Aspergillus niger Ochratoxinase Is a Highly Specific, Metal-Dependent Amidohydrolase Suitable for OTA Biodetoxification in Food and Feed.

Microbial enzymes can be used as processing aids or additives in food and feed industries. Enzymatic detoxification of ochratoxin A (OTA) is a promising method to reduce OTA content. Here, we characterize the full-length enzyme ochratoxinase (AnOTA), an amidohydrolase from Aspergillus niger. AnOTA hydrolyzes OTA and ochratoxin B (OTB) mycotoxins efficiently and also other substrates containing phenylalanine, alanine, or leucine residues at their C-terminal position, revealing a narrow specificity profile. AnOTA lacks endopeptidase or aminoacylase activities. The structural basis of the molecular recognition by AnOTA of OTA, OTB, and a wide array of model substrates has been investigated by molecular docking simulation. AnOTA shows maximal hydrolytic activity at neutral pH and high temperature (65 °C) and retained high activity after prolonged incubation at 45 °C. The reduction of OTA levels in food products by AnOTA has been investigated using several commercial plant-based beverages. The results showed complete degradation of OTA with no detectable modification of beverage proteins. Therefore, the addition of AnOTA seems to be a useful procedure to eliminate OTA in plant-based beverages. Moreover, computational predictions of in vivo characteristics indicated that AnOTA is neither an allergenic nor antigenic protein. All characteristics found for AnOTA supported the suitability of its use for OTA detoxification in food and feed.

Aurasperone A Inhibits SARS CoV-2 In Vitro: An Integrated In Vitro and In Silico Study.

Several natural products recovered from a marine-derived Aspergillus niger were tested for their inhibitory activity against SARS CoV-2 in vitro. Aurasperone A (3) was found to inhibit SARS CoV-2 efficiently (IC50 = 12.25 µM) with comparable activity with the positive control remdesivir (IC50 = 10.11 µM). Aurasperone A exerted minimal cytotoxicity on Vero E6 cells (CC50 = 32.36 mM, SI = 2641.5) and it was found to be much safer than remdesivir (CC50 = 415.22 µM, SI = 41.07). To putatively highlight its molecular target, aurasperone A was subjected to molecular docking against several key-viral protein targets followed by a series of molecular dynamics-based in silico experiments that suggested Mpro to be its primary viral protein target. More potent anti-SARS CoV-2 Mpro inhibitors can be developed according to our findings presented in the present investigation.

Biological activity of chitosan inducing resistance efficiency of rice (Oryza sativa L.) after treatment with fungal based chitosan.

Reduced pathogen resistance and management of the left-over rice stubble are among the most important challenges faced in rice cultivation. A novel and eco-friendly strategy to synthesise 'Fungal Chitosan' (FC) from Aspergillus niger using rice straw could serve as a sustainable treatment approach to improve both disease resistance and yields, while also effectively managing the rice stubble waste. The FC treatment promoted germination as well as growth parameters in rice varieties, TN1 (high yielding-susceptible) and PTB33 (low yielding-resistant) better than a commercial chitosan (PC). Treatments of exogenously applied FC to plants produced direct toxicity to Xoo, and reduced the BLB disease index by 39.9% in TN1. The capability of FC to trigger a cascade of defense pathways was evident from the measurable changes in the kinetics of defense enzymes, peroxidase (POD) and polyphenol oxidase (PPO). FC treatment increased levels of POD in TN1 by 59.4%, which was 35.3% greater than that of untreated PTB33. Therefore, the study demonstrated the effectiveness of FC treatments for use in agriculture as a potential biostimulant as well as protective agent against bacterial leaf blight, BLB, of rice (Oryza sativa) that could be produced from stubble waste and improve rice stubble management strategies.

Biochemical characterization of xylanase GH11 isolated from Aspergillus niger BCC14405 (XylB) and its application in xylooligosaccharide production.

OBJECTIVE: To develop an endo-ß-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. RESULTS: The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg-1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g-1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. CONCLUSION: The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.

In Silico Analysis of Fungal and Chloride-Dependent α-Amylases within the Family GH13 with Identification of Possible Secondary Surface-Binding Sites.

This study brings a detailed bioinformatics analysis of fungal and chloride-dependent α-amylases from the family GH13. Overall, 268 α-amylase sequences were retrieved from subfamilies GH13_1 (39 sequences), GH13_5 (35 sequences), GH13_15 (28 sequences), GH13_24 (23 sequences), GH13_32 (140 sequences) and GH13_42 (3 sequences). Eight conserved sequence regions (CSRs) characteristic for the family GH13 were identified in all sequences and respective sequence logos were analysed in an effort to identify unique sequence features of each subfamily. The main emphasis was given on the subfamily GH13_32 since it contains both fungal α-amylases and their bacterial chloride-activated counterparts. In addition to in silico analysis focused on eventual ability to bind the chloride anion, the property typical mainly for animal α-amylases from subfamilies GH13_15 and GH13_24, attention has been paid also to the potential presence of the so-called secondary surface-binding sites (SBSs) identified in complexed crystal structures of some particular α-amylases from the studied subfamilies. As template enzymes with already experimentally determined SBSs, the α-amylases from Aspergillus niger (GH13_1), Bacillus halmapalus, Bacillus paralicheniformis and Halothermothrix orenii (all from GH13_5) and Homo sapiens (saliva; GH13_24) were used. Evolutionary relationships between GH13 fungal and chloride-dependent α-amylases were demonstrated by two evolutionary trees-one based on the alignment of the segment of sequences spanning almost the entire catalytic TIM-barrel domain and the other one based on the alignment of eight extracted CSRs. Although both trees demonstrated similar results in terms of a closer evolutionary relatedness of subfamilies GH13_1 with GH13_42 including in a wider sense also the subfamily GH13_5 as well as for subfamilies GH13_32, GH13_15 and GH13_24, some subtle differences in clustering of particular α-amylases may nevertheless be observed.

Acetylcholine esterase inhibitory activity of green synthesized nanosilver by naphthopyrones isolated from marine-derived Aspergillus niger.

Aspergillus niger metabolites exhibited a wide range of biological properties including antioxidant and neuro-protective effects and some physical properties as green synthesis of silver nanoparticles AgNP. The present study presents a novel evidence for the various biological activities of green synthesized AgNPs. For the first time, some isolated naphtho-γ-pyrones from marine-derived Aspergillus niger, flavasperone (1), rubrofusarin B (2), aurasperone A (3), fonsecinone A (4) in addition to one alkaloid aspernigrin A (7) were invistigated for their inhibitory activity of acetylcholine esterase AChE, a hallmark of Alzheimer's disease (AD). The ability to synthesize AgNPs by compounds 3, 4 and 7 has been also tested for the first time. Green synthesized AgNPs were well-dispersed, and their size was ranging from 8-30 nm in diameter, their morphology was obviously spherical capped with the organic compounds. Further biological evaluation of their AChE inhibitory activity was compared to the parent compounds. AgNps dramatically increased the inhibitory activity of Compounds 4, 3 and 7 by 84, 16 and 13 fold, respectively to be more potent than galanthamine as a positive control with IC50 value of 1.43 compared to 0.089, 0.311 and 1.53 of AgNPs of Compounds 4, 3 and 7, respectively. Also compound 2 showed moderate inhibitory activity. This is could be probably explained by closer fitting to the active sites or the synergistic effect of the stabilized AgNPs by the organic compouds. These results, in addition to other intrinsic chemical and biological properties of naphtho-γ-pyrones, suggest that the latter could be further explored with a view towards other neuroprotective studies for alleviating AD.

Saccharomyces Cerevisiae as an Untapped Source of Fungal Chitosan for Antimicrobial Action.

Despite being widely available, Saccharomyces cerevisiae has not been widely explored for direct extraction of chitosan biopolymer for antimicrobial applications. In our study, S. cerevisiae from Baker's yeast and Aspergillus niger from moldy onion extracts are studied as alternative sources of chitosan; and S cerevisiae chitosan tested for antimicrobial efficacy. The properties of S. cerevisiae chitosan are compared with moldy onion chitosan and shrimp chitosan extracted from shrimp shells. Chitosan extracted from S. cerevisiae is tested for antimicrobial efficacy against Staphylococcus Aureus. The maximum yields of fungal chitosan are 20.85 ± 0.35 mg/g dry S. cerevisiae biomass at 4th day using a culture broth containing sodium acetate, and 16.15 ± 0.95 mg/g dry A. niger biomass at 12th day. The degree of deacetylation (DD%) of the extracted fungal chitosan samples from S. cerevisiae and A. niger is found to be 63.4%, and 61.2% respectively, using Fourier Transform Infrared Spectroscopy. At a concentration of 2 g/L, S. cerevisiae chitosan shows the maximum inhibition zone diameter of 15.48 ± 0.07 mm. Baker's yeast S cerevisiae biomass and A. niger from moldy onions has not been previously explored as a source of extractible fungal chitosan. This study gives insight that S. cerevisiae and A. niger from agricultural or industrial wastes could be a potential biomass source for production of the chitosan biopolymer. The S. cerevisiae chitosan displayed effective antimicrobial properties against S aureus, indicating the viablitiy of S cerevisae as a resource for extraction of high-quality chitosan.

Production of Protocatechuic Acid from p-Hydroxyphenyl (H) Units and Related Aromatic Compounds Using an Aspergillus niger Cell Factory.

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great pharmaceutical interest. Much research has been performed in creating bacterial protocatechuic acid production strains, but no protocatechuic acid-producing fungal cell factories have been described. The filamentous fungus Aspergillus niger can produce protocatechuic acid as an intermediate of the benzoic acid metabolic pathway. Recently, the p-hydroxybenzoate-m-hydroxylase (phhA) and protocatechuate 3,4-dioxygenase (prcA) of A. niger have been identified. It has been shown that the prcA deletion mutant is still able to grow on protocatechuic acid. This led to the identification of an alternative pathway that converts protocatechuic acid to hydroxyquinol (1,3,4-trihydroxybenzene). However, the gene involved in the hydroxylation of protocatechuic acid to hydroxyquinol remained unidentified. Here, we describe the identification of protocatechuate hydroxylase (decarboxylating) (PhyA) by using whole-genome transcriptome data. The identification of phyA enabled the creation of a fungal cell factory that is able to accumulate protocatechuic acid from benzyl alcohol, benzaldehyde, benzoic acid, caffeic acid, cinnamic acid, cinnamyl alcohol, m-hydroxybenzoic acid, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-anisyl alcohol, p-anisaldehyde, p-anisic acid, p-coumaric acid, and protocatechuic aldehyde. IMPORTANCE Aromatic compounds have broad applications and are used in many industries, such as the cosmetic, food, fragrance, paint, plastic, pharmaceutical, and polymer industries. The majority of aromatic compounds are synthesized from fossil sources, which are becoming limited. Plant biomass is the most abundant renewable resource on Earth and can be utilized to produce chemical building blocks, fuels, and bioplastics through fermentations with genetically modified microorganisms. Therefore, knowledge about the metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable aromatic compounds such as protocatechuic acid. Protocatechuic acid has many pharmaceutical properties but also can be used as a chemical building block to produce polymers and plastics. Here, we show that the fungus Aspergillus niger can be engineered to produce protocatechuic acid from plant-derived aromatic compounds and contributes to creating alternative methods for the production of platform chemicals. .

Naphtho-γ-pyrone Dimers from an Endozoic Aspergillus niger and the Effects of Coisolated Monomers in Combination with Cisplatin on a Cisplatin-Resistant A549 Cell Line.

Chemotherapy resistance is one of the main causes of lung cancer treatment failure, and a combination regimen may be an effective way to overcome this. Here we report 5 new (1-3, 7, and 9) and 15 known polyketides, isolated from an endozoic Aspergillus niger. The structures of the new compounds were determined by the interpretation of IR, HRESIMS, NMR, and ECD spectra. The ESI-MS/MS fragmentation of the isolated naphtho-γ-pyrone isomers in positive mode is discussed. The effects of isolated compounds in combination with cisplatin (DDP) on a DDP-resistant A549 cell line (A459/DDP) are investigated. The most active compound, 12, could reduce the ratio of GSH/GSSG, promote the generation of intracellular ROS, and cooperate with DDP to down-regulated levels of Nrf2, Akt, HO-1, and NQO1, suggesting that inhibition of Nrf2 and Akt pathways might be involved in the combined effect of 12 and DDP in A549/DDP cells.

The chemical profile of activated secondary metabolites by overexpressing LaeA in Aspergillus niger.

Although the mechanisms of regulating secondary metabolism by LaeA remains unclear, the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation. In our previous sutdy, RNA-seq data has showed that the transcriptional level of many SM backbone genes could be upregulated by overexpressing LaeA. Herein, we analyzed the chemical profile of activated secondary metabolites in the variant of A. niger FGSC A1279 by overexpressing LaeA (OElaeA). 14 compounds were activated in A. niger FGSC A1279 OElaeA variant in the WATM medium. Chemical workup of organic extracts of the culture broth from the A. niger OElaeA mutant identified three pure compounds, flaviolin, orlandin and kotanin. The structures of these compounds were confirmed by HR-ESIMS, 1D/2D NMR, and computer assisted structure elucidation (CASE). Based on homologous alignment and comparison of literatures, the biosynthetic gene cluster (fla) of flaviolin was identified. The in vivo function of the backbone gene, flaA, encoding a multidomain non-reducing polyketide synthase (SAT-KS-AT-PT-ACP), was verified via gene knockout and chemical analysis. Finally, a biosynthetic model for fungal flaviolin was proposed.

The fucose-specific lectin ANL from Aspergillus niger possesses anti-cancer activity by inducing the intrinsic apoptosis pathway in hepatocellular and colon cancer cells.

The L-fucose-specific lectin from Aspergillus niger (ANL), isolated from the corneal smears of a keratitis patient was reported earlier. Here, we examined the interaction of ANL with human hepatocellular and colon cancer cells, evaluated its anti-cancer activity and diagnostic potential to detect aberrantly glycosylated tumour-associated serum glycoproteins such as alpha-fetoprotein (AFP). We observed that ANL strongly bound to both HepG2 and HT-29 cell-lines and this interaction was effectively blocked with L-fucose and mucin in a dose and time-dependent manner with an IC50 of 1.25 and 5 µg/mL for HepG2 and HT-29 cells respectively at 48 hours. ANL treatment increased hypodiploidy and decreased the number of HepG2 cell in G0 -G1 phase at both 24 and 48 hours. Furthermore, ANL increased the level of apoptosis in both HepG2 and HT-29 cells in a time-dependent manner via enhanced production of reactive oxygen species and altered mitochondrial membrane potential, indicative of intrinsic apoptotis pathway activation. Immunoblot analysis confirmed the time-dependent elevation of levels of cytochrome c, initiator caspase-9 and activation of caspase-3. ANL immunohistochemistry on colon cancer tissue and quantification of AFP in HCC patient serum samples by developing an ANL-anti-AFP antibody sandwich enzyme-linked immunosorbent assay confirmed the diagnostic potential of ANL. Here, interaction of ANL with AFP could be effectively blocked in the presence of competing fucose-bearing glycans. We found ANL to be more sensitive than Lens culinaris lectin, a well-known fucose-specific lectin and currently used diagnostic agent. ANL can be further explored as a diagnostic and anti-cancer agent.

Label-free surface enhanced Raman scattering spectroscopy for discrimination and detection of dominant apple spoilage fungus.

Fungal infection is one of the main causes of apple corruption. The main dominant spoilage fungi in causing apple spoilage are storage mainly include Penicillium Paecilomyces paecilomyces (P. paecilomyces), penicillium chrysanthemum (P. chrysogenum), expanded Penicillium expansum (P. expansum), Aspergillus niger (Asp. niger) and Alternaria. In this study, surface-enhanced Raman spectroscopy (SERS) based on gold nanorod (AuNRs) substrate method was developed to collect and examine the Raman fingerprints of dominant apple spoilage fungus spores. Standard normal variable (SNV) was used to pretreat the obtained spectra to improve signal-to-noise ratio. Principal component analysis (PCA) was applied to extract useful spectral information. Linear discriminant analysis (LDA) and non-linear pattern recognition methods including K nearest neighbor (KNN), Support vector machine (SVM) and back propagation artificial neural networks (BPANN) were used to identify fungal species. As the comparison of modeling results shown, the BPANN model established based on the characteristic spectra variables have achieved the satisfactory result with discrimination accuracy of 98.23%; while the PCA-LDA model built using principal component variables achieved the best distinguish result with discrimination accuracy of 98.31%. It was concluded that SERS has the potential to be an inexpensive, rapid and effective method to detect and identify fungal species.

Citric acid from Aspergillus niger: a comprehensive overview.

Microbial citric acid has high economic importance and widely used in beverage, food, detergents, cosmetics and pharmaceutical industries. The filamentous fungus Aspergillus niger is a work horse and important cell factory in industry for the production of citric acid. Although in-depth literatures and reviews have been published to explain the biochemistry, biotechnology and genetic engineering study of citric acid production by Aspergillus niger separately but the present review compiled, all the aspects with upto date brief summary of the subject describing microorganisms, substrates and their pre-treatment, screening, fermentation techniques, metabolic engineering, biochemistry, product recovery and numerous biotechnological application of citric acid for simple understanding of microbial citric acid production. The availability of genome sequence of this organism has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism. An attempt has been also made to address the molecular mechanisms and application of recent advanced techniques such as CRISPR/Cas9 systems in enhancement of citric acid production.

Aspergillus niger lectin elicits MyD88 dependent proliferation and apoptosis at lower and higher doses in immortalized human corneal epithelial cells leading to pathogenesis.

An L-fucose lectin, ANL from the corneal smears of a mycotic keratitis patient was reported earlier. Interaction of ANL with immortalized Human Corneal Epithelial Cells (HCECs) was studied in order to assign the role of ANL in pathogenesis. ANL showed strong binding to HCECs which could be blocked by L-fucose and mucin. At concentrations below 0.6 µg/mL ANL showed proliferative effect and highest at 0.07 µg/mL leading to expression of proinflammatory cytokines IL-6 and IL-8. ANL induced proinflammatory response is mediated by TLR-2,-4, MyD88, NFkB and C-Jun dependent signaling. In contrast, ANL at concentrations above 0.6 µg/mL showed growth inhibitory effect at 48 h with an IC50 of 2.75 µg/mL. Western blot analysis revealed that HCECs treated with ANL at lower concentration induced the expression of proinflammatory signaling proteins TLR-2, 4, MyD88, NFkB and C-Jun which maintain high cell proliferating state. At higher concentration ANL induced apoptotic effect in HCECs with an increase in early apoptotic population as demonstrated by Annexin V-PI assay. ANL induced the expression of apoptotic proteins FADD, Caspase 8 and -3 mediated by MyD88. These findings demonstrate implication of ANL in pathogenesis and the findings are of clinical significance in developing strategy for controlling the infection leading to mycotic keratitis.

Chitin-glucan complex - Based biopolymeric structures using biocompatible ionic liquids.

This work explores the novelty of dissolving chitin-glucan complex (CGC), from two fungal strains, Komagataella pastoris (CGCP) and Aspergillus niger (CGCKZ) (KiOnutrime-CG™), using biocompatible ionic liquids (ILs). Three cholinium-based ILs were tested, choline acetate, choline propionate and choline hexanoate. Although all tested ILs resulted in the dissolution of the co-polymer at a concentration of 5 % (w/w), distinct polymeric structures, films or gels, were obtained from CGCP and CGCKZ, respectively. CGCP films were dense, flexible and elastic, with high swelling capacity (> 200 %). The IL anion alkyl chain length influenced the polymeric structures' properties, namely, the CGCP films elongation at break and swelling degree. CGCKZ resulted in weak gels. For both polymeric structures, exposure to the ILs under the dissolution conditions caused significant changes in the co-polymers' chemical structure, namely, reduction of their glucan moiety and reduction of the degree of acetylation, thus yielding chitosan-glucan complexes (ChGC) enriched in glucosamine (53.4 ± 0.3-60.8 ± 0.3 %).

Expression and function of an Hac1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae.

Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. However, low levels of foreign protein expression by S. cerevisiae have limited their widespread application. The ability of the endoplasmic reticulum (ER) to fold and process foreign proteins is an important factor restricting the expression of foreign proteins. In the current study, the effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were then examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL.

Potent Fungal Chitinase for the Bioconversion of Mycelial Waste.

Aspergillus niger mycelial waste is a good raw material for production of N-acetyl-d-glucosamine (GlcNAc). In this study, AnChiB, an A. niger chitinase which is upregulated during autolysis, was found to degrade A. niger mycelial waste with high efficiency. It could produce 1.45 mM (GlcNAc)2 in 8 h from raw mycelial waste, outperforming other chitinases, including bacterial SmChiA, human HsCht, and insect OfChtI and OfChi-h. The crystal structure of AnChiB was determined, and residues Trp106 and Trp118 were found to be important for the activity of AnChiB toward mycelial waste; mutation of either Trp106 or Trp118 into phenylalanine or alanine resulted in dramatically decreased activity. A recombinant strain of Bacillus subtilis was constructed to extracellularly produce AnChiB, and the culture supernatant was used to treat mycelial waste. This eco-friendly strategy could produce 3.7 mM of GlcNAc from 10 g of mycelial waste in 94 h with a yield of 71.3%.

High Performance Liquid Chromatography and Metabolomics Analysis of Tannase Metabolism of Gallic Acid and Gallates in Tea Leaves.

Tannase (E.C. 3.1.1.20) is hypothesized to be involved in the metabolism of gallates and gallic acid (GA) in pu-erh tea fermentation. In this work, we measured tannase in Aspergillus niger fermented tea leaves and confirmed the production of fungal tannase during pu-erh tea fermentation. A decrease in catechin and theaflavin gallates and a significant increase in GA content and the relative peak areas of ethyl gallate, procyanidin A2, procyanidin B2, procyanidin B3, catechin-catechin-catechin, epiafzelechin, and epicatechin-epiafzelechin [variable importance in the projection (VIP) > 1.0, p < 0.05, and fold change (FC) > 1.5] were observed using high performance liquid chromatography (HPLC) and metabolomics analysis of tea leaves fermented or hydrolyzed by tannase. In vitro assays showed that hydrolysis by tannase or polymerization of catechins increased the antioxidant activity of tea leaves. In summary, we identified a metabolic pathway for gallates and their derivatives in tea leaves hydrolyzed by tannase as well as associated changes in gallate and GA concentrations caused by fungal tannase during pu-erh tea fermentation.

Molecular wiring of glucose oxidase enzyme with Mn polypyridine complex on MWCNT modified electrode surface and its bio-electrocatalytic oxidation and glucose sensing.

A simple method for molecular wiring of glucose oxidase (GOx) enzyme with a low cost Mn polypyridine complex, Mn(phen)2Cl2, carboxylic acid functionalized multiwalled carbon nanotube (f-MWCNT) and Nafion (Nf), which is useful for glucose oxidation and sensing application in pH 7 phosphate buffer solution, has been demonstrated. In the typical preparation, f-MWCNT, Mn(phen)2Cl2, Nafion and GOx solution/suspension were successfully drop-casted as layer-by-layer on a cleaned glassy carbon electrode and potential cycled using cylic voltametric (CV) technique. In this preparation procedure, the Mn(phen)2Cl2 complex is in-situ converted as a dimer complex, Mn2(phen)2(O)(Cl2). A cooperative interaction based on π-π, covalent, ionic, hydrophilic and hydrophobic are operated in the bioelectrode for molecular wiring and electron-transfer shutting reaction. The modified electrode is designated as GCE/f-MWCNT@Mn2(phen)2(O)(Cl2)-Nf@GOx. CV response of the bioelectrode showed a defined redox peak current signal at an apparent standard electrode potential, E°'=0.55V vs Ag/AgCl. Upon exposure of glucose, the modified electrode showed a current linearity in a range, 0-6mM with a current sensitivity value, 349.4µAmM-1cm-2 by CV and a current linearity in a window, 50-550µM with a current sensitivity, 316.8µAmM-1cm-2 at applied biased potential, 0.65V vs Ag/AgCl by amperometric i-t methods. Obtained glucose oxidation current sensitivity values are relatively higher than Os-complex based transducer systems.

Nano-immobilized cellulases for biomass processing with application in biofuel production.

Several researches have focused on the enzymatic pretreatment of lignocellulose biomass to produce fermentable sugars that can lead to ethanol production thus facilitating pathways for sustainable biofuel production. Enzymes are fundamental to the pretreatment process, however, are required in larger quantities during pretreatment process thus influencing biofuel production cost. Immobilization of enzymes to a suitable support/matrix could enhance its stability, and reusability thus containing cost. This chapter focuses on developing an advanced technology for immobilizing enzymes to nanomaterials; variety of nanomaterials used for immobilization, nature of enzyme/protein nanomaterial interactions, methods of enzyme immobilization, and factors affecting mode of interaction for achieving hydrolysis of microcrystalline cellulose and natural cellulosic substrate. The binding of enzyme (94%) to a nanomaterial was established by spectroscopy techniques. The kinetics study, conducted at optimum pH (pH 4) and temperature (50°C for free and 60°C immobilized enzyme), exhibited improvement in immobilized enzyme properties. The immobilized enzyme retained up to 50% of its enzyme activity in five consecutive cycles. This chapter advocates the use of nano-immobilized enzymes in biomass hydrolysis for biofuel production.