Characterization of bioactive compounds produced by endophytic fungi isolated from Gynura procumbens (Sambung Nyawa).

This research evaluates the bioactivity of twelve endophytic fungi successfully isolated and characterised from Gynura procumbens. The fungal extracts displayed inhibitory activity against Staphylococcus aureus, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Salmonella typhi with the MIC and MBC of 5000 µg/mL. High antioxidant activity using DPPH free radical scavenging assay with inhibition of 86.6% and IC50 value of 104.25 ± 18.51 µg/mL were exhibited by ethyl acetate extract of Macrophomina phaseolina SN6. In contrast, the highest scavenging activity percentage of methanolic extract was exhibited by Mycoleptodiscus indicus SN4 (50.0%). Besides that, the highest ferric reducing antioxidant power (FRAP) value of ethyl acetate and methanolic extract was recorded from M. phaseolina SN6 (239.9 mg Fe (II)/g) and M. indicus SN4 (44.7 mg Fe (II)/g), respectively. Total phenolic content (TPC) and total flavonoid content (TFC) of ethyl acetate and methanolic fungal extracts were determined using Folin-Ciocalteu and aluminium chloride, respectively. The highest TPC for ethyl acetate and methanolic extracts were exhibited by Colletotrichum gloeosporioides SN11 (87.0 mg GAE/g) and M. indicus SN4 (35.0 mg GAE/g), whereas the highest TFC of ethyl acetate and methanolic extracts were showed by M. phaseolina SN6 (122.8 mg QCE/g) and M. indicus SN4 (60.4 mg QCE/g), respectively. Bioactive metabolites of isoelemicin (50.8%), terpinen-4-ol (21.5%), eucalyptol (24.3%), oleic acid (19.8%) and ß-pinene (10.9%) were detected. Owing to the higher content of phytochemicals represented in the ethyl acetate extract of M. phaseolina, SN6 is therefore identified to be a superior candidate in exhibiting strong antioxidant and antimicrobial properties be fit for further pharmaceutical studies.

Fatty Acid Profile and Antioxidant Capacity of Dabai (Canarium odontophyllum L.): Effect of Origin and Fruit Component.

In the present work, the influence of geographical location on the fatty acid profiles, antioxidant potential, as well as cytotoxicity of edible dabai fruit fractions (kernel, skin, and pulp) were analyzed. The fatty acid profiles were determined by Gas Chromatography (GC), and the antioxidant activity was quantified with free 2,2-diphenyl-1-picr/ylhdrazyl, while the cytotoxicity was assessed by the brine shrimp lethality test. The results showed that the samples from Sibu, Serian, and Kapit geographical locations had a high content of the saturated fatty acids, ranging from 46.63% to 53.31% in the three fractions. The highest mono-saturated fatty acids (MUFA) content was found in Sibu. Serian and Kapit kernel fractions MUFA, however, ranged from 21.2% to 45.91%. No fatty acid composition was detected in Bentong and Kanowit. The fatty acid composition and DPPH free radical scavenging antioxidant activity of dabai were statistically independent using a multivariate analysis in different localities in Malaysia. The skin fraction had a more appreciable antioxidant potential and toxicity level than the pulp and kernel fractions. The highest antioxidant activity (EC50 198.76 ± 1.06 µg/mL) with an LC50 value of 1387.22 µg/mL was obtained from the Sibu skin fraction. Therefore, the fatty acid composition, antioxidant, as well as cytotoxicity analyses of the extracts from different localities indicated that "geographical location" remarkably influenced fatty acid composition, antioxidant activity, and toxicity.

Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9.

The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.

Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence.

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Polymer Based Protein Therapeutics.

Proteins have played a very important role in the drug industry for developing treatments of various diseases such as auto-immune diseases, cancer, diabetes, mental disorder, metabolic disease, and others. Therapeutic proteins have high activity and specificity but they have some limitations such as short half-life, poor stability, low solubility and immunogenicity, so they cannot prolong their therapeutic activity. These shortcomings have been rectified by using polymers for the conjugation with proteins. The conjugates of protein-polymer improves the half-lives, stability and makes them non-immunogenic. Poly(ethylene glycol) (PEG), is widely used in the delivery of proteins because it is the current gold standard for stealth polymers in the emerging field of polymer-based delivery as compared to various biodegradable polymers. PEGylation enhances the retention of therapeutic proteins, effectively alters the pharmacokinetics and enhances the pharmaceutical value. Smart polymer have been used to cope with the pathophysiological environment of target site and have imposed less toxic effects.The contents of this article are challenges in formulation of therapeutic proteins, synthetic routes of conjugates, smart polymer-protein conjugates and also some advantages/disadvantages of polymers as a carrier system of proteins.

Decolourisation Capabilities of Ligninolytic Enzymes Produced by Marasmius cladophyllus UMAS MS8 on Remazol Brilliant Blue R and Other Azo Dyes.

Marasmius cladophyllus was examined for its ability to degradatively decolourise the recalcitrant dye Remazol Brilliant Blue R (RBBR) and screened for the production of ligninolytic enzymes using specific substrates. Monitoring dye decolourisation by the decrease in absorbance ratio of A592/A500 shows that the decolourisation of RBBR dye was associated with the dye degradation. Marasmius cladophyllus produces laccase and lignin peroxidase in glucose minimal liquid medium containing RBBR. Both enzyme activities were increased, with laccase activity recorded 70 times higher reaching up to 390 U L-1 on day 12. Further in vitro RBBR dye decolourisation using the culture medium shows that laccase activity was correlated with the dye decolourisation. Fresh RBBR dye continuously supplemented into the decolourised culture medium was further decolourised much faster in the subsequent round of the RBBR dye decolourisation. In vitro dye decolourisation using the crude laccase not only decolourised 76% of RBBR dye in just 19 hours but also decolourised 54% of Orange G and 33% of Congo red at the same period of time without the use of any exogenous mediator. This rapid dye decolourisation ability of the enzymes produced by M. cladophyllus thus suggested its possible application in the bioremediation of dye containing wastewater.

Optimization of Physiochemical Parameters during Bioremediation of Synthetic Dye by Marasmius cladophyllus UMAS MS8 Using Statistical Approach.

In many industrial areas such as in food, pharmaceutical, cosmetic, printing, and textile, the use of synthetic dyes has been integral with products such as azo dye, anthrax, and dyestuffs. As such, these industries produce a lot of waste by-products that could contaminate the environment. Bioremediation, therefore, has become an important emerging technology due to its cost-sustainable, effective, natural approach to cleaning up contaminated groundwater and soil via the use of microorganisms. The use of microorganisms in bioremediation requires the optimisation of parameters used in cultivating the organism. Thus the aim of the work was to assess the degradation of Remazol Brilliant Blue R (RBBR) dye on soil using Plackett-Burman design by the basidiomycete, M. cladophyllus UMAS MS8. Biodegradation analyses were carried out on a soil spiked with RBBR and supplemented with rice husk as the fungus growth enhancer. A two-level Plackett-Burman design was used to screen the medium components for the effects on the decolourization of RBBR. For the analysis, eleven variables were selected and from these four parameters, dye concentration, yeast extract concentration, inoculum size, and incubation time, were found to be most effective to degrade RBBR with up to 91% RBBR removal in soil after 15 days.

Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris.

A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.

Application of Response Surface Methodology for Optimizing Process Parameters in the Production of Amylase by Aspergillus flavusNSH9 under Solid State Fermentation

Amylase is recognized as one of the important commercial enzymes. This group of enzymes has the ability in hydrolyzing starch into smaller oligosacharides. The present work aimed to determine the optimum fermentation conditions for maximum production of crude amylase enzyme by Aspergillus flavus NSH9 employing response surface methodology (RSM).Central composite design (CCD) was applied to determine the optimal fermentation condition with respect to the four main process parameters such as temperature, initial moisture content, pH and the incubation period. Solid state fermentation (SSF) was performed using 5.0 g of sago hampas inoculated with 1x107sporesmL-1following the experimental design obtained using CCD and further optimized by RSM. The initial moisture, pH and temperature showed significant effect on the amylase production (p<0.05). The maximum amylase activity produced was achieved and recorded as 1.055 ± 0.03U mL-1after four days of fermentation period with 100% (v/v) moisture holding capacity, pH 6.5 and temperature at 28°C. The optimum fermentation conditions for amylase production was determined with A. flavusNSH9 on sago hampas.

Bioremediation of PCP by Trichoderma and Cunninghamella Strains Isolated from Sawdust

Four fungal isolates, SD12, SD14, SD19 and SD20 isolated from the aged sawdust grew on agar plates supplemented with PCP up to a concentration of 100 mg L-1. At high PCP concentration, isolate SD12 showed the highest radial growth rate of 10 mm day-1, followed by SD14 and SD19 both with 4.5 mm day-1 and SD20 with 4.2 mm day-1. Ultrastructural study on the effect of PCP on the PCP tolerant fungi using scanning electron microscope showed that high concentration of PCP caused the collapse of both fungal hyphae and spores. Among the four PCP tolerant fungi examined, isolate SD12 showed the least structural damage at high PCP concentration of 100 mg L-1. This fungal isolate was further characterized and identified as Cunninghamella sp. UMAS SD12. Preliminary PCP biodegradation trial performed in liquid minimal medium supplemented with 20 mg L-1 of PCP using Cunninghamella sp. UMAS SD12 showed that the degradation up to 51.7% of PCP in 15 days under static growth condition.

Decolourisation of Synthetic Dyes by Endophytic Fungal Flora Isolated from Senduduk Plant (Melastoma malabathricum).

A total of twenty endophytic fungi successfully isolated from Melastoma malabathricum (Senduduk) were examined for their ability to decolourise azo dyes: Congo red, Orange G, and Methyl red and an anthraquinone dye, Remazol Brilliant Blue R. Initial screening on the glucose minimal media agar plates amended with 200 mg L(-1) of each respective dye showed that only isolate MS8 was able to decolourise all of the four dyes. The isolate decolourised completely both the RBBR and Orange G in the agar medium within 8 days. Further quantitative analysis of the dye decolourisation by isolate MS8 in aqueous minimal medium showed that isolate MS8 was able to decolourise all the tested dyes at varying levels. Dye decolourisation by the isolate MS8 was determined to be 97% for RBBR, 33% for Orange G, 48% for Congo red, and 56% for Methyl red, respectively, within a period of 16 days. Molecular identification of the fungal isolate MS8 using primer ITS1 and ITS4 showed that isolate MS8 shared 99% sequence similarity with Marasmius cladophyllus, a Basidiomycete. The ability to decolourise different types of dyes by isolate MS8 thus suggested a possible application of this fungus in the decolourisation of dyestuff effluents.