Application of ANN-MOGA for nutrient sequestration for wastewater remediation and production of polyunsaturated fatty acid (PUFA) by Chlorella sorokiniana MSP1.

Chlorella bears excellent potential in removing nutrients from industrial wastewater and lipid production enriched with polyunsaturated fatty acids. However, due to the changing nutrient dynamics of wastewater, growth and metabolic activity of Chlorella are affected. In order to sustain microalgal growth in wastewater with concomitant production of PUFA rich lipids, RSM (Response Surface Methodology) followed by heuristic hybrid computation model ANN-MOGA (Artificial Neural Network- Multi-Objective Genetic Algorithm) were implemented. Preliminary experiments conducted taking one factor at a time and design matrix of RSM with process variables viz. Sodium chloride (1 mM-40 mM), Magnesium sulphate (100 mg-800 mg) and incubation time (4th day to 20th day) were validated by ANN-MOGA. The study reported improved biomass and lipid yield by 54.25% and 12.76%, along with total nitrogen and phosphorus removal by 21.92% and 18.72% respectively using ANN-MOGA. It was evident from FAME results that there was a significantly improved concentration of linoleic acid (19.1%) and γ-linolenic acid (21.1%). Improved PUFA content makes it a potential feedstock with application in cosmeceutical, pharmaceutical and nutraceutical industry. The study further proves that C. sorokiniana MSP1 mediated industrial wastewater treatment with PUFA production is an effective way in providing environmental benefits along with value addition. Moreover, ANN-MOGA is a relevant tool that could control microalgal growth in wastewater.

Recent advances in enhancing the production of long chain omega-3 fatty acids in microalgae.

Omega-3 fatty acids have gained attention due to numerous health benefits. Eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) are long chain omega-3 fatty acids produced from precursor ALA (α-linolenic acid) in humans but their rate of biosynthesis is low, therefore, these must be present in diet or should be taken as supplements. The commercial sources of omega-3 fatty acids are limited to vegetable oils and marine sources. The rising concern about vegan source, fish aquaculture conservation and heavy metal contamination in fish has led to the search for their alternative source. Microalgae have gained importance due to the production of high-value EPA and DHA and can thus serve as a sustainable and promising source of long chain omega-3 fatty acids. Although the bottleneck lies in the optimization for enhanced production that involves strategies viz. strain selection, optimization of cultivation conditions, media, metabolic and genetic engineering approaches; while co-cultivation, use of nanoparticles and strategic blending have emerged as innovative approaches that have made microalgae as potential candidates for EPA and DHA production. This review highlights the possible strategies for the enhancement of EPA and DHA production in microalgae. This will pave the way for their large-scale production for human health benefits.

Molecular interaction between plants and Trichoderma species against soil-borne plant pathogens.

Trichoderma spp. (Hypocreales) are used worldwide as a lucrative biocontrol agent. The interactions of Trichoderma spp. with host plants and pathogens at a molecular level are important in understanding the various mechanisms adopted by the fungus to attain a close relationship with their plant host through superior antifungal/antimicrobial activity. When working in synchrony, mycoparasitism, antibiosis, competition, and the induction of a systemic acquired resistance (SAR)-like response are considered key factors in deciding the biocontrol potential of Trichoderma. Sucrose-rich root exudates of the host plant attract Trichoderma. The soluble secretome of Trichoderma plays a significant role in attachment to and penetration and colonization of plant roots, as well as modulating the mycoparasitic and antibiosis activity of Trichoderma. This review aims to gather information on how Trichoderma interacts with host plants and its role as a biocontrol agent of soil-borne phytopathogens, and to give a comprehensive account of the diverse molecular aspects of this interaction.

Arsenic causing gallbladder cancer disease in Bihar.

In recent times Gallbladder cancer (GBC) incidences increased many folds in India and are being reported from arsenic hotspots identified in Bihar. The study aims to establish association between arsenic exposure and gallbladder carcinogenesis. In the present study, n = 200 were control volunteers and n = 152 confirmed gallbladder cancer cases. The studied GBC patient's biological samples-gallbladder tissue, gallbladder stone, bile, blood and hair samples were collected for arsenic estimation. Moreover, n = 512 gallbladder cancer patients blood samples were also evaluated for the presence of arsenic to understand exposure level in the population. A significantly high arsenic concentration (p < 0.05) was detected in the blood samples with maximum concentration 389 µg/L in GBC cases in comparison to control. Similarly, in the gallbladder cancer patients, there was significantly high arsenic concentration observed in gallbladder tissue with highest concentration of 2166 µg/kg, in gallbladder stones 635 µg/kg, in bile samples 483 µg/L and in hair samples 6980 µg/kg respectively. Moreover, the n = 512 gallbladder cancer patient's blood samples study revealed very significant arsenic concentration in the population of Bihar with maximum arsenic concentration as 746 µg/L. The raised arsenic concentration in the gallbladder cancer patients' biological samples-gallbladder tissue, gallbladder stone, bile, blood, and hair samples was significantly very high in the arsenic exposed area. The study denotes that the gallbladder disease burden is very high in the arsenic exposed area of Bihar. The findings do provide a strong link between arsenic contamination and increased gallbladder carcinogenesis.

Nanotechnological approaches for management of soil-borne plant pathogens.

Soil borne pathogens are significant contributor of plant yield loss globally. The constraints in early diagnosis, wide host range, longer persistence in soil makes their management cumbersome and difficult. Therefore, it is crucial to devise innovative and effective management strategy to combat the losses caused by soil borne diseases. The use of chemical pesticides is the mainstay of current plant disease management practices that potentially cause ecological imbalance. Nanotechnology presents a suitable alternative to overcome the challenges associated with diagnosis and management of soil-borne plant pathogens. This review explores the use of nanotechnology for the management of soil-borne diseases using a variety of strategies, such as nanoparticles acting as a protectant, as carriers of actives like pesticides, fertilizers, antimicrobials, and microbes or by promoting plant growth and development. Nanotechnology can also be used for precise and accurate detection of soil-borne pathogens for devising efficient management strategy. The unique physico-chemical properties of nanoparticles allow greater penetration and interaction with biological membrane thereby increasing its efficacy and releasability. However, the nanoscience specifically agricultural nanotechnology is still in its toddler stage and to realize its full potential, extensive field trials, utilization of pest crop host system and toxicological studies are essential to tackle the fundamental queries associated with development of commercial nano-formulations.

Advances in Nanotechnology as a Potential Alternative for Plant Viral Disease Management.

Plant viruses cause enormous losses in agricultural production accounting for about 47% of the total overall crop losses caused by plant pathogens. More than 50% of the emerging plant diseases are reported to be caused by viruses, which are inevitable or unmanageable. Therefore, it is essential to devise novel and effective management strategies to combat the losses caused by the plant virus in economically important crops. Nanotechnology presents a new tendency against the increasing challenges in the diagnosis and management of plant viruses as well as plant health. The application of nanotechnology in plant virology, known as nanophytovirology, includes disease diagnostics, drug delivery, genetic transformation, therapeutants, plant defense induction, and bio-stimulation; however, it is still in the nascent stage. The unique physicochemical properties of particles in the nanoscale allow greater interaction and it may knock out the virus particles. Thus, it opens up a novel arena for the management of plant viral diseases. The main objective of this review is to focus on the mounting collection of tools and techniques involved in the viral disease diagnosis and management and to elucidate their mode of action along with toxicological concerns.

Current Perspectives on Chitinolytic Enzymes and Their Agro-Industrial Applications.

Chitinases are a large and diversified category of enzymes that break down chitin, the world's second most prevalent polymer after cellulose. GH18 is the most studied family of chitinases, even though chitinolytic enzymes come from a variety of glycosyl hydrolase (GH) families. Most of the distinct GH families, as well as the unique structural and catalytic features of various chitinolytic enzymes, have been thoroughly explored to demonstrate their use in the development of tailor-made chitinases by protein engineering. Although chitin-degrading enzymes may be found in plants and other organisms, such as arthropods, mollusks, protozoans, and nematodes, microbial chitinases are a promising and sustainable option for industrial production. Despite this, the inducible nature, low titer, high production expenses, and susceptibility to severe environments are barriers to upscaling microbial chitinase production. The goal of this study is to address all of the elements that influence microbial fermentation for chitinase production, as well as the purifying procedures for attaining high-quality yield and purity.

Characterization of the starch synthase under terminal heat stress and its effect on grain quality of wheat.

Terminal heat stress (HS) is a key barrier for wheat grain yield and quality. Various physiochemical and molecular parameters such as photosynthetic rate, expression analysis and activity of starch synthase (SS), total starch, amylose and amylopectin content, total amylolytic activity, and total antioxidant capacity (TAC) were analysed in wheat cvs.HD3059 (thermotolerant) and BT-Schomburgk (thermosusceptible) at grain-filling stage under HS (32 °C and 40 °C, 1 h). The decrease in photosynthetic rate was observed under HS. Expression analysis of the SS gene at transcript level showed downregulation in both the wheat cvs.HD3059 and BT-Schombugk under HS (32 °C and 40 ºC, 1 h) as compared to the control. Although the downregulation of SS gene transcript expression was less in HD3059 than BT-Schombugk. Both the cultivars showed decrease in starch synthase activity and starch content under HS and the overall content was higher in HD3059, compared to BT-Schomburgk. Higher total amylolytic activity and amylose content were observed in BT-Schomburgk. Scanning electron microscopy (SEM) showed un-structured starch granules under HS. Total antioxidant capacity (TAC) was found higher in HD3059 (14.07 mM FeSO4 gm-1 FW) compared to BT-Schomburgk (8.89 mM FeSO4 gm-1 FW) under HS (40 ºC, 1 h). Findings suggest that HS during grain filling stage had more severe impact on the overall physiochemical properties of the wheat grain. Thus the starch bisynthesis pathway associated gene(s) could be exploit to enhance the yield and quality of wheat under heat stress.

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents.

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55 °C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72 h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.

Cytosolic tryparedoxin of Leishmania donovani modulates host immune response in visceral leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by the unicellular protozoan parasite of genus Leishmania. Tryparedoxin (TXN) is a low molecular mass dithiol protein belonging to oxidoreductases super-family; which function in concert with tryparedoxin peroxidase (TXNPx) as a system in protozoan parasites including Leishmania. Leishmanial hydroperoxides detoxification cascade uses trypanothione as electron donor to reduce hydroperoxide inside the macrophages during infection. However, the mechanism by which tryparedoxin can contribute in progression of visceral leishmaniasis (VL) and its impact on host's cellular immune response during infection in Indian VL patient is unknown. In this study, we purified a ∼17 kDa recombinant cytosolic tryparedoxin (cTXN) protein of Leishmania donovani (rLdcTXN) and investigated its immunological responses in peripheral blood monocytes (PBMC) isolated from VL patients. The protein significantly enhanced the promastigotes count after 96 h of culture showing a direct correlation with parasite growth. Furthermore, stimulation of PBMC isolated from VL patients with rLdcTXN resulted in up-regulation of IL-4 and IL-10 production whereas IL-12 and IFN-γ was significantly down-regulated suggesting a pivotal role of cTXN in provoking the immune suppression during VL. Our study demonstrates the importance of cTXN protein which can potentially modulate the outcome of disease through suppressing host protective Th1 response in VL patients.

Bioprospecting microbes for single-cell oil production from starchy wastes.

Production of lipid from oleaginous yeast using starch as a carbon source is not a common practice; therefore, the purpose of this investigation was to explore the capability of starch assimilating microbes to produce oil, which was determined in terms of biomass weight, productivity, and lipid yield. Saccharomyces pastorianus, Rhodotorula mucilaginosa, Rhodotorula glutinis, and fungal isolate Ganoderma wiiroense were screened for the key parameters. The optimization was also performed by one-factor-at-a-time approach. Considering the specific yield of lipid and cell dry weight yield, R. glutinis and R. mucilaginosa showed superiority over other strains. G. wiiroense, a new isolate, would also be a promising strain for starch waste utilization in terms of extracellular and intracellular specific yield of lipids. Extracellular specific yield of lipid was highest in R. glutinis culture (0.025 g g-1 of biomass) followed by R. mucilaginosa (0.022 g g-1 of biomass) and G. wiiroense (0.020 g g-1 of biomass). Intracellular lipid was again highest in R. glutinis (0.048 g g-1 of biomass). The most prominent fatty acid methyl esters among the lipid as detected by GC-MS were saturated lipids mainly octadecanoic acid, tetradecanoate, and hexadecanoate. Extracellular lipid produced on starch substrate waste would be a cost-effective alternative for energy-intensive extraction process in biodiesel industry.

Potential of ionic liquids for inhibiting the growth and ß-lactamase production by Bacillus cereus EMB20.

Present work reports the inhibition of Bacillus cereus EMB20 ß-lactamase by a deep eutectic solvent, maline in an uncompetitive manner. Far-UV CD and intrinsic fluorescence spectroscopy revealed a disrupted secondary as well as tertiary structure as a function of maline concentration. The effect of individual components of maline on ß-lactamase inhibition showed that malonic acid was mainly responsible for inhibiting the ß-lactamase. Structural and docking studies found that malonic acid led to major perturbations in the secondary and tertiary structure of the enzyme while H-bonding with the active site residues. Further the antibacterial and cytotoxic studies also confirmed the potential of maline as a potent growth inhibitor of ß-lactamase producing B. cereus EMB20.

Biodegradation of waste grease by Penicillium chrysogenum for production of fatty acid.

The aim of present work was to effectively remediate grease waste by Penicillium chrysogenum. For efficient degradation, grease waste was pre-treated using various lipases, among them lipolase was the best. The pretreated grease was used as a substrate by P. chrysogenum resulting into the production of fatty acids. Process was optimized by response surface methodology (RSM) using four variables viz; FeCl2 (mM), spore concentration (spores/ml), time period (days) and amount of grease (g). The optimized conditions viz; FeCl2 1.25mM, culture amount 5×1011spores/ml and time period 16days led to the production of 6.6mg/g fatty acid from 10.0g of pre-treated grease mixed with 5.0g wheat bran in 10.0ml czapek-dox medium under solid state fermentation. The fermented media was extracted with hexane and subjected to GCMS analysis, which showed the presence of higher amount of palmitic acid. It was purified by crystallization method and 2.8g of palmitic acid was recovered from 1.0kg grease waste in tray fermentation.

Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts.

Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability.

Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology.

Lipase catalyzes hydrolysis of fats in lipid water interphase and perform variety of biotransformation reactions under micro aqueous conditions. The major sources include microbial lipases; among these yeast and fungal lipases are of special interest because they can carry out various stereoselective reactions. These lipases are highly diverse and are categorized into three classes on the basis of oxyanion hole: GX, GGGX and Y. The detailed phylogenetic analysis showed that GX family is more diverse than GGGX and Y family. Sequence and structural comparisons revealed that lipases are conserved only in the signature sequence region. Their characteristic structural determinants viz. lid, binding pocket and oxyanion hole are hotspots for mutagenesis. Few examples are cited in this review to highlight the multidisciplinary approaches for designing novel enzyme variants with improved thermo stability and substrate specificity. In addition, we present a brief account on biotechnological applications of lipases. Lipases have also gained attention as virulence factors, therefore, we surveyed the role of lipases in yeast physiology related to colonization, adhesion, biofilm formation and pathogenesis. The new genomic era has opened numerous possibilities to genetically manipulate lipases for food, fuel and pharmaceuticals.

Functional characterisation of novel enantioselective lipase TALipA from Trichosporon asahii MSR54: sequence comparison revealed new signature sequence AXSXG among yeast lipases.

A gene encoding lipase TALipA from Trichosporon asahii MSR54 was successfully isolated, cloned and expressed in Pichia pastoris X-33. It was purified to homogeneity by affinity chromatography with 1.7 purification fold. SDS-PAGE revealed it as a monomeric 27-kDa protein. Sequence comparison showed that it has close affinity with bacterial and actinobacterial lipases. It has unique oxyanion hole "GL" and conserved pentapeptide AHSMG where alanine is present instead of glycine, which is unique to yeast lipase database. The temperature and pH optima for activity were 60 °C and pH 8.0, respectively. It is thermostable with t1/2 of 68 min at 70 °C. It hydrolyzed p-np esters with better specificity on p-np palmitate, which was again confirmed during hydrolysis of triacylglyceride mixture. The enzyme was found to be regioselective during hydrolysis of triolein. It exhibited enantio preference during esterification of phenylethanol depending upon solvent used. It was S-enantioselective in 1,4-dioxane and R-selective in isopropanol and hexane. It is a magnesium-activated metalloenzyme inhibited by 10-mM EDTA. It was stable towards most of the polar and non-polar solvents.

Functional characterization of a novel aspartic acid rich lipase, TALipC, from Trichosporon asahii MSR54: solvent-dependent enantio inversion during esterification of 1-phenylethanol.

A novel lipase gene TAlipC was isolated from Trichosporon asahii MSR54 and functionally expressed in Pichia pastoris. The protein was His-tagged and purified to homogeneity by affinity chromatography. Sequence comparison revealed a high homology with lipases from Cryptococcus sp. It had a GX type oxyanion hole and a GHSLG-type conserved penta-peptide similar to those in the lipases from Yarrowia lipolytica. The enzyme had optimal activity at pH 8 and 50 °C. It was specific for long chain fatty acid groups of p-nitrophenol esters and triacylglycerols, showing regio- and enantio-selectivity. It was activated by Mg(2+) ions (20 mM) and had a predicted Mg-binding domain at the aspartic acid-rich C-terminal. Solvent-based enantio- inversion was the key feature of the enzyme where it showed (S)-selectivity in 1,4-dioxane and 2-propanol and (R)-selectivity in hexane during chiral separation of (±)1-phenylethanol by esterification.

Novel strategy of using methyl esters as slow release methanol source during lipase expression by mut+ Pichia pastoris X33.

One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut+ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression.

Phenylalanine to leucine point mutation in oxyanion hole improved catalytic efficiency of Lip12 from Yarrowia lipolytica.

In lipases, oxyanion hole has crucial role in the stabilisation of enzyme-substrate complex. Majority of lipases from Yarrowia lipolytica consist of two oxyanion hole residues viz.; Thr and Leu. However, Lip12 has Phe instead of Leu at second oxyanion hole residue. It was observed that Lip12 has lower specific activity and catalytic efficiency than other lipases of Yarrowia. In silico analysis of Phe to Leu mutation revealed improved binding energy of Lip12 for p-np palmitate. This was validated by Phe148 to Leu point mutation where, specific activity of mutant was 401U/mg on olive oil, which was two fold higher in comparison to wild-type. Kcat, remained unaltered, while decrease in Km was predominant for all the substrates used in the study. Improved catalytic efficiency of mutant was a function of chain length in case of p-np esters, with 73% improvement for p-np stearate. However, hydrolysis of triacylglycerides improved by 20%, irrespective of chain length. Decrease in activation energy for all the substrates, was observed in mutant in comparison to wild-type, indicating better stabilisation of transition state complex. Further, unaltered differential activation energy for mutant depicts that substrate specificity of enzyme remained same after mutation.

Comparative biochemical characterization and in silico analysis of novel lipases Lip11 and Lip12 with Lip2 from Yarrowia lipolytica.

Novel lipases lip11 and lip12 from Yarrowia lipolytica MSR80 were cloned and expressed in E. coli HB101 pEZZ18 system along with lip2. These enzymes were constitutively expressed as extracellular proteins with IgG tag. The enzymes were purified by affinity chromatography and analyzed by SDS-PAGE with specific activity of 314, 352 and 198 U/mg for Lip2, Lip11 and Lip12, respectively on olive oil. Biochemical characterization showed that all were active over broad range of pH 4.0-9.0 and temperature 20-80 °C with optima at pH 7 and 40 °C. All the three lipases were thermostable up to 80 °C with varying t(½). Activity on various substrates revealed that they were most active on oils > triacylglycerides > p-np-esters. Relatively Lip2 and Lip11 showed specificity for mid to long chain fatty acids, while Lip12 was mid chain specific. GC analysis of triolein hydrolysis by these lipases revealed that Lip2 and Lip11 are regioselective, while Lip12 is not. Effect of metal ions showed that Lip2 and Lip12 were activated by Ca²âº whereas Lip11 by Mg²âº. All were thiol activated and inhibited by PMSF and N-bromosuccinimide. All were activated by non polar solvents and inhibited by polar solvents. Detailed sequence analysis and structural predictions revealed Lip11 and Lip12 shared 61 and 62 % homology with Lip2 (3O0D) and three dimensional superimposition revealed Lip2 was closer to Lip11 than to Lip12 as was observed during biochemical characterization. Finally, thermostability and substrate specificity has been explained on the basis of detailed amino acid analysis.