-dependent TLC analysis of hydrolyzed products of beechwood xylan released by PsGH10A showed the release of xylose, xylobiose and xylotetraose as end products confirming the endolytic mode of action. PsGH10A hydrolyzed products of xylan and substituted xylan indicate production of series of short-chain xylooligosaccharides and arabinoxylo-saccharides. PsGH10A also showed saccharification of AFEX pretreated poplar and sugarcane bagasse. Therefore, it can be used for various biotechnological applications such as for prebiotic xylooligosaccharides and bioethanol production from pretreated agrowaste biomass. This is the first report on β-1,4-xylanase cloned from Pseudopedobacter saltans.

The structure and biophysical properties of endo β-1,4-xylanase (PsGH10A) of family 10 glycoside hydrolase were characterized. The modeled PsGH10A structure showed classical (β/α)8-barrel fold. Ramachandran plot displayed 99.1% residues in favored and 0.3% in the generously allowed region and only 0.6% residues in disallowed region. The secondary structure analysis of PsGH10A by CD revealed 31.75% α-helices 20.0% β-strands and 48.25% random coils. Protein melting study of PsGH10A showed complete unfolding at 60°C and did not require any metal ion for its stability. Structural superposition and docking analysis confirmed the involvement of Glu156 and Glu263 residues in catalysis. SAXS analysis displayed that PsGH10A is monomeric in nature showing fully folded state in solution form. Guinier analysis gave the radius of gyration (Rg) 2.23-2.29nm. Kratky plot indicated that the protein is fully folded globular shaped and flexible in solution form. The ab initio derived dummy model of PsGH10A displayed chicken thigh like shape. The ab initio derived dummy model superposed well with its comparative modeled structure except the N-terminal His-tag region.

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K m and Vmax, for PsPL12a were 4.6 ± 0.5 mg/ml and 70 ± 2 U/mg, respectively. Ten millimolars of each Mg2+ and Mn2+ ions enhanced PsPL12a activity by 80%, whereas Ni2+ inhibited by 75% and Co2+ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg2+ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination.

Pectin methylesterase (CtPME) from Clostridium thermocellum of family 8 carbohydrate esterase (CE8) belongs to pectin methylesterase super family (E.C.3.1.1.11). BLAST analysis of CtPME showed 38% sequence identity with PME from Erwinia chrysanthemi. Multiple sequence alignment of CtPME with other known structures of pectin methylesterase revealed the conserved and semi-conserved amino acid residues. Homology modelling of CtPME structure revealed a characteristic right handed parallel β-helices. The energy of modelled structure was minimized by using YASARA software. The Ramachandran plot of CtPME shows 83.7% residues in non-glycine and non-proline residues in most-favorable region, 13.8% in additional allowed region and 1.4% in generously allowed region, indicating that CtPME has a stable conformation. The secondary structure of CtPME predicted using PSI-Pred software and confirmed by the circular dichroism (CD) showed α-helices (3.1%), β-sheets (40.1%) and random coils (56.9%). Small Angle X-ray Scattering (SAXS) analysis demonstrated the overall shape and structural characterization of CtPME in solution form. Guinier analysis gave the radius of gyration (Rg) 2.28 nm for globular shape and 0.74 nm for rod shape. Kratky plot gave the indication that protein is fully folded in solution. The ab initio derived dummy atom model of CtPME superposed well on modelled CtPME structure.

Enzymes that participate in the hydrolysis of complex carbohydrates display a modular architecture, although the significance of enzyme modularity to flexibility and catalytic efficacy is not fully understood. α-L-arabinofuranosidase from

Endo-β-1,4-mannanase named as

The structure of heparinase II/III belonging to family 12 polysaccharide lyase (

Glucuronoxylan-β-1,4-xylanohydrolase from Clostridium thermocellum (CtXynGH30) hydrolyzes β-1,4-xylosidic linkages in 4-O-Methyl-D-glucuronoxylan. CtXynGH30 comprises an N-terminal catalytic domain, CtXyn30A, joined by a typical linker sequence to a family 6 carbohydrate-binding module, termed CtCBM6. ITC, mass spectrometric and enzyme activity analyses of CtXyn30A:CtCBM6 (1:1 M ratio), CtXyn30A and CtXynGH30 showed that the linker peptide plays a key role in connecting and orienting CtXyn30A and CtCBM6 modules resulting in the enhanced activity of CtXynGH30. To visualize the disposition of the two protein domains of CtXynGH30, SAXS analysis revealed that CtXynGH30 is monomeric and has a boot-shaped molecular envelope in solution with a Dmax of 18 nm and Rg of 3.6 nm. Kratky plot displayed the protein in a fully folded and flexible state. The ab initio derived dummy atom model of CtXynGH30 superposed well with the modelled structure.

Enzymes that participate in the hydrolysis of complex carbohydrates display a modular architecture, although the significance of enzyme modularity to flexibility and catalytic efficacy is not fully understood. α-L-arabinofuranosidase from Clostridium thermocellum (CtAraf43) catalyzes the release of α-1,2-, α-1,3-, or α-1,5- linked L-arabinose from arabinose decorated polysaccharides. CtAraf43 comprises an N-terminal catalytic domain (CtAbf43A) connected with two family 6 carbohydrate-binding modules (CBMs), termed as CtCBM6A and CtCBM6B, through flexible linker peptides. Here, we modeled the structure of CtAraf43 revealing that the module, CtAbf43A displays a 5-fold β-propeller fold and the CBMs the typical jellyroll type β-sandwich folds. Ramachandran plot showed 98.5% residues in the favored region and 1.5% residues in the disallowed region. Molecular dynamics simulation analysis of CtAraf43 revealed significant flexibility that is more expressive in the C-terminal CtCBM6B module in terms of structure and orientation. Small angle X-ray scattering analysis of CtAraf43 revealed its elongated structure. CtAraf43 at 1.2 mg/mL demonstrated the monomeric nature and a multi-modular shaped molecular envelope in solution with a Dmax of 12 nm. However, at 4.7 mg/mL, CtAraf43 displayed a dimeric structure and elongated molecular envelope. Kratky plot analysis revealed the folded state of CtAraf43 with limited flexibility at both concentrations. The data revealed higher flexibility at the C-terminal of CtAraf43 suggesting a coordinated action of the N-terminal catalytic module CtAbf43A and the internal CtCBM6A. AbbreviationCBMsCarbohydrate Binding ModulesCtAraf43α-L-arabinofuranosidaseGHsGlycoside HydrolasesMDMolecular DynamicsRMSDRoot Mean Square DeviationRMSFRoot Mean Square FluctuationSAXSSmall angle X-ray scattering Carbohydrate Binding Modules α-L-arabinofuranosidase Glycoside Hydrolases Molecular Dynamics Root Mean Square Deviation Root Mean Square Fluctuation Small angle X-ray scattering Communicated by Ramaswamy H. Sarma

The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure–function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure ofClostridium thermocellumarabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported.CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure ofCtAbf43A comprises a five-bladed β-propeller fold typical of GH43 enzymes.CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis ofCtAbf43A along with the other member of GH43 subfamily 16 with known structure, theBacillus subtilisarabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.

The development of new food products, improvement in food quality, and ease of food production process is of prime concern with the growing world population and rapidly rising demand for functional foods. These concerns make it imperative, the use of various enzymes such as glycoside hydrolases, lipases, proteases, transglutaminases, etc., in the processing of food and food ingredients. Crops and fruits used in food and brewing industry contain considerable amount of xylan. Xylan is a branched heteropolysaccharide and its main chain is composed of xylose subunits linked by β-(1 → 4) glycosidic bonds and contains different substitutions in the side chain. Xylanase cleaves β-(1 → 4) glycosidic bonds in heteroxylan randomly and converts it into xylooligosaccharides. In the last decade, xylanase has received appreciable attention owing to its applications in various food processing industries such as cereal food processing for the improvement of gluten agglomeration, baking industry for the improved texture of bread and cookies, clarification of fruit juices, production of xylooligosaccharide or arabinoxylooligosaccharides as prebiotic food supplements. This chapter presents a comprehensive overview of xylanase, its sources, production, and applications in food production and processing, with a particular focus on recent developments.

Cellulose, hemicellulose, pectin, and lignin are the major components of plant cell wall. Hemicellulose is the second most abundant carbohydrate polymer on the earth. Hemicelluloses are branched, hetero-polysaccharides formed by β-(1 → 4)-linked backbones of hexoses like glucose (xyloglucan), galactose (galactan), mannose (mannan) or pentoses like xylose (xylan), and arabinose (arabinan). Xylan contains the backbone of 1,4-linked-β-d-xylopyranose with various side-chain substitutions such as arabinose, acetic acid, glucuronic acid, ferulic, acid, and p-coumaric acid. l-arabinose side chain is found in hemicelluloses like arabinan, arabinoxylan, oat spelt xylan, and arabinogalactan. The extent of side-chain substitution depends on the source of the xylan, which makes its structure complex and hinders its enzymatic hydrolysis. α-l-arabinofuranosidase hydrolyzes arabinose side chain present at α-1,2-, α-1,3-, and α-1,5-positions in arabinoxylan, thus potentiating other xylanolytic enzymes to act efficiently on the backbone. Therefore, α-l-arabinofuranosidase has potential application in agro-industrial processes because of its functioning synergistically with other hemicellulases. α-l-arabinofuranosidases are used for improving bread quality, for wine flavor, for clarification of fruit juices, as supplement for feedstock for enhancing digestion, in the production of medicinal compounds, and in the production of oligosaccharide and modification of their side chains. This chapter presents a comprehensive overview of α-l-arabinofuranosidase, sources, production, and its applications in food processing.

The bulk properties such as lattice constants, bulk moduli, and cohesive energies of alkali, alkaline-earth, and transition metals are studied within the framework of the recently developed meta-GGA (meta-Generalized Gradient Approximation) level semilocal exchange-correlation functionals. To establish the applicability, broadness, and accuracy of meta-GGA functionals, we also put the results obtained using PBE (Perdew-Burke-Ernzerhof) [J. P. Perdew et al., Phys. Rev. Lett. 77, 3865 (1996)] and PBE reparameterized for solid [J. P. Perdew et al., Phys. Rev. Lett. 100, 136406 (2008)] GGA functionals. The interesting feature of the present paper is that it measures the accuracy of the recently developed TM (Tao-Mo), TMTPSS [TM exchange with Tao-Perdew-Staroverov-Scuseria (TPSS)] [J. Tao and Y. Mo, Phys. Rev. Lett. 117, 073001 (2016)] correlation, and strongly constrained and appropriately normed [J. Sun et al., Phys. Rev. Lett. 115, 036402 (2015)] functionals to calculate the aforementioned properties. Not only that, we also include other (popular) meta-GGA functionals in order to have a closer look at the performance of the meta-GGA functionals too. The present systematic investigation shows that the TM functional is accurate in describing the lattice constants while for cohesive energies and bulk moduli, the PBE and modified TPSS perform better compared to others.

A revised version of the semilocal exchange-correlation functional [Phys. Rev. Lett. 117, 073001 (2016)] (TM) is proposed by incorporating the modifications to its correlation content obtained from the full high-density second-order gradient expansion as proposed in the case of revised Tao-Perdew-Staroverov-Scuseria (revTPSS) [Phys. Rev. Lett. 103, 026403 (2009)] functional. The present construction improves the performance of TM functional over a wide range of quantum chemical and solid-state properties (thermochemistry and structural). More specifically, the cohesive energies, jellium surface exchange-correlation energies, and real metallic surface energies are improved by preserving the accuracy of the solid-state lattice constants and bulk moduli. The present proposition is not only physically motivated but also enhances the applicability of the TM functional. New physical insights with proper exemplification of the present modification which is presented here can further serve for more realistic non-empirical density functional construction.

β-mannanases have been shown to play an important role in various biological processes such as the cell wall component degradation, defence signalling in plants, the mobilization of storage reserves and in various industrial processes. To date, glycoside hydrolases (GHs) have been divided into 135 families and 14 clans from A to N based upon their sequence, overall structural fold and function. β-mannanases belong glycoside hydrolases and exist under four different glycoside hydrolase families, GH5, GH26, GH113 and GH134. GH5 and GH26 are combined in clan GH-A. GH5 and GH26 contain hydrolases which follow the retaining type reaction mechanism. Structural survey of β-mannanases of GH5 and GH26, suggests that both families contain similar TIM barrel fold. In addition, they also share common catalytic residues and their location in the structure. Despite these structural similarities, a distinct difference lies between the substrate binding sub-sites which define substrate specificity. This review summarizes the recent reports on the structure and function perspectives of β-mannanases of GH5 and GH26 and highlights the similarities and differences between them.