Dinitrosalicylic Acid Research Articles

Optimization of Alpha-Amylase Production by a Local Bacillus paramycoides Isolate and Immobilization on Chitosan-Loaded Barium Ferrite Nanoparticles

We set out to isolate alpha-amylase producers from soil samples, optimize the production, and immobilize the enzyme on chitosan-loaded barium ferrite nanoparticles (CLBFNPs). Alpha-amylase producers were isolated on starch agar plates and confirmed by dinitrosalicylic acid assay. The potent isolate was identified by phenotypic methods, 16S-rRNA sequencing, and phylogenetic mapping. Sequential optimization of α-amylase production involved the use of Plackett–Burman (P–BD) and central composite designs (CCD), in addition to exposing the culture to different doses of gamma irradiation. Alpha-amylase was immobilized on CLBFNPs, and the nanocomposite was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy, with energy-dispersive analysis of X-ray analysis. Forty-five α-amylase producers were isolated from 100 soil samples. The highest activity (177.12 ± 6.12 U/mg) was detected in the MS009 isolate, which was identified as Bacillus paramycoides. The activity increased to 222.3 ± 5.07 U/mg when using the optimal culture conditions identified by P–BD and CCD, and to 319.45 ± 4.91 U/mg after exposing the culture to 6 kGy. Immobilization of α-amylase on CLBFNPs resulted in higher activity (246.85 ± 6.76 U/mg) compared to free α-amylase (222.254 ± 4.89 U/mg), in addition to retaining activity for up to five cycles of usage. Gamma irradiation improved α-amylase production, while immobilization on CLBFNPs enhanced activity, facilitated enzyme recovery, and enabled its repetitive use.

Field Study and Chemical Analysis of Plant Waste in the Fez-Meknes Region, Morocco

Throughout the entire world, the biomass plant remains an important source of renewable energy. However, in Morocco, the energy recovery of this biomass is little or badly exploited compared to other solar, hydraulic, and wind resources. The aim of this study is to know the extent to which Moroccan companies are involved in the valorization of green waste and to identify among the latter those that have great energy and industrial value. The field investigation was carried out with the use of a questionnaire to different sectors of activity. The chemical analyses of the waste samples were carried out by different methods: Van Soest to investigate the fiber content, dinitrosalicylic acid and phenol-sulfuric acid to determine sugars, while the Folin–Ciocalteu method was employed for the determination of phenolic compounds. These are the ASTM standard methods to determine elemental, proximate composition, and calorific value (CV). The results of this survey showed that solid vegetable waste is diverse and represents 68.4% of the total green waste, of which 98% is not treated. Moreover, the chemical analysis displayed that forestry waste (FW), extracted parts wastes (EPW), and unused parts wastes (UPW) of medicinal and aromatic plants have high contents of cellulose (respectively 34.75, 48.44, and 54.19%) and hemicelluloses (28.44, 27.19 and 28.50%) and containing low amounts of lignin and phenolic compounds compared to olive waste (OW), olive pomace (OP), and household waste (HW). Almost all biomass wastes, except HW, have a low moisture (<12%), ash content less than 5.1%, a significant percentage of C and H, and CV between 14.5 and 21.6 MJ/Kg. The PCA analysis showed a discrepancy in terms of components between the set formed by FW, UPW, and EPW with other solid waste. In conclusion, FW, UPW, and EPW, specially can be potentially energetic biomass and valorized together in the form of a mixture.

Production of Cellulase by A. Niger and T. Reesei Under Solid State Fermentation Using Bagasse as Substrate

Cellulase is a very important enzyme for ethanol production from lignocellulose and others. Bagasse is very good to be chosen as lignocellulosic material for cellulase production because it is cheap and already available in a huge amount. Solid-state fermentation has advantages than submerged fermentation because of its higher yield and activity. Cellulase production using solid-state fermentation has been researched by many people, but for different substrate, different pretreatment and different fungi producer, it is likely to have a different condition. It is very important to perform specific study to optimize cellulase production with specific substrate and specific fungi which available in Indonesia. Because large amount of fungi strains available so it is wise full if they are screened using effective and efficient methods. This research is started by initial screening of three strain of Trichoderma reesei and five strain of Aspergillus niger using Congo red dye staining on CMC agar plate. It is shown that a strain of Trichoderma reesei and two strains of Aspergillus niger has better activity than the others. Further screening is performed by conducting solid-state fermentation using bagasse by three best strains for five days. The activity of the cellulase is measured every 24-hour using Ghose method by dinitrosalicylic acid as reagent. The highest activity is shown by A.niger ITBCC L74 on third day at 0.525 Unit/gram

Preparation of fusarium oxysporum-inhibiting chito-oligosaccharide by chitin/chitosan-hydrolyzing enzyme from actinomycetes

Chito-oligosaccharide (COS), one of remakable derivatives of chitosan, has almost chitosan’s biological function but better activities in antibacteria, antifungi and plant elicitor… Producing COS by biological enzymes shows many advantages in comparision with physical and chemical methods. In our study, COS was prepared using chitin/chitosan-hydrolyzing enzyme from actinomycetes. By analysis of 16S rDNA sequence, the enzyme producer VTCC 940003 belonged to Streptomyces macrosporeus. The strain’s ability to synthesize the enzyme having chitin/chitosan-hydrolyzing activity was first detected by agar plate method and then its activity equivalent to chitinase activity was determined using dinitrosalicylic acid (DNS). The enzyme activity in the strain culture was 24 ± 0.05 U/ml. The COS was prepared by using the enzyme from S. macrosporeus VTCC 940003 to hydrolyze chitosan. The degree of polymerization (DP) of the obtained COS was mainly DP3-DP5. At the concentration of 0,3‰ the COS inhibited 98% of spore germination of Fusarium oxysporum after 24 hours and this activity remained after 48 hours.

GC-MS analysis of bioactive phytochemicals of gum exudates of Mangifera indica. linn.

The aim of this study was to verify the potential of Mangifera indica gum (MIG) as a bioactive agent. Plant gum was collected from the bark of Mangifera indica and was, purified and characterized with Fourier-transform infrared spectroscopy and gas chromatography (GC) analyses. Purified gum was tested for its antimicrobial, antioxidant, antidiabetic, and anti-inflammatory. IR and GC analyses of gum showed the presence of several types of bioactive components in Mangifera indica gum. The gum showed potent antibacterial activity against Gram-positive bacteria Staphylococcus aureus (63%) and Enterococcus faecalis (58%). The antioxidant activity of MOG was investigated by 2,2- diphenyl 2- picryl hydrazyl hydrate (DPPH) radical scavenging and the MIG shows good activity when compared with the standard ascorbic acid. The anti-inflammatory activity of the MIG was estimated by the bovine serum albumin denaturation (BSA)method and showed significant activity against the standard drug. The anti-diabetic activity of the MIG was determined by the dinitro salicylic acid (DNSA)method displayed significant anti-diabetic activity. These findings confirmed that MIG has a potential source for the formulation of new therapeutic drugs.

Optimization of Acid-Catalyst Hydrolysis Process in Lactic Acid Production from Rice Husk by Using Lactobacillus bacteria

Lactic acid is an important component of manufacturing polylactic acid (PLA), which can be produced with high-lignocellulosic biomass, still few of them have utilized that. The production process starts with Alkaline Hydrogen Peroxide (AHP) pretreatment, acid-catalyzed (H2SO4) hydrolysis followed by post-hydrolysis, and fermentation using a mixture of Lactobacillus brevis + Lactobacillus rhamnosus and single Lactobacillus delbrueckii bacteria. The effectiveness of pretreatment results was analyzed by using Thermal Gravity Analysis (TGA), which resulted in the removal of lignin and cellulose are 32.60% and 43.18%, respectively. Lignin reduction results are supported by Scanning Electron Microscope (SEM) for morphology analysis; it is shown the surface of rice husk becomes rough and cracks after pretreatment. Cellulose reduction was analyzed by using X-Ray Diffraction (XRD) and shown the Crystallinity Index (CrI) decrease after chemical pretreatment. The optimum operating condition of hydrolysis was also studied using Face Centered Composite Design (FCCD) by Response Surface Methodology (RSM), and get the optimum conditions at a 4 M H2SO4 for 30.7 minutes 63.1 . Followed by post-hydrolysis for 10 minutes at 121 , it resulted in sugar concentration of 15.3056 g/L, which was analyzed by using dinitrosalicylic-acid (DNS) with Root Mean Square Error of 0.78009. The last process is fermentation which is carried out at 125 rpm 37 , for 48 hours. Through High-Performance Liquid Chromatography (HPLC) analysis, the lactic acid concentration of a single bacterium and a mixture was 24.595 and 24.975%, respectively. Similar research has been previously carried out using palm waste raw materials with the enzymatic hydrolysis method without optimization of RSM. This study investigates the effect of AHP pretreatment in the lignin removal process, the effectiveness of acid-catalyzed hydrolysis in the optimized sugar reduction through FCCD, and compares lactic acid concentrations obtained from the fermentation process with L. delbrueckii and culture L. rhamnosus + L. brevis.

Facile Cellulase Immobilisation on Bioinspired Silica.

Cellulases are enzymes with great potential for converting biomass to biofuels for sustainable energy. However, their commercial use is limited by their costs and low reusability. Therefore, the scientific and industrial sectors are focusing on finding better strategies to reuse enzymes and improve their performance. In this work, cellulase from Aspergillus niger was immobilised through in situ entrapment and adsorption on bio-inspired silica (BIS) supports. To the best of our knowledge, this green effect strategy has never been applied for cellulase into BIS. In situ entrapment was performed during support synthesis, applying a one-pot approach at mild conditions (room temperature, pH 7, and water solvent), while adsorption was performed after support formation. The loading efficiency was investigated on different immobilisation systems by Bradford assay and FTIR. Bovine serum albumin (BSA) was chosen as a control to optimize cellulase loading. The residual activity of cellulase was analysed by the dinitro salicylic acid (DNS) method. Activity of 90% was observed for the entrapped enzyme, while activity of ~55% was observed for the adsorbed enzyme. Moreover, the supported enzyme systems were recycled five times to evaluate their reuse potential. The thermal and pH stability tests suggested that both entrapment and adsorption strategies can increase enzyme activity. The results highlight that the entrapment in BIS is a potentially useful strategy to easily immobilise enzymes, while preserving their stability and recycle potential.

Optimization of Amylase Production from <i>Lactobacillus plantarum</i> CS by Submerged Fermentation Using Agro Wastes Substrates

Amylases are known to be of great importance worldwide. Most of their production using refined carbon and nitrogen substrates are expensive, resulting in the production of expensive goods which are not easily affordable by end users in our country Nigeria. This necessitates the call for the use of cost-effective substrates under optimized condition for maximum amylase yield. Hence, the present study was focused on the optimization of amylase production from <i>Lactobacillus plantarum</i> CS using agro wastes from local substrates. The parameters assayed were carbon and nitrogen substrates from agro wastes, fermentation period (24-168h), pH (3.5-11) and temperature (30°C-70°C). Others included solvents for preparation of fermentation medium and metal ions of Mg²⁺, Mn²⁺, K⁺, Na⁺, Hg²⁺, Fe²⁺, Cu²⁺ and Pb²⁺ as supplements. The amylase produced via shake-flask fermentation was assayed using Dinitro salicylic acid method. The result revealed 3% sweet potato and 1% bambara nut wastes as the best carbon and nitrogen components of the required fermentation medium of pH 6.5. The optimal amylase produced (38.6U/ml) was achieved within 48h at 35°C with 1% inoculum and Mn²⁺/ Mg²⁺ as a co-factor. Relatively, Cu²⁺, Pb²⁺ and Hg²⁺ reduced the amylase yield to < 68.13% as against Mn²⁺ and Mg²⁺ which enhanced the production to 121.66% and 101.09% respectively. Natural rain water used as solvent for the preparation of fermentation medium significantly enhanced amylase production (24.65 U/ml) in relation to other test waters at P>0.05. Comparatively, there was a significant increase in amylase produced under optimized condition than not. Conclusively, the obtained results which revealed high amylase yield with available and cost effective agro-wastes is promising for industrial application in Nigeria.

Polysaccharide hydrolyzing enzyme activity of bacteria, native to Apis florea gut

Introduction and Aim: Apis florea commonly known as “dwarf honey bee” harbors enormous gut bacteria that can digest complex carbohydrates and other food components. In this regard, the present investigation was focused on analyzing the polysaccharide degrading ability of bacteria isolated from the gut of honeybee, for their possible application in nutraceutical and pharmaceutical industries. Materials and Methods: Nine bacterial isolates were screened for carbohydrate degrading enzymes viz., amylase, pectinase, cellulase, tannase and laccase, using respective substrate by plate assay method. Further activities of amylase and pectinase were measured quantitatively by dinitrosalicylic acid (DNS) method. Results: All the nine selected isolates exhibited amylase and pectinase activities. However, only two isolates exhibited lignolytic and cellulolytic activity. None of the isolates showed tannin degradation. Maximum amylase activity (4.95 U/mg) was observed in Bacillus halotolerans af-M9 followed by Klebsiella oxytoca af-G4 (4.62 U/mg). With respect to pectinase activity Klebsiella pneumoniae af-E17 displayed higher activity (0.24 U/mg) followed by Klebsiella oxytoca af-G4 (0.20 U/mg). Conclusion: Habitat-specific innovations are being explored for novel compounds for therapeutic applications. This study throws a light on selection of carbohydrate degrading bacteria from a new source i.e., GUT of honeybee.

Preparation of Ethanolic Extract of Cassia auriculata and Its Anti-Diabetic Activity

Introduction: Diabetes has caused a major burden to the health sector in the developing countries and has shown an increasing trend among the urban population. It is estimated that most patients are with type II diabetes which could be easily treated with dietary changes, exercise, and medication. Sri Lanka carries a long history of ayurvedic medicine where it uses the plant for treating many diseases. Therefore it is important to screen medicinal plants scientifically so they could be used safely and effectively in the traditional medical system and also be used for further investigations. Cassia auriculata is a plant used in the Ayurvedic medical system in Sri Lanka for treating many diseases including diabetics. We evaluated the anti-diabetic properties and the antioxidant properties of Cassia auriculata leaves.&#x0D; Methods: The methanol extract of the leaves was sequentially extracted with petroleum ether and thereafter was partitioned between EtOAc, and water. The α-amylase inhibition assay was performed using the 3,5- dinitrosalicylic acid method. The antioxidant activities were measured using the DPPH free radical scavenging activity and the total phenolic content using Folin-Ciocalteu’s reagent. The cytotoxicity of the extract was evaluated using the Brine shrimp bioassay.&#x0D; Results: The extract shows very good anti-diabetic activity for the Cassia auriculata extract by using BSA and EAA Assay.&#x0D; Conclusion: The leaf extracts of cassia auriculata exhibit remarkable α-amylase inhibitory activity in the crude methanolic extract. Hence leaves of cassia auriculata have a potential to be used as a regular green vegetable and also be investigated further in isolating pure compounds with anti-diabetic activity.

Studies on cellulases of some cellulose-degrading soil fungi.

In this study, novel cellulolytic fungal strains were isolated, purified, and identified. The ability of these isolates to biodegrade cellulosic materials was examined. The potential of the isolates to produce cellulolytic enzymes and optimize the cellulose degradation process was also investigated. The nylon net bag technique was used to separate cellulose-degrading fungi from soil. A total of 37 species were isolated and tested for their capacity to break down rice straw as a cellulose source. Qualitative assays for cellulase were carried out by Congo red plate assay, while quantitative assays were carried out using the dinitrosalicylic acid (DNS) method at 546nm. Following morphological and molecular identification, the isolates that exhibited the highest cellulase activities were identified as Neurospora intermedia (Assiut University Mycological Center (AUMC) 14359), Fusarium verticillioides (AUMC 14360), and Rhizopus oryzae (AUMC 14361). For N. intermedia, the optimal conditions for maximum activity were a 2% carboxymethylcellulose (CMC) concentration, beef extract and peptone as nitrogen sources, pH 7, 30°C, and an inoculum size of 2.5 (v/v). For F. verticillioides, the optimal conditions were 2.5% CMC concentration, yeast extract as a nitrogen source, pH 6, 30°C, and an inoculum size of 2 (v/v). For R. oryzae, the optimal conditions were 2% CMC concentration, beef extract as a nitrogen source, pH 7, 30°C and an inoculum size of 2.5 (v/v). The identified strains represent new starter strains with high efficiency under optimum conditions for prospective large-scale application in cellulose degradation and nanocellulose production-dependent water purification.

Transmission of the effect of Mercurius corrosivus 30 CH on -amylase in cell free medium through water.

Abstract&#x0D; Background&#x0D; Mercurius corrosivus 30CH promoted -amylase activity in a cell free medium invitro. -amylase causes hydrolysis of starch. The activity of the enzyme is measured in terms of the amount of maltose liberated due to breakdown of starch. In a number of experimental studies it has been demonstrated that the effect of homeopathic potency would be transmitted from one plant to another through water. Here one leaf of a pair of plants was dipped in water in a beaker. The two beakers were connected by a water filled polythene tube. The effect of treatment of one plant with homeopathic potency would be observed in the directly treated plant as well as the connected plant. Two groups of toads were kept in water in two different containers. The two containers were connected by a water filled polythene tube. The effect of treatment of one group of toads with homeopathic potency would be observed in both the directly treated group as well as the connected group.&#x0D; Objectives&#x0D; The purpose of the present study is to see whether the effect of Mercurius corrosivus 30 CH on -amylase in one test tube would be transmitted to another test tube connected with the former by water filled capillary tube.&#x0D; Methods&#x0D; Mercurius corrosivus 30 CH was diluted with distilled water (1:100). Two hard glass test tubes each containing -amylase were connected with a water filled capillary tube while one test tube received Mercurius corrosivus 30CH solution, the other only the control solution. The control solution consisted of equal amount of 90% ethanol diluted with water (1:100).There were two more test tubes, one containing same amount of distilled water instead of Mercurius corrosivus 30 CH solution and the other test tube the same amount of 90% ethanol (1:100) as in the control set. After 10 mins starch solution in Sodium acetate buffer was added to each test tube. The enzyme in each test tube was allowed to react with starch for 15 mins and then it was stopped by DNSA (Dinitro salicylic acid) solution .The activity of -amylase was measured by standard biochemical process. The breakdown product maltose in each test tube was quantified by a standard curve prepared by measuring the optical density of the maltose solution at 560 nm in a UV-VIS Spectrophotometer. This experiment was repeated 20 times.&#x0D; Results&#x0D; Activity of -amylase was expressed in terms of the amount of maltose liberated from breakdown of starch with standard errors in 15mins at a fixed temperature. The data were analyzed statistically using t-test. Mercurius corrosivus 30 CH enhanced the enzyme activity significantly in the directly treated test tube as well as the connected one (p

Cloning, biochemical characterization and molecular docking of novel thermostable β-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin

This study reports a novel BglA9 gene of 1345 bp encoding β-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were Km (0.28 mM), Vmax (43.8 μmol/min/mg), kcat (38.43 s−1) and kcat/Km (135.5 s−1 mM−1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 μmol/min/mg, 18.28 s−1and 3.27 s−1 mM−1 for Km, Vmax, kcat, and kcat/Km values, respectively. The Ki value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

Inhibition of α-amylase activity of Chamaecrista nigricans (Vahl) Greene (Fabaceae-Caesalpinioideae) and Pseudocedrela kotschyi (Schweinf.) Harms (Meliaceae) water extracts

Introduction: Antioxidants and glucosidases inhibitors are interesting in diabetes mellitus prevention and its management. However, conventional drugs have a multitude of unenviable side effects. Thus, research is turning towards herbal remedies, hence this study that was interested in two plants used in the traditional treatment of diabetes in Bobo-Dioulasso. Objective: The aim was to evaluate Chamaecrista (C.) nigricans and Pseudocedrela (P.) kotschyi roots water extracts effect on sorghum α-amylase activity.&#x0D; Methodology: Total phenolic and flavonoid contents were quantified using Folin-Ciocalteu reagent and aluminum trichloride, respectively. FRAP, DPPH and ABTS methods were used to estimate the extracts antioxidant activity. The α-amylase inhibition tests were carried out using the glucose assay method with dinitrosalicylic acid.&#x0D; Results: The C. nigricans and P. kotschyi extracts have shown strong antioxidant activities, 7031.52±15 and 8762.35±51 µmol AAE.g-1E respectively, referring to trolox using ABTS method. These extracts have also shown an inhibitory effect of 98.57% and 97.67% respectively on α-amylase activity.&#x0D; Conclusion/perspectives: These plants have in vitro an interesting anti-amylasic potential. Further investigations will be necessary using a pure enzyme and a wider range of concentration of the extracts in order to determine the kinetic parameters and the fractionation by chromatography to determine the most effective fraction(s). Finally, an in vivo evaluation of the anti-diabetic effect of the extracts could be considered.&#x0D; Keywords: anti α-amylase, antioxidant, Chamaecrista nigricans, Pseudocedrela kotschyi.

Isolation and Identification of Cellulase Producing and Sugar Fermenting Bacteria for Second-Generation Bioethanol Production

Over the last decades, the negative impacts of fossil fuel on the environment and increasing demand for energy due to the unavoidable depletion of fossil fuels, has transformed the world’s interests towards alternative fuels. In particular, bioethanol production from cellulosic biomass for the transportation sector has been incrementing since the last decade. The bacterial pathway for bioethanol production is a relatively novel concept and the present study focused on the isolation of potential “cellulase-producing” bacteria from cow dung, compost soil, and termite gut and isolating sugar fermenting bacteria from palm wine. To select potential candidates for cellulase enzyme production, primary and secondary assays were conducted using the Gram’s iodine stain in Carboxy Methyl Cellulose (CMC) medium and the Dinitrosalicylic acid (DNS) assays, respectively. Durham tube assay and Solid-Phase Micro-Extraction (SPME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) was used to evaluate the sugar fermenting efficiency of the isolated bacteria. Out of 48 bacterial isolates, 27 showed cellulase activity where Nocardiopsis sp. (S-6) demonstrated the highest extracellular crude enzyme activity of endoglucanase (1.56±0.021 U) and total cellulase activity (0.93±0.012 U). The second-highest extracellular crude enzyme activity of endoglucanase (0.21±0.021 U) and total cellulase activity (0.35±0.021 U) was recorded by Bacillus sp. (T-4). Out of a total of 8 bacterial isolates, Achromobacter sp. (PW-7) was positive for sugar fermentation resulting in 3.07% of ethanol in broth medium at 48 h incubation. The results of the study revealed that Nocardiopsis sp. (S-6) had the highest cellulase enzyme activity. However, the highest ethanol percentage was achieved with by having both Bacillus sp. (T-4) and Achromobacter sp. (PW-7) for the simultaneous saccharification and fermentation (SSF) method, as compared to separate hydrolysis and fermentation (SHF) methodologies.

Isolation and selection of amylase-producing microbes isolated from ragi tape and cassava tape available on the markets

Amylase has an important role in biotechnology development and occupies an important position in the world enzyme market, as a biocatalyst in various industrial fields. This study has the goal to find microbial isolates that have the ability to produce amylase enzymes. The study was conducted in two stages, namely: 1) Isolation and selection of microbes that can produce amylase enzymes using starch as substrate, was incubated for 4-7 days at 30°C. Microbial isolates that can produce amylase enzymes are characterized by the presence of clear zones around the colony after the addition of an iodine solution of 1% in the overgrown media of microbes, 2) Test the activity of amylase enzymes using a dinitrosalicylic acid reagent test. The activity of the amylase enzyme is determined by measurement using a spectrophotometer at a wavelength of 540 nm. The sample used comprised of 7 types of ragi tape and 2 samples from cassava tape that has been fermented for 5-7 days. The results obtained in the first stage were 65 microbial isolates, 16 of which had clear zones, consisting of 7 isolates from ragi tape samples and 9 isolates from cassava tape samples. In the enzyme activity test, there are several isolates that have the potential to produce amylase enzymes, these include R5I4 (0.897 ± 0.018 U/mL), R2I5.1 (0.814 ± 0.011 U/mL), R5I3 (0.727 ± 0,042 U/mL) (derived from cassava ragi tape samples) and T2I2.2 (0.812 ± 0.013 U/mL), T2I6.1 (0.817 ± 0.010 U/mL), T2I2.1 (0.735 ± 0.023 U/mL), T1I4 (0.755 ± 0.020 U/mL) (derived from cassava tape samples). The isolate with the highest enzyme activity is the R5I4 which has the value enzyme activity of 0.897 ± 0.018 U/mL and with a fairly high or moderate category, while the lowest enzyme activity is the T1I1.1 isolate of 0.284 ± 0.020 U/mL.

Thermoxylanolytic and Thermosaccharolytic Potential of a Heat Adapted Bacterial Consortium Developed From Goat Rumen Contents

Thermoactive xylanases have important applications in the industrial deconstruction of lignocellulosic plant biomass, due to their sustained activity even at high temperature conditions of industrial bioreactors. We herein report the development of a thermoactive xylanolytic microbial consortium from the semi-digested contents of goat rumen and characterization of the xylanolytic enzyme cocktail produced by it. The consortium exhibited maximum endoxylanase activity at pH6 and at 60°C. Zymogram analysis revealed the production of multiple xylanases. The xylanase cocktail was stable over a pH range of 5–9 after pre-incubation for 3 h. It retained 74% activity after pre-incubation (60°C) for 50 min. It’s activity was enhanced in presence of β-mercaptoethanol, NH4+, Mg2⁺ and Ca2⁺, whereas Hg2⁺ had an inhibitory effect. The xylanolytic cocktail was further utilized for the saccharification of alkali pre-treated rice straw and mushroom spent rice straw. Saccharification was studied quantitatively using the dinitrosalicylic acid method and qualitatively using scanning electron microscopy. Results indicated the potential of the xylanolytic cocktail for the saccharification of rice straw and highlighted the significance of chemical and/or biological pre-treatment in improving the accessibility of the substrate towards the xylanase cocktail.

A comprehensive evaluation of pectinase, pectinmethylesterase and pectolyase activity

Pectin polysaccharide has galacturonic acid with linear chains of α-(1–4)-linked D- galacturonic acid. Rhamnogalacturonan I pectins (RG-I) shows the existence of the repeating disaccharide 4-α-D-galacturonic acid-(1,2)-α-L- rhamnose, which acts as a backbone. Chiefly, D-galactose, L-arabinose, and D-xylose are the sugars types and its proportions of neutral sugars are varied according to the origin of pectin. Pectinase, pectinmethylesterase, and pectolyase enzymes have important applications in food, textile and agricultural industries. These enzymes play an important role in the breakdown of the central part of the plant cell wall. Pectin forms the center part of the plant cell wall. Pectins are termed as structural polysaccharide that has integrity for the steadiness of the plant cell wall. Citrate buffer of molarity 0.1 utilized to verify optimal pH along with temperature, for standardising enzyme activity of pectinase, pectolyase, and pectinmethylesterase by the dinitrosalicylic acid reagent method. Confirmatory check of enzyme’s activity was performed on plant leaves dried particles. Impact of catechin presence in enzyme reaction was too studied. Results delve into degradation of the plant polysaccharide by applying these enzymes. An increase in the monosaccharide galacturonic acid quantity was also significant. The highest release of the polyphenols was found due to pectolyase followed by pectinmethylesterase and pectinase. Pectinmethylesterase effect showed the maximum release of the flavonoids followed by pectinase and pectolyase which was remarkable.

Analytical study on Fungal Cellulase Produced by Penicillium Expansum grown on Malus Domestica (Apple Fruits)

The rise in world industrialization and the cost of importing enzyme by local industries have led to arise in the search for novel and native enzyme producing microorganisms. Cellulase is an enzyme that catalyzes the breaking down of carbon chains in cellulose and hemicellulose, this research therefore aimed at studying fungal cellulase produced by Penicillium expansum grown on malus domestica (apple fruits). Fresh apple fruit was allowed to deteriorate under laboratory condition until there was visible mould growth. The mould with desired features of the organism of interest was subcultured by direct plating on PDA plates to which 10 % streptomycin has been added to prevent bacterial contaminants. The plates were incubated at 28±2 0C for 7 days until a visible mass of blue mycelia appear. The isolate was further subcultured onto freshly prepared media until pure culture was obtained. Characterization and identification of isolate were done using macroscopy and microscopy techniques. The isolate was re-inoculated into healthy apple fruits and the fruits were incubated at temperature of 28±2 oC for 8 days. Cellulolytic activity was examined every day throughout the incubation period. Crude enzyme was extracted each day using standard methods. Carboxyl methyl cellulose was used as standard for the crude cellulase activity assay after extraction from the infected apple fruits using Dinitrosalicylic acid (DNSA). Culture parameters like pH and temperature were also optimized to determine their effect on cellulolytic activity of the fungus. Cellulase activity was defined as the amount of glucose produced in μmol/mg/min under the assay condition. The highest cellulase activity of 86.84±0.52 μmol/mg/min was observed on day 6 of incubation at 28±2 oC and at pH 7. In conclusion, it is evident from this research that P. expansum isolated could be used as potential novel organism for industrial production of cellulase under optimized fermentation conditions.

Saccharification of Hazelnut and Rhododendron Biomasses Using β-xylanase from Thermotoga naphthophila

Enzymes can be used in various biotechnological applications due to the easy and cheap production. Since xylanase enzymes are preferred in various industries, researchon this enzyme is extensively being carried out. In this study, the β-xylanase gene was cloned from Thermotoga naphthophila, a thermophilic organism. The expression vector pET21a(+) was expressed in Escherichia coli BL21 (DE3). As a result of the studies, the pH, temperature and IPTG concentration of the enzyme were optimized to obtain highest expression. Dinitrosalicylic acid (DNS) was used to determine sugar content of the enzyme. The molecular mass of the purified β-xylanase enzyme was determined using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular mass of the enzyme was calculated to be 38 kDa. Enzymatic hydrolysis of hazelnut shell, rhododendron branch and rhododendron leaves was performed. Released reducing sugar contents from the enzymatic hydrolysis were calculated as 0.8461 mg mL-1, 0.6976 mg mL-1 and 0.3605 mg mL-1 for hazelnut shell, rhododendron branch, and rhododendron leaf respectively. In conclusion, β-xylanase enzyme can be an effective source for enzymatic hydrolysis to produce fermentable sugars from such biomasses.