Search results for: enzyme production from fungi

Authors: Kais Rtibi, Slimen Selmi, Jamel El-Benna, Lamjed Marzouki, Hichem Sebai

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Background: Myeloperoxidase (MPO) is a hemic enzyme found in high concentrations in the primary neutrophils granules. In addition to its peroxidase activity, it has a chlorination activity, using hydrogen peroxide and chloride ions to form hypochlorous acid, a strong oxidant, capable of chlorinating molecules. Bioactive compounds contained in medicinal plants could limit the action of this enzyme to reduce the reactive oxygen species production and its chlorination activity. The purpose of this study is to evaluate the effect of the carob aqueous extract (CAE) on the release of MPO by human neutrophils in vitro and its activity following stimulation of these cells by PMA. Methods: Neutrophils were isolated by simple sedimentation using the Dextran/Ficoll method. After stimulation with phorbol 12-myristate 13-acetate (PMA), neutrophils release the MPO by degranulation. The effect of CAE on the release of MPO was analyzed by the Western blot technique, while, its activity was determined by biochemical method using the method of 3,3', 5,5'- Tetramethylbenzidine (TMB) and hydrogen peroxide. The data were expressed as mean ± SEM. Results: The carob aqueous extract causes a decrease in MPO quantity and activity in a concentration-dependent manner which leads to a reduction of the production of the ROS (reactive oxygen species) and the protection of the molecules against oxidation and chlorination mechanisms. Conclusion: Thanks to its richness in bioactive compounds, the aqueous extract of carob could limit the development of damages related to the uncontrolled activity of MPO.

Keywords: carob, MPO, myeloperoxidase, neutrophils, PMA, phorbol 12-myristate 13-acetate

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Authors: Evgeniya Y. Yuzbasheva, Tigran V. Yuzbashev, Natalia I. Perkovskaya, Elizaveta B. Mostova

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Cell-surface display of lipase is of great interest as it has many applications in the field of biotechnology owing to its unique advantages: simplified product purification, and cost-effective downstream processing. One promising area of application for whole-cell biocatalysts with surface displayed lipase is biodiesel synthesis. Biodiesel is biodegradable, renewable, and nontoxic alternative fuel for diesel engines. Although the alkaline catalysis method has been widely used for biodiesel production, it has a number of limitations, such as rigorous feedstock specifications, complicated downstream processes, including removal of inorganic salts from the product, recovery of the salt-containing by-product glycerol, and treatment of alkaline wastewater. Enzymatic synthesis of biodiesel can overcome these drawbacks. In this study, Lip2p lipase was displayed on Yarrowia lipolytica cells via C- and N-terminal fusion variant. The active site of lipase is located near the C-terminus, therefore to prevent the activity loosing the insertion of glycine-serine linker between Lip2p and C-domains was performed. The hydrolytic activity of the displayed lipase reached 12,000–18,000 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. In case of C-terminal fusion variant, the leakage was occurred due to the proteolytic cleavage within the linker peptide. In case of N-terminal fusion variant, the leaking enzyme was presented as three proteins, one of which corresponded to the whole hybrid protein. The calculated number of recombinant enzyme displayed on the cell surface is approximately 6–9 × 105 molecules per cell, which is close to the theoretical maximum (2 × 106 molecules/cell). Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, cell-bound lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. It retained 74% of original activity at 60°C for 5 min of incubation, and 83% of original activity after incubation at 50°C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1–% and 71.0–% methyl esters after 33–h and 45–h reactions, respectively.

Keywords: biodiesel, cell-surface display, lipase, whole-cell biocatalyst

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Authors: Jung-En Kuan, Whei-Fen Wu

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In this study, a symbiotic bacteria from yellow mealworm's (Tenebrio molitor) mid-gut was isolated with characteristics of growth on minimal-tributyrin medium. After a PCR-amplification of its 16s rDNA, the resultant nucleotide sequences were then analyzed by schemes of the phylogeny trees. Accordingly, it was designated as Pseudomonas nitroreducens D-01. Next, by searching the lipolytic enzymes in its protein data bank, one of those potential lipolytic α/β hydrolases was identified, again using PCR-amplification and nucleotide-sequencing methods. To construct an expression of this lipolytic gene in plasmids, the target-gene primers were then designed, carrying the C-terminal his-tag sequences. Using the vector pET21a, a recombinant lipolytic hydrolase D gene with his-tag nucleotides was successfully cloned into it, of which the lipolytic D gene is under a control of the T7 promoter. After transformation of the resultant plasmids into Eescherichia coli BL21 (DE3), an IPTG inducer was used for the induction of the recombinant proteins. The protein products were then purified by metal-ion affinity column, and the purified proteins were found capable of forming a clear zone on tributyrin agar plate. Shortly, its enzyme activities were determined by degradation of p-nitrophenyl ester(s), and the substantial yellow end-product, p-nitrophenol, was measured at O.D.405 nm. Specifically, this lipolytic enzyme efficiently targets p-nitrophenyl butyrate. As well, it shows the most reactive activities at 40°C, pH 8 in potassium phosphate buffer. In thermal stability assays, the activities of this enzyme dramatically drop when the temperature is above 50°C. In metal ion assays, MgCl₂ and NH₄Cl induce the enzyme activities while MnSO₄, NiSO₄, CaCl₂, ZnSO₄, CoCl₂, CuSO₄, FeSO₄, and FeCl₃ reduce its activities. Besides, NaCl has no effects on its enzyme activities. Most organic solvents decrease the activities of this enzyme, such as hexane, methanol, ethanol, acetone, isopropanol, chloroform, and ethyl acetate. However, its enzyme activities increase when DMSO exists. All the surfactants like Triton X-100, Tween 80, Tween 20, and Brij35 decrease its lipolytic activities. Using Lineweaver-Burk double reciprocal methods, the function of the enzyme kinetics were determined such as Km = 0.488 (mM), Vmax = 0.0644 (mM/min), and kcat = 3.01x10³ (s⁻¹), as well the total efficiency of kcat/Km is 6.17 x10³ (mM⁻¹/s⁻¹). Afterwards, based on the phylogenetic analyses, this lipolytic protein is classified to type IV lipase by its homologous conserved region in this lipase family.

Keywords: enzyme, esterase, lipotic hydrolase, type IV

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Authors: Y. Moldes-Diz, M. Gamallo, G. Eibes, C. Vazquez-Vazquez, G. Feijoo, J. M. Lema, M. T. Moreira

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Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are excellent biocatalysts for biotechnological and environmental applications because of their high activity, selectivity, and specificity. Specifically, these characteristics allow them to perform the oxidation of recalcitrant compounds with simple requirements for the catalysis (presence of molecular oxygen). Nevertheless, the low stability under unfavorable conditions (pH, inactivating agents or temperature) and high production costs still limits their use for practical applications. Immobilization of enzymes has proven particularly valuable to avoid some of the aforementioned drawbacks. Magnetic nanoparticles (MNPs) have received increasing attention as carriers for enzyme immobilization since they can potentially provide an easy recovery of the biocatalyst from the reaction medium under an external magnetic field. In the present work, silica-coated magnetic nanoparticles (Fe3O4@SiO2) were prepared, characterized and used for laccase immobilization by covalent binding. The synthesis of Fe3O4@SiO2 was performed in a two-step procedure: co-precipitation and reverse microemulsion. The influence of immobilization conditions: concentrations of the functionalization agent (3-aminopropyl-triethoxy-silane) and the cross-linker (glutaraldehyde) as well as the influence of pH, T or inactivating agents were evaluated. In general, immobilized laccase showed superior stability compared to that of free enzyme. The reusability of the biocatalyst was demonstrated in successive batch reactions, where enzyme activity was maintained above 65% after 8 cycles of oxidation of the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate).

Keywords: silica-coated magnetic nanoparticles, laccase, immobilization, regeneration

Procedia PDF Downloads 187

Authors: Khalida Boutemak, Nasssima Benali, Nadji Moulai-Mostefa

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Effect of enzyme on the yield and chemical composition of Artemisia campestris essential oil is reported in the present study. It was demonstrated that enzyme facilitated the extraction of essential oil with increase in oil yield and did not affect any noticeable change in flavour profile of the volatile oil. Essential oil was tested for antibacterial activity using Escherichia coli; which was extremely sensitive against control with the largest inhibition (29mm), whereas Staphylococcus aureus was the most sensitive against essential oil obtained from enzymatic pre-treatment with the largest inhibition zone (25mm). The antioxidant activity of the essential oil with hemicellulase pre-treatment (EO2) and control sample (EO1) was determined through reducing power. It was significantly lower than the standard drug (vitamin C) in this order: vitamin C˃EO2˃EO1.

Keywords: Artemisia campestris, enzyme pre-treatment, hemicellulase, antibacterial activity, antioxidant activity

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Authors: Rashida Rahmat Zohra, Afsheen Aman, Shah Ali Ul Qader

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Commercially important biopolymer, dextran, is enzymatically degraded into lower molecular weight fractions of vast industrial potential. Various organisms are associated with dextranase production, among which fungal, yeast and bacterial origins are used for commercial production. Dextranases are used to remove contaminating dextran in sugar processing industry and also used in oral care products for efficient removal of dental plaque. Among the hydrolytic products of dextran, isomaltooligosaccharides have prebiotic effect in humans and reduces the cariogenic effect of sucrose in oral cavity. Dextran derivatives produced by hydrolysis of high molecular polymer are also conjugated with other chemical and metallic compounds for usage in pharmaceutical, fine chemical industry, cosmetics, and food industry. Owing to the vast application of dextran and dextranases, current study focused on purification and analysis of kinetic parameters of dextranase from a newly isolated strain of Bacillus licheniformis KIBGE-IB25. Dextranase was purified up to 35.75 folds with specific activity of 1405 U/mg and molecular weight of 158 kDa. Analysis of kinetic parameters revealed that dextranase performs optimum cleavage of low molecular weight dextran (5000 Da, 0.5%) at 35ºC in 15 min at pH 4.5 with a Km and Vmax of 0.3738 mg/ml and 182.0 µmol/min, respectively. Thermal stability profiling of dextranase showed that it retained 80% activity up to 6 hours at 30-35ºC and remains 90% active at pH 4.5. In short, the dextranase reported here performs rapid cleavage of substrate at mild operational conditions which makes it an ideal candidate for dextran removal in sugar processing industry and for commercial production of low molecular weight oligosaccharides.

Keywords: Bacillus licheniformis, dextranase, gel permeation chromatograpy, enzyme purification, enzyme kinetics

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Authors: Itti Bist, Snehasis Bhakta, Di Jiang, Tia E. Keyes, Aaron Martin, Robert J. Forster, James F. Rusling

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DNA damage from metabolites of lipophilic drugs and pollutants, generated by enzymes, represents a major toxicity pathway in humans. These metabolites can react with DNA to form either 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is the oxidative product of DNA or covalent DNA adducts, both of which are genotoxic and hence considered important biomarkers to detect cancer in humans. Therefore, detecting reactions of metabolites with DNA is an effective approach for the safety assessment of new chemicals and drugs. Here we describe a novel electrochemiluminescent (ECL) sensor array which can detect DNA oxidation and chemical DNA damage in a single array, facilitating a more accurate diagnostic tool for genotoxicity screening. Layer-by-layer assembly of DNA and enzyme are assembled on the pyrolytic graphite array which is housed in a microfluidic device for sequential detection of two type of the DNA damages. Multiple enzyme reactions are run on test compounds using the array, generating toxic metabolites in situ. These metabolites react with DNA in the films to cause DNA oxidation and chemical DNA damage which are detected by ECL generating osmium compound and ruthenium polymer, respectively. The method is further validated by the formation of 8-oxodG and DNA adduct using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by liquid chromatography-mass spectrometry (LC-MS). Hence, this combined DNA/enzyme array/LC-MS approach can efficiently explore metabolic genotoxic pathways for drugs and environmental chemicals.

Keywords: biosensor, electrochemiluminescence, DNA damage, microfluidic array

Procedia PDF Downloads 335

Authors: Amtul Jamil Sami

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Apis mellifera is an insect of immense economic importance lives on rich carbohydrate diet including cellulose, nectar, honey and pollen. The carbohydrate metabolism in A mellifera has not been understood fully, as there are no data available, on the functional expression of cellulase gene. The cellulose hydrolyzing enzyme is required for the digestion of pollen cellulose wall, to release the important nutrients (amino acids, minerals, vitamins etc.) from the pollen. A dissection of Apis genome had revealed that there is one gene present for the expression of endo-beta-1,4-glucanase, for cellulose hydrolysis. In the presented work, functional expression of endo-beta-1,4 glucanase gene is reported. Total soluble proteins of the honey bee were isolated and were tested cellulose hydrolyzing enzyme activity, using carboxy-methyl cellulose, as a substrate. A mellifera proteins were able to hydrolyze carboxy-methyl cellulose, confirming its endo- type mode of action. Endo beta-1,4 glucanase enzyme was only present in the gut tissues, no activity was detected in the salivary glands. The pH optima of the enzyme were in the acidic pH range of 4-5-5-0, indicating its metabolic role in the acidic stomach of A mellifera. The reported enzyme is unique, as endo-beta- 1,4 glucanase was able to generate non reducing sugar, as an end product. The results presented, are supportive to the information that the honey bee is capable of producing its novel endo-beta-1,4 glucanase. Further it could be helpful, in understanding, the carbohydrate metabolism in A mellifera.

Keywords: honey bees, Endo-beta 1, 4- glucanase, Apis mellifera, functional expression

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Authors: Susan Mwai, Mary Muchane, Peter Wachira, Sheila Okoth, Muchai Muchane, Halima Saado

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Tropical forests harbor a wide range of biodiversity and rich macro-fungi diversity compared to the temperate regions in the World. However, biodiversity is facing the threat of extinction following the rate of forest loss taking place before proper study and documentation of macrofungi is achieved. The present study was undertaken to determine the effect of converting indigenous habitat to plantation forest on macrofungi diversity. To achieve the objective of this study, an inventory focusing on macro-fungi diversity was conducted within Kereita block in Kikuyu Escarpment forest which is on the southern side of Aberdare mountain range. The macrofungi diversity was conducted in the indigenous forest and in more than 15 year old Patula plantation forest , during the wet (long rain season, December 2014) and dry (Short rain season, May, 2015). In each forest type, 15 permanent (20m x 20m) sampling plots distributed across three (3) forest blocks were used. Both field and laboratory methods involved recording abundance of fruiting bodies, taxonomic identity of species and analysis of diversity indices and measures in terms of species richness, density and diversity. R statistical program was used to analyze for species diversity and Canoco 4.5 software for species composition. A total number of 76 genera in 28 families and 224 species were encountered in both forest types. The most represented taxa belonged to the Agaricaceae (16%), Polyporaceae (12%), Marasmiaceae, Mycenaceae (7%) families respectively. Most of the recorded macro-fungi were saprophytic, mostly colonizing the litter 38% and wood 34% based substrates, which was followed by soil organic dwelling species (17%). Ecto-mycorrhiza fungi (5%) and parasitic fungi (2%) were the least encountered. The data established that indigenous forests (native ecosystems) hosts a wide range of macrofungi assemblage in terms of density (2.6 individual fruit bodies / m2), species richness (8.3 species / plot) and species diversity (1.49/ plot level) compared to the plantation forest. The Conversion of native forest to plantation forest also interfered with species composition though did not alter species diversity. Seasonality was also shown to significantly affect the diversity of macro-fungi and 61% of the total species being present during the wet season. Based on the present findings, forested ecosystems in Kenya hold diverse macro-fungi community which warrants conservation measures.

Keywords: diversity, Indigenous forest, macro-fungi, plantation forest, season

Procedia PDF Downloads 189

Authors: Kanchan Vishwakarma, Shivesh Sharma, Nitin Kumar

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Soil is incoherent matter on Earth’s surface having organic and mineral content. The spatial variation of 4 soil enzyme activities and microbial biomass were assessed for two seasons’ viz. monsoon and winter along the latitudinal gradient in North-central India as the area of this study is fettered with respect to national status. The study was facilitated to encompass the effect of climate change, enzyme activity and biomass on nutrient cycling. Top soils were sampled from 4 sites in North-India. There were significant correlations found between organic C, N & P wrt to latitude gradient in two seasons. This distribution of enzyme activities and microbial biomass was consequence of alterations in temperature and moisture of soil because of which soil properties change along the latitude transect.

Keywords: latitude gradient, microbial biomass, moisture, soil, organic carbon, temperature

Procedia PDF Downloads 364

Authors: Alabi Olushola John, Ogbiko Anthonia, Tsado Daniel Nma, Mbajiorgu Ejike Felix, Adama Theophilus Zubairu

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A total of thirty (30) goats weighting between 5.8 and 7.3 kg were used to determine the growth performance, body linear measurements and body condition score of Semi intensively manged Savanna Brown goats fed enzyme treated sawdust diets (ETSD). They divided into five dietary treatments (T) groups with three replications using a completely randomized design. Treatment one (1) comprises of animals fed diet on 0 % enzyme treated sawdust while Treatment 2 (T2), Treatment 3 (T3), Treatment 4 (T4) and Treatment 5 (T5) comprises of animals fed diets containing 10, 20, 30 and 40 % enzyme treated sawdust diets, respectively. The study lasted 16 weeks. Data on growth performance parameters, body linear measurement (height at wither, body length, chest girth, hind leg length, foreleg length, facial length) and body condition score were collected and analyzed using one way analysis of variance. No significant difference (p>0.05) was observed in the all growth performance parameters and linear body measurements. However, significant difference was observed in body length and daily body length gains with highest value observed in animals fed the control diets (7.38 and 0.08 cm respectively) and animals on 30 % ETSD (7.25 and 0.07 cm respectively) and lowest values (4.75 and 0.05 cm respectively) were observed in animals fed 10 % ETSD among the treatment groups. It was, therefore, concluded that enzyme treated sawdust can be used in the diets of Savanna Brown goats up to 40 % replacement for maize offal since this treatment improved the body length and daily body length gains.

Keywords: performance, sawdust, enzyme treated, semi-intensively, replacement

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Authors: Ayuko Itsuki, Sachiyo Aburatani

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Soil enzyme activities in Kasuga-yama Hill Primeval Forest (Nara, Japan) were examined to determine levels of mineralization and metabolism. Samples were selected from the soil surrounding laurel-leaved (B_B-1) and Carpinus japonica (B_B-2 and P_w) trees for analysis. Cellulase, β-xylosidase, and protease activities were higher in B_B-1 samples those in B_B-2 samples. These activity levels corresponded to the distribution of cellulose and hemicellulose in the soil horizons. Cellulase, β-xylosidase, and chymotrypsin activities were higher in soil from the P_w forest than in that from the B_B-2 forest. The relationships between the soil enzymes calculated by Spearman’s rank correlation indicate that the interactions between enzymes in B_B-2 samples were more complex than those in P_w samples.

Keywords: comparative analysis, enzyme activities, forest soil, Spearman's rank correlation

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Authors: Varsha Shinde, Belle Damodara Shenoy

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Tarballs are crude oil remnants found in oceans after long term weathering process and are a global concern since several decades as potential marine pollutant. Being complicated in structure microbial remediation of tarballs in natural environment is a slow process. They are rich in high molecular weight alkanes and poly aromatic hydrocarbons which are resistant to microbial attack and other environmental factors, therefore remain in environment for long time. However, it has been found that many bacteria and fungi inhabit on tarballs for nutrients and shelter. Many of them are supposed to be oil degraders, while others are supposed to be getting benefited by byproducts formed during hydrocarbon metabolism. Thus tarballs are forming special interesting ecological niche of microbes. This work aimed to study diversity of bacteria and fungi from tarballs and to see their potential application in crude oil degradation. The samples of tarballs were collected from Betul beach of south Goa (India). Different methods were used to isolate culturable fraction of bacteria and fungi from it. Those were sequenced for 16S rRNA gene and ITS for molecular level identification. The 16S rRNA gene sequence analysis revealed the presence of 13 bacterial genera/clades (Alcanivorax, Brevibacterium, Bacillus, Cellulomonas, Enterobacter, Klebsiella, Marinobacter, Nitratireductor, Pantoea, Pseudomonas, Pseudoxanthomonas, Tistrella and Vibrio), while the ITS sequence analysis placed the fungi in 8 diverse genera/ clades (Aspergillus, Byssochlamys, Monascus, Paecilomyces, Penicillium, Scytalidium/ Xylogone, Talaromyces and Trichoderma). All bacterial isolates were screened for oil degradation capacity. Potential strains were subjected to crude oil degradation experiment for quantification. Results were analyzed by GC-MS-MS.

Keywords: bacteria, biodegradation, crude oil, diversity, fungi, tarballs

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Authors: Mohammad A. M. Kewisha

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Xerophilic fungi , which are responsible for many cases of biodeterioration monuments, have been known as an interesting source of antimicrobial compounds. Sixty nine fungal strains, isolated from different localities and species inside Egyptian museums, were screened for antimicrobial activity against some bacterial species and unicellular fungi. The most potent antimicrobial activity was obtained by Asperigillus fumigatus which was identified by ITS4 ……. and showed activity against Staphylococcus aureus with 20 mm and C. albicans with18 mm of inhibition zone. Different parameters were optimized to enhance this activity. The culture grown under stationary conditions for 8 days at 30°C and pH 8 gave the best antimicrobial activity. Moreover, both starch and yeast extract showed the most suitable carbon and nitrogen sources, respectively. The antimicrobial compound was purified and subjected to spectroscopic characterization, which revealed that the antimicrobial compound might be 5,7 ethoxy, 4\,5\ methoxy isorhamnetin -3- O- galactoside. This study suggests that Aspergillus fumagates as a potential candidate offering a better scope for the production, purification and isolation of broad-spectrum antimicrobial compounds. These findings will facilitate the scale-up and further purification to ascertain the compounds responsible for antimicrobial activity, which can be exploited for the treatment of biodeterioration monuments and pharmaceutical applications.

Keywords: antimicrobial activity, asperigillus fumigatus, Identification by ITS4, Staphylococcus aureus, C.albicans

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Authors: Ofonime U. M. John, Justina I. R. Udotong, Victor O. Nwaugo, Ime R. Udotong

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Oily wastes from oil and gas exploration and production (O&G E&P) activities were remediated for twelve weeks using Soil-Poultry dropping amendment. Culture-dependent microbiological, chemical and enzymatic techniques were employed to assess the efficacy of remediation process. Microbiological activities of the remediated wastes showed increased hydrocarbonoclastic microbial populations with increased remediation time; 2.7±0.1 x 105cfu/g to 8.3 ± 0.04 x106cfu/g for hydrocarbon utilizing bacteria, 1.7 ± 0.2 x103cfu/g to 6.0 ± 0.01 x 104cfu/g for hydrocarbon utilizing fungi and 2.2 ± 0.1 x 102cfu/g to 6.7 ± 0.1 x 103cfu/g for hydrocarbon utilizing actinomycetes. Bacteria associated with the remediated wastes after the remediation period included the genera Bacillus, Psuedomonas, Beijerinckia, Acinetobacter, Alcaligenes and Serratia. Fungal isolates included species of Penicillium, Aspergillus and Cladosporium, while the Actinomycetes included species of Rhodococcus, Nocardia and Streptomyces. Slight fluctuations in pH values between 6.5± 0.2 and 7.1 ± 0.08 were recorded throughout the process, while total petroleum hydrocarbon (TPH) content decreased from 89, 900 ± 0.03mg/kg to 425 ± 0.1 mg/kg after twelve weeks of remediation. The polycyclic aromatic hydrocarbon (PAH) levels decreased with increased remediation time; naphthalene, flourene, pheneanthrene, anthracene, pyrene, chrysene and benzo(b)flouranthene showed decreased values < 0.01 after twelve weeks of remediation. Enzyme activities revealed increased dehydrogenase and urease activities with increased remediation time and decreased phenol oxidase activity with increased remediation period. There was a positive linear correlation between densities of hydrocarbonoclastic microbes and dehydrogenase activity. On the contrary, phenol oxidase and urease activities showed negative correlation with microbial population. Results of this study confirmed that remediation of oily wastes using soil-poultry dropping amendment can result in eco-friendly O&G E&P wastes. It also indicates that urease and phenol oxidase activities can be reliable indices/tools to monitor PAH levels and rates of petroleum hydrocarbon degradation.

Keywords: dehydrogenase activity, oily wastes, remediation, soil-poultry dropping amendment

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Authors: I. Ahmad, A. Hannan, S. Ahmad, M. Asif, M. F. Nawaz, M. A. Tanvir, M. F. Azhar

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During this research, a comprehensive survey of tree growing areas of Faisalabad district of Pakistan was conducted to observe the symptoms, spectrum, occurrence and severity of A. nilotica and E. camaldulensis decline. Objective of current research was to investigate specific fungal pathogens involved in decline of A. nilotica and E. camaldulensis. For this purpose, infected roots, bark, neck portion, stem, branches, leaves and infected soils were collected to identify associated fungi. Potato dextrose agar (PDA) and Czepak dox agar media were used for isolations. Identification of isolated fungi was done microscopically and different fungi were identified. During survey of urban locations of Faisalabad, disease incidence on Kikar and Eucalyptus was recorded as 3.9-7.9% and 2.6-7.1% respectively. Survey of Agroforest zones of Faisalabad revealed decline incidence on kikar 7.5% from Sargodha road while on Satiana and Jhang road it was not planted. In eucalyptus trees, 4%, 8% and 0% disease incidence was observed on Jhang road, Sargodha road and Satiana road respectively. The maximum fungus isolated from the kikar tree was Drechslera australiensis (5.00%) from the stem part. Aspergillus flavus also gave the maximum value of (3.05%) from the bark. Alternaria alternata gave the maximum value of (2.05%) from leaves. Rhizopus and Mucor spp. were recorded minimum as compared to the Drechslera, Alternaria and Aspergillus. The maximum fungus isolated from the Eucalyptus tree was Armillaria luteobubalina (5.00%) from the stem part. The other fungi isolated were Macrophamina phaseolina and A. niger.

Keywords: decline, frequency of mycoflora, A. nilotica and E. camaldulensis, Drechslera australiensis, Armillaria luteobubalina

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Authors: Razieh Karimi Aghcheh, Christian Kubicek, Joseph Strauss, Gerhard Braus

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Filamentous fungi are of considerable economic and social significance for human health, nutrition and in white biotechnology. These organisms are dominant producers of a range of primary metabolites such as citric acid, microbial lipids (biodiesel) and higher unsaturated fatty acids (HUFAs). In particular, they produce also important but structurally complex secondary metabolites with enormous therapeutic applications in pharmaceutical industry, for example: cephalosporin, penicillin, taxol, zeranol and ergot alkaloids. Several fungal secondary metabolites, which are significantly relevant to human health do not only include antibiotics, but also e.g. lovastatin, a well-known antihypercholesterolemic agent produced by Aspergillus. terreus, or aflatoxin, a carcinogen produced by A. flavus. In addition to their roles for human health and agriculture, some fungi are industrially and commercially important: Species of the ascomycete genus Hypocrea spp. (teleomorph of Trichoderma) have been demonstrated as efficient producer of highly active cellulolytic enzymes. This trait makes them effective in disrupting and depolymerization of lignocellulosic materials and thus applicable tools in number of biotechnological areas as diverse as clothes-washing detergent, animal feed, and pulp and fuel productions. Fungal LaeA/LAE1 (Loss of aflR Expression A) homologs their gene products act at the interphase between secondary metabolisms, cellulase production and development. Lack of the corresponding genes results in significant physiological changes including loss of secondary metabolite and lignocellulose degrading enzymes production. At the molecular level, the encoded proteins are presumably methyltransferases or demethylases which act directly or indirectly at heterochromatin and interact with velvet domain proteins. Velvet proteins bind to DNA and affect expression of secondary metabolites (SMs) genes and cellulases. The dynamic interplay between LaeA/LAE1, velvet proteins and additional interaction partners is the key for an understanding of the coordination of metabolic and morphological functions of fungi and is required for a biotechnological control of the formation of desired bioactive products. Aspergilli and Trichoderma represent different biotechnologically significant species with significant differences in the LaeA/LAE1-Velvet protein machinery and their target proteins. We, therefore, performed a comparative study of the interaction partners of this machinery and the dynamics of the various protein-protein interactions using our robust proteomic and mass spectrometry techniques. This enhances our knowledge about the fungal coordination of secondary metabolism, cellulase production and development and thereby will certainly improve recombinant fungal strain construction for the production of industrial secondary metabolite or lignocellulose hydrolytic enzymes.

Keywords: cellulases, LaeA/1, proteomics, secondary metabolites

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Authors: Patsarawadee Paojinda, Waranya Imprasittichai, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, can be stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to 3 orders of magnitude, while the Km was not much affected and remained at low µM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivaties and metabolic functions on molecular evolution.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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Authors: Palwinder Singh, Baljit Kaur, Sukhmeet Kaur

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Amongst a library of rationally designed small peptides, (H)Gly-Gly-Phe-Leu(OMe) was identified, reducing prostaglandin production of COX-2 with IC50 60 nM vs. 6000 nM for COX-1. The 5 mg Kg-1 dose of this compound rescued albino mice by 80% from capsaicin-induced paw licking and recovered it by 60% from carrageenan-induced inflammation. The mode of action of the compound for targeting COX-2, iNOS, and VGSC was investigated by using substances P, L-arginine, and veratrine, respectively, as the biomarkers. The interactions of the potent compound with COX-2 were supported by the isothermal calorimetry experiments showing Ka 6.10±1.10x104 mol-1 and ΔG -100.3 k J mol-1 in comparison to Ka 0.41x103 ±0.09 mol-1 and ΔG -19.2±0.06 k J mol-1 for COX-1. This compound did not show toxicity up to 2000 mg Kg-1 dose. Furthermore, beyond the conventional mode of working with anti-inflammatory agents through enzyme inhibition, COX-2 was provided with a peptide-based alternate substrate. Proline-centered pentapeptide iso-conformational to arachidonic acid exhibited appreciable selectivity for COX-2 overcoming acetic acid and formalin-induced pain in rats to almost 80% and was treated as a substrate by the enzyme. Hence, we suggest small peptides as highly potent and promising candidates for their further development into an anti-inflammatory drug.

Keywords: small peptides, cyclooxygenase, inflammation, substrate

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Authors: Gauravi Agarkar, Mahendra Rai

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Endophytic fungi from medicinal plants are important source of numerous pharmacologically important compounds. In the present investigation, the endophytic fungi were isolated from three medicinal plants; Andrographis paniculata, Rauwolfia serpentina and Tridax procumbens. Endophytic Colletotrichum sp. were identified on the basis of cultural and morphological characteristics as well as internal transcribed spacer (ITS) sequence analysis. Antibacterial and antifungal activity of the ethyl acetate and methanol extract of endophytic Colletotrichum sp. was evaluated against seven different human pathogenic bacteria and six Candida sp. The extracts were effective and showed significant activity against all the test pathogens. In case of yeast Candida, the combined effect of extracts and standard antibiotic was enhanced greatly showing synergistic activity. Further, the extracts were assayed for Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) where, MIC values were in the range of 100-250 μg/ml. These results suggest that the endophytic Colletotrichum sp. isolated from the medicinal plants are capable of producing promising antimicrobial metabolites.

Keywords: antimicrobial, colletotrichum, endophytic fungi, medicinal plants

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Authors: Jyosthna Khanna Goli, Shaik Naseeruddin, Hameeda Bee

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Employing lignocellulosic biomass in fermentative production of xylitol and bioethanol is gaining interest as it is renewable, cheap, and abundantly available. Xylitol is a polyol, gaining its importance in the food and pharmacological industry due to its low calorific value and anti-cariogenic nature. Bioethanol from lignocellulosic biomass is widely accepted as an alternative fuel for transportation with reduced CO₂ emissions, thus reducing the greenhouse effect. Typha latifolia, an aquatic weed, was found to be promising lignocellulosic substrate as it posses a high amount of sugars and does not compete with arable lands and interfere with food and feed competition. In the present study, xylose from hemicellulosic fraction of typha is converted to xylitol by isolate Jfh5 (Candida. tropicalis) and cellulose part to ethanol using Saccharomyces cerevisiaeVS3. Initially, alkali pretreatment of typha using sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, sodium bisulphate and sodium dithionate for overnight (18h) at room temperature (28 ± 2°C), resulted in maximum delignification of 75% with 2% (v/v) sodium bisulphate. Later, pretreated biomass was subjected to acid hydrolysis with 1%, 1.5%, 2%, and 3% H₂SO₄ at 110 °C and 121°C for 30 and 60 min, respectively. 2% H₂SO₄ at 121°C for 60 min was found to release 13.5 g /l sugars, which on detoxification and fermentation produced 8.1g/l xylitol with yield and productivity of 0.65g/g and 0.112g/l/h respectively. Further enzymatic hydrolysis of the residual substrate obtained after acid hydrolysis released 11g/l sugar, which on fermentation with VS3 produced 4.9g/l ethanol with yield and productivity of 0.22g/g and 0.136g/l/h respectively.

Keywords: delignification, xylitol, bioethanol, acid hydrolysis, enzyme hydrolysis

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Authors: Ahlem Nefzi, Rania Aydi Ben Abdallah, Hayfa Jabnoun-Khiareddine, Ammar Nawaim, Rabiaa Haouala, Mejda Daami-Remadi

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Seven endophytic fungi were isolated from the wild Solanaceous species Lycium arabicum growing in the Tunisian Centre-East and were assessed for their ability to suppress Fusarium Crown and Root Rot disease caused by Fusarium oxysporum f. sp. radicis lycopersici (FORL) and to enhance plant growth. Fungal isolates were shown able to colonize tomato cv. Rio Grande roots, crowns, and stems. A significant promotion in all studied growth parameters (root length, shoot height, and roots and shoots fresh weight) was recorded in tomato plants treated with fungal conidial suspensions or their cell-free culture filtrates compared to FORL-inoculated or pathogen-free controls. I15 and I18 isolates were shown to be the most effective leading to 85.7-87.5 and 93.6-98.4% decrease in leaf and root damage index and the vascular discoloration extent, respectively, over FORL-inoculated and untreated control. These two bioactive and growth-promoting isolates (I15 and I18) were morphologically characterized and identified using rDNA sequencing gene as being Alternaria alternata (MF693801) and Fusarium fujikuroi (MF693802). These fungi significantly suppressed FORL mycelial growth and showed chitinolytic, proteolytic and amylase activities whereas only F. fujikuroi displayed a lipolytic activity. This study clearly demonstrated the potential use of fungi naturally associated with L. arabicum as biocontrol and bio-fertilizing agents.

Keywords: biocontrol, endophytic fungi, Fusarium oxysporum f. sp. radicis-lycopersici, tomato promotion, Lycium arabicum

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Authors: Anna Trusek-Holownia, Andrzej Noworyta

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Using an enzyme of known specificity the hydrolysis of protein was carried out in a controlled manner. The aim was to obtain oligopeptides being the so-called active peptides or their direct precursors. An original way of expression of the protein hydrolysis kinetics was introduced. Peptide bonds contained in the protein were recognized as a diverse-quality substrate for hydrolysis by the applied protease. This assumption was positively verified taking as an example the hydrolysis of albumin by thermolysin. Peptide linkages for this system should be divided into at least four groups. One of them is a group of bonds non-hydrolyzable by this enzyme. These that are broken are hydrolyzed at a rate that differs even by tens of thousands of times. Designated kinetic constants were k'F = 10991.4 L/g.h, k'M = 14.83L/g.h, k'S about 10-1 L/g.h for fast, medium and slow bonds, respectively. Moreover, a procedure for unfolding of the protein, conducive to the improved susceptibility to enzymatic hydrolysis (approximately three-fold increase in the rate) was proposed.

Keywords: peptide bond hydrolysis, kinetics, enzyme specificity, biologically active peptides

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Authors: Nisha Singh, Munish Puri, Collin Barrow, Deepak Tuli, Anshu S. Mathur

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The biological conversion of lignocellulosic biomass to ethanol is a promising strategy to solve the present global crisis of exhausting fossil fuels. The existing bioethanol production technologies have cost constraints due to the involvement of mandate pretreatment and extensive enzyme production steps. A unique process configuration known as consolidated bioprocessing (CBP) is believed to be a potential cost-effective process due to its efficient integration of enzyme production, saccharification, and fermentation into one step. Due to several favorable reasons like single step conversion, no need of adding exogenous enzymes and facilitated product recovery, CBP has gained the attention of researchers worldwide. However, there are several technical and economic barriers which need to be overcome for making consolidated bioprocessing a commercially viable process. Finding a natural candidate CBP organism is critically important and thermophilic anaerobes are preferred microorganisms. The thermophilic anaerobes that can represent CBP mainly belong to genus Clostridium, Caldicellulosiruptor, Thermoanaerobacter, Thermoanaero bacterium, and Geobacillus etc. Amongst them, Clostridium thermocellum has received increased attention as a high utility CBP candidate due to its highest growth rate on crystalline cellulose, the presence of highly efficient cellulosome system and ability to produce ethanol directly from cellulose. Recently with the availability of genetic and molecular tools aiding the metabolic engineering of Clostridium thermocellum have further facilitated the viability of commercial CBP process. With this view, we have specifically screened cellulolytic and xylanolytic thermophilic anaerobic ethanol producing bacteria, from unexplored hot spring/s in India. One of the isolates is a potential CBP organism identified as a new strain of Clostridium thermocellum. This strain has shown superior avicel and xylan degradation under unoptimized conditions compared to reported wild type strains of Clostridium thermocellum and produced more than 50 mM ethanol in 72 hours from 1 % avicel at 60°C. Besides, this strain shows good ethanol tolerance and growth on both hexose and pentose sugars. Hence, with further optimization this new strain could be developed as a potential CBP microbe.

Keywords: Clostridium thermocellum, consolidated bioprocessing, ethanol, thermophilic anaerobes

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Authors: Li Na Zhao, Arieh Warshel

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The design of enzymes is an extremely challenging task, and this is also true for metalloenzymes. In the case of naturally evolved enzymes, one may consider the active site residues as the musicians in the enzyme orchestra, while the metal can be considered as their concertmaster. Together they catalyze reactions as if they performed a masterpiece written by nature. The Lactonase can be thought as a member of the amidohydrolase family, with two concertmasters, Fe and Zn, at its active site. It catalyzes the quorum sensing signal- N-acyl homoserine lactones (AHLs or N-AHLs)- by hydrolyzing the lactone ring. This process, known as quorum quenching, provides a strategy in the treatment of infectious diseases without introducing selection pressure. However, the activity of lactonase is metal-dependent, and this dependence hampers the clinic usage. In our study, we use the empirical valence bond (EVB) approach to evaluate the catalytic contributions decomposing them to electrostatic and other components.

Keywords: enzyme redesign, empirical valence bond, lactonase, quorum quenching

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Authors: Wayde Veldman, Ozlem T. Bishop, Igor Polikarpov

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In bacteria, mono and disaccharides are phosphorylated during uptake into the cell via the widely used phosphoenolpyruvate (PEP)-dependent phosphotransferase transport system. As an initial step in the phosphorylated disaccharide metabolism pathway, certain glycoside hydrolase family 1 (GH1) enzymes play a crucial role in releasing phosphorylated and non-phosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. GH1 enzymes are known to have a wide array of functions. According to the CAZy database, there are twenty-one different enzymatic activities in the GH1 family. Here, the structure and substrate specificity of a GH1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was investigated. The sequence of the enzyme BlBglH was compared to the sequences of other characterized GH1 enzymes using sequence alignment, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-β-glucosidase activity enzymes. Additionally, motif and structure comparisons of the three most commonly studied GH1 enzyme-activities revealed a shared loop amongst the different structures that consist of different sequence motifs – this loop is thought to guide specific substrates (depending on activity) towards the active-site. To further affirm BlBglH enzyme activity, molecular docking and molecular dynamics simulations were performed. Docking was carried out using 6-phospho-β-glucosidase enzyme-activity positive (p-Nitrophenyl-beta-D-glucoside-6-phosphate) and negative (p-Nitrophenyl-beta-D-galactoside-6-phosphate) control ligands, followed by 400 ns molecular dynamics simulations. The positive-control ligand maintained favourable interactions within the active site until the end of the simulation. The negative-control ligand was observed exiting the enzyme at 287 ns. Binding free energy calculations showed that the positive-control complex had a substantially more favourable binding energy compared to the negative-control complex. Jointly, the findings of this study suggest that the BlBglH enzyme possesses 6-phospho-β-glucosidase enzymatic activity.

Keywords: 6-P-β-glucosidase, glycoside hydrolase 1, molecular dynamics, sequence analysis, substrate specificity

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Authors: Shady S. Hassan, Brijesh K. Tiwari, Gwilym A. Williams, Amit K. Jaiswal

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EU is known as the destination with the highest rate of the coffee consumption per capita in the world. Spent coffee grounds (SCG) are the main by-product of coffee brewing. SCG is either disposed as a solid waste or employed as compost, although the polysaccharides from such lignocellulosic biomass might be used as feedstock for fermentation processes. However, SCG as a lignocellulose have a complex structure and pretreatment process is required to facilitate an efficient enzymatic hydrolysis of carbohydrates. However, commonly used pretreatment methods, such as chemical, physico-chemical and biological techniques are still insufficient to meet optimal industrial production requirements in a sustainable way. Ultrasound is a promising candidate as a sustainable green pretreatment solution for lignocellulosic biomass utilization in a large scale biorefinery. Thus, ultrasound pretreatment of SCG without adding harsh chemicals investigated as a green technology to enhance enzyme hydrolysis. In the present work, ultrasound pretreatment experiments were conducted on SCG using different ultrasound frequencies (25, 35, 45, 130, and 950 kHz) for 60 min. Regardless of ultrasound power, low ultrasound frequency is more effective than high ultrasound frequency in pretreatment of biomass. Ultrasound pretreatment of SCG (at ultrasound frequency of 25 kHz for 60 min) followed by enzymatic hydrolysis resulted in total reducing sugars of 56.1 ± 2.8 mg/g of biomass. Fourier transform Infrared Spectroscopy (FTIR) was employed to investigate changes in functional groups of biomass after pretreatment, while high-performance liquid chromatography (HPLC) was employed for determination of glucose. Pretreatment of lignocellulose by low frequency ultrasound in water only was found to be an effective green approach for SCG to improve saccharification and glucose yield compared to native biomass. Pretreatment conditions will be optimized, and the enzyme hydrolysate will be used as media component substitute for the production of ethanol.

Keywords: lignocellulose, ultrasound, pretreatment, spent coffee grounds

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Authors: Vicente A. Hernandez, Felipe Galleguillos, Rene Thibaut, Alejandro Muller

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Natural pigments have been proposed as an eco-friendly alternative to artificial pigments. Among the diverse organisms able to synthesize natural pigments, several wood colonizing fungi produce extracellular pigments which have been tested to dye fabrics at laboratory conditions with good results. However, the dyeing conditions used at laboratory level not necessary meet the real conditions in which dyeing of fabrics is conducted at industrial level. In this work, yellow and red pigments from the fungi Penicillium murcianum and Talaromyces australis, respectively, were used to dye yarn and linen fabrics using dyeing processes optimized according to the standard conditions used at industrial level. After dyeing treatments, fabrics were tested for color fastness to wash and to wet and dry rubbing, but also to tensile strength tests. Satisfactory result was obtained with both yellow and red pigments in yarn and linen, when used alone or mixed to different proportions. According to these results, natural pigments synthesized by both wood colonizing fungi have a great potential to be used in dyeing processes at industrial level.

Keywords: natural pigments, fungal pigments, yarn, linen

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Authors: Shivankar Agrawal, Indira Sarangthem

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Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the major causes of healthcare-associated and community-onset infections worldwide. Hence the search for non-toxic natural compounds having antibacterial activity has intensified for future drug development. The exploration of less studied niches of Earth can highly increase the possibility to discover novel bioactive compounds. Therefore, in this study, the cultivable fraction of fungi from the sediments of natural hot springs has been studied to mine potential fungal candidates with antibacterial activity against the human pathogen Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. We isolated diverse strains of thermophilic fungi from a collection of samples from sediment. Following a standard method, we isolated a promising thermophilic fungus strain IBSD19, identified as Acrophialophora levis, possessing the potential to produce an anti-Staphylococcus aureus agent. The growth conditions were optimized and scaled to fermentation, and its produced extract was subjected to chemical extraction. The ethyl acetate fraction was found to display significant activity against Staphylococcus aureus and MRSA with a minimum inhibitory concentration (MIC) of 0.5 mg/ml and 4 mg/ml, respectively. The cell membrane integrity assay and SEM suggested that the fungal metabolites cause bacteria clustering and further lysis of the cell.

Keywords: antibacterial activity, antioxidant, fungi, Staphylococcus aureus, MRSA, thermophiles

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Authors: R. Pravin Kumar, L. Roopa

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Enzymes are molecular machines used in various industries such as pharmaceuticals, cosmetics, food and animal feed, paper and leather processing, biofuel, and etc. Nevertheless, this has been possible only by the breath-taking efforts of the chemists and biologists to evolve/engineer these mysterious biomolecules to work the needful. Main agenda of this enzyme engineering project is to derive screening and selection tools to obtain focused libraries of enzyme variants with desired qualities. The methodologies for this research include the well-established directed evolution, rational redesign and relatively less established yet much faster and accurate insilico methods. This concept was initiated as a Receptor Rependent-4Dimensional Quantitative Structure Activity Relationship (RD-4D-QSAR) to predict kinetic properties of enzymes and extended here to study transaminase by a 7D QSAR approach. Induced-fit scenarios were explored using Quantum Mechanics/Molecular Mechanics (QM/MM) simulations which were then placed in a grid that stores interactions energies derived from QM parameters (QMgrid). In this study, the mutated enzymes were immersed completely inside the QMgrid and this was combined with solvation models to predict descriptors. After statistical screening of descriptors, QSAR models showed > 90% specificity and > 85% sensitivity towards the experimental activity. Mapping descriptors on the enzyme structure revealed hotspots important to enhance the enantioselectivity of the enzyme.

Keywords: QMgrid, QM/MM simulations, RD-4D-QSAR, transaminase

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