Correlation between physicochemical characteristics of lignin deposited on autohydrolyzed wood chips and their cellulase enzymatic hydrolysis.

Enzymatic hydrolysis is a method to generate biofuel from biomass, and autohydrolysis is a popular method to pretreat biomass prior to enzymatic hydrolysis. The primary aim of the present study was to determine the role of lignin produced in the autohydrolysis process on the enzymatic hydrolysis of biomass. The HSQC and 31P NMR analyses confirmed that ß-O-4 of lignin was reduced, while ß-5, ß-ß, and S/G-ratio of lignin were increased with intensifying the hydrolysis intensity.The increase in the hydrolysis intensity significantly enhanced the condensed and non-condensed phenolic OH group of lignin. Interestingly, the cellulase enzyme adsorbed more on lignin that had more phenolic content, and its association with lignin reduced its activity for hydrolyzing cellulose microcrystals. Strong negative correlations were observed between the enzymatic hydrolysis yield and the condensed S-OH (r2 = 0.978) and G-OH (r2 = 0.961) of lignin generated in the autohydrolysis process.

Bioactive compounds guided diversity of endophytic fungi from Baliospermum montanum and their potential extracellular enzymes.

Baliospermum montanum (Willd.) Muell. Arg, a medicinal plant distributed throughout India from Kashmir to peninsular-Indian region is extensively used to treat jaundice, asthma, and constipation. In the current study, 203 endophytic fungi representing twenty-nine species were isolated from tissues of B. montanum. The colonization and isolation rate of endophytes were higher in stem followed by seed, root, leaf and flower. The phytochemical analysis revealed 70% endophytic isolates showed alkaloids and flavonoids, 13% were positive for phenols, saponins and terpenoids. Further, these endophytes produced remarkable extracellular enzymes such as amylase, cellulase, phosphates, protease and lipase. The most promisive three endophytic fungi were identified by ITS region and secreted metabolites were identified by gas chromatography-mass spectrometry (GC-MS/MS). The GC-MS profile detected twenty-five bioactive compounds from ethyl acetate extracts. Among endophytic fungi, Trichoderma reesei isolated from flower exhibited nine bioactive compounds namely, 2-Cyclopentenone, 2-(4-chloroanilino)-4-piperidino, Oxime-methoxy-Phenyl, Methanamine N-hydroxy-N-methyl, Strychane, Cyclotetrasiloxane, Octamethyl and 1-Acetyl-20a-hydroxy-16-methylene. The endophyte, Aspergillus brasiliensis isolated from root and Fusarium oxysporum isolated from seed produced nine and seven bioactive compounds, respectively. Overall, a significant contribution of bioactive compounds was noticed from the diverse endophytic fungi associated with B. montanum and could be explored for development of novel drug with commercial values.

A Need for Improved Cellulase Identification from Metagenomic Sequence Data.

Improved sequencing technologies and the maturation of metagenomic approaches allow the identification of gene variants with potential industrial applications, including cellulases. Cellulase identification from metagenomic environmental surveys is complicated by inconsistent nomenclature and multiple categorization systems. Here, we summarize the current classification and nomenclature systems, with recommendations for improvements to these systems. Addressing the issues described will strengthen the annotation of cellulose-active enzymes from environmental sequence data sets-a rapidly growing resource in environmental and applied microbiology.

Real-Time Visualization of Cellulase Activity by Microorganisms on Surface.

A variety of methods to detect cellulase secretion by microorganisms has been developed over the years, none of which enables the real-time visualization of cellulase activity on a surface. This visualization is critical to study the interaction between soil-borne cellulase-secreting microorganisms and the surface of plant roots and specifically, the effect of surface features on this interaction. Here, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable the real-time tracking of cellulase activity of microorganisms on a surface. A surface was formed using pure CMC with acridine orange dye incorporated in it. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization, since the common need for post hydrolysis dyeing was negated. Using root-knot nematode (RKN) as a model organism that penetrates plant roots, we showed that it was possible to follow microorganism cellulase secretion on the surface. Furthermore, the addition of natural additives was also shown to be an option and resulted in an increased RKN response. This method will be implemented in the future, investigating different microorganisms on a root surface microstructure replica, which can open a new avenue of research in the field of plant root-microorganism interactions.

Dynamic changes in the phenolic composition and antioxidant activity of oats during simultaneous hydrolysis and fermentation.

Solid-state fermentation (SSF) is the preferred method of enhancing the phenolic content of oats, while scientific optimization for improving specific phenolic compounds is limited. In this study, sequential targeting of phenolic conversion in simultaneous hydrolysis and fermentation (SHF) of oats was investigated. The results revealed that SHF with adding cellulase at 0, 6 and 12 days could increase the total phenolic content by 4.4%, 67.8% and 59.1%, respectively, over that of SSF. The α-amylase and CMCase activity were highly correlated with the soluble and insoluble phenolic contents in SHF (-6 and -12) systems (r > 0.8, p < 0.05). Interestingly, the content of phenolic fraction, such as ferulic acid, was up-regulated, whereas sinapic acid was down-regulated. These results indicated that the phenolic conversion occurred in SHF, resulting in variation in DPPH and ABTS+ radical scavenging abilities. This research provided metabolic understanding of the optimization of phenolic compounds to increase the functional ingredient of oats.

Sustainable use of the spent mushroom substrate of Pleurotus florida for production of lignocellulolytic enzymes.

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57-74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08.

Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts.

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

Integration of World-to-Chip Interfaces with Digital Microfluidics for Bacterial Transformation and Enzymatic Assays.

Digital microfluidics (DMF) represents an alternative to the conventional microfluidic paradigm of transporting fluids in enclosed channels. One of the major benefits of DMF is that fluid motion and control is achieved without external pumps. The automation component of DMF have pushed the barriers of this "lab-on-chip" technology. However, integration with external components (i.e., "world-to-chip") interfaces have been a challenge. Two common "world-to-chip" challenges are (1) delivering biological samples to DMF devices and (2) accurately controlling temperatures on device. To address these challenges, this work describes two "world-to-chip" interface features that have been integrated on a DMF platform: a reagent delivery system and a thermal control apparatus. This platform enables a variety of biological or chemical experiments to be conducted on-chip while reducing manual intervention. Specifically, our platform increases reagent volumes available to device reservoirs volume by at least 50-fold eliminating the need to manually refill reservoirs while improving droplet dispensing reproducibility. In addition, we have integrated a closed-loop temperature control system that offers precise temperature control on-chip. To validate our "world-to-chip" interface, we have automated bacterial transformation and enzymatic assay protocols, showing that such a system enhances DMF performance. Overall, we propose that this system will improve biological experimentation which requires fluidic and temperature control integrated on DMF platforms.

A simplified and miniaturized glucometer-based assay for the detection of ß-glucosidase activity.

ß-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of ß-glucosidase activity. Single-factor experiments showed that optimum ß-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of ß-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from ß-dextranase, snailase, ß-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for ß-glucosidase activity. The easy-to-operate method was successfully used to detect a series of ß-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of ß-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Population structure and virulence analysis of Alternaria carthami isolates of India using ISSR and SSR markers.

Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. Diversity among 95 isolates of A. carthami was determined using virulence assays, enzyme assays, dominant (ISSR) and co-dominant (SSR) markers. Collections and isolations were made from three major safflower producing states of India. The virulence assays categorised the population into four groups based on level of virulence. Estimation of activities of cell wall degrading enzymes (CWDE) yielded concurrent results to virulence assays with maximum CWDE activities in most virulent group. Eighteen ISSR primers were used and 23 polymorphic microsatellite markers were developed to assess the genetic diversity and determine the population structure of A. carthami. Analysis of ISSR profiles revealed high genetic diversity (Nei's Genetic diversity index; h = 0.36). Microsatellite markers produced a total of 56 alleles with an average of 2.43 alleles per microsatellite marker and Nei's genetic diversity index as h = 0.43. Unweighted Neighbor-joining and population structure analysis using both the marker systems differently arranged the isolates into three clusters. Distance analysis of the marker profiles provided no evidence for geographical clustering of isolates, indicating that isolates are randomly spread across India, signifying high potential of the fungus to adapt to diverse regions. Microsatellite markers clustered the isolates in consonance to the virulence groups in the dendrogram. This implies that the fungus has a high potential to adapt to resistant cultivars or fungicides. The information can aid in the breeding and deployment of A. carthami resistant varieties, and in early blight disease management in all safflower growing regions of the world.

Assessing the cellulase enzyme heterogeneity of bacterial strains and their feedback to cattle manure degradation in a greenhouse model of in vivo pond ecosystem.

The responses of cellulase enzymes of three bacterial isolates and their impacts on cattle manure decomposition were assessed in a greenhouse model in vivo pond ecosystem. Fifty grams of fresh cattle manure was placed in a fastened nylon bag (mesh size ~ 50 µm dia.) and placed in triplicate in a plastic bucket with 10 l of pond water which was hung inside the enclosed polyhouse, semi-closed and open systems for 4 weeks. Samples of manure residue directly from nylon bag and water from manure leached bucket water, water, and soil from the enclosed polyhouse were collected for enzymatic assays, enumeration of aerobic cellulose decomposing and heterotrophic bacteria, and determination of water and soil quality parameters. Responses of cellulases to different temperatures in situ were also elucidated. The values of test bacteria, endoglucanase, exoglucanase and ß-glucosidase, and organic carbon were significantly (P Ë‚ 0.05) higher in the closed system compared to semi-closed or open system. Priming of all the enzymes coupled with the peak of aerobic cellulose decomposing bacteria and heterotrophic bacterial populations occurred on the day 14 or 21 in vivo. Since the peaks of three cellulases of bacterial isolates (KUPH1, KUPH6, and KUPH8) were demonstrated between 35 and 40 °C, and that temperature coincided with temperature of the greenhouse model, this temperature range appeared to favor the growth of cellulose decomposing bacterial populations and involved cellulase enzymes.

Preparation of a novel soluble inducer by cellobiase-release microcapsules and its application in cellulase production.

Effective and low-cost inducer is a prerequisite for efficiency improvement in cellulase production by Trichoderma reesei. In this study, spores of Aspergillus niger enriched with cellobiase were embedded in Ca-alginate microcapsules, attaining a sustained cellobiase-release system (named CRMA) which was then used to catalyze the conversion of glucose into glucose-sophorose sugar mixture via transglycosylation. Further, the process and optimal parameters of transglycosylation reaction were studied in a 5 L reactor and the sophorose concentration reached 70.55 g/L at 50 °C, pH 4.0 with CRMA loading of 200 mL after 72 h of reaction. The obtained sugar mixture was subsequently used as both carbon source and inducer in cellulase production by Trichoderma reesei under fed-batch process. The filter paper activity (FPA) reached 90.28 IU/mL at 144 h fermentation time, 1.85-fold higher than that induced by lactose. These results indicate that the newly established strategy for soluble inducer synthesis has the potential for efficiency improvement and cost reduction in cellulase production.

Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

A bacterial pioneer produces cellulase complexes that persist through community succession.

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, 'Candidatus Reconcilibacillus cellulovorans', possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the 'Ca. Reconcilibacillus cellulovorans' multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.

Antibacterial activity and probiotic characterization of autochthonous Paenibacillus polymyxa isolated from Anabas testudineus (Bloch, 1792).

Microbial fish pathogens are prevalent in aquaculture. Control of bacterial fish pathogens is important and bio control of pathogenic bacteria is a novel field of study. The aim of this study was to evaluate the antagonistic activity of bacteria isolated from Anabas testudineus against potent fish pathogens. The cellular components/preparations and filtered cell free culture supernatants were effective against six fish pathogens. Altogether 110 strains were isolated from fish proximal and distal intestine, out of which 10 strains were selected through well diffusion method. From them a strain HGA4C having prominent antimicrobial activity was selected as candidate probiotic strain. The whole-cell product, heat-killed whole-cell product and the filtered broth were exhibited bactericidal activity against the tested pathogens. Among them cell free culture supernatant showed maximum inhibition. In addition, isolated candidate probiotic bacterium was capable of producing extracellular enzymes important for the digestion of food ingredients and was effectively grown in fish mucus obtained from Oreochromis niloticus. The strain tolerated gradient of bile juice secreted by the host and effectively produced biofilm. Analysis of 16S rDNA sequence revealed that isolated strain HGA4C was Paenibacillus polymyxa (MF457398.1). Furthermore intraperitoneal injection of the bacterium did not induce any pathological anomalies or mortalities in Oreochromis niloticus and disclosed the safety of this bacterium as a candidate probiotic in aquaculture.

Constitutive Expression of Chimeric Transcription Factors Enables Cellulase Synthesis under Non-Inducing Conditions in Penicillium oxalicum.

Industrial production of cellulase by filamentous fungi is largely dependent on cellulose, which serves as a natural inducer of cellulase expression. However, insoluble cellulose is unfavorable to submerged fermentation and thus limits the production level of cellulase. The possibility of cellulase production under non-inducing conditions is explored in Penicillium oxalicum by overexpressing two chimeric transcription factors. The chimeric transcription factors contain the DNA binding domain of cellulase transcriptional activator ClrB linked to the C-terminal sequences of XlnRA871V , a constitutively active mutant of hemicellulase transcriptional activator. The obtained recombinant mutants exhibited dramatically improved basal production of cellulase, which was not observed with the overexpression of intact ClrB. When cultivated in a complex cellulosic medium, one of these mutants, OE-CXC -S-1, displayed a 7.3-fold increase in cellulase production (2.8 U mL-1 ) relative to the parent strain. The results demonstrate that the dependence of cellulase synthesis on cellulose could be reduced by the overexpression of artificially designed chimeric transcription factors, and offers a potential strategy to engineer fungal strains for improving cellulase production.

Regular enzyme recovery enhances cellulase production by Trichoderma reesei in fed-batch culture.

OBJECTIVE: To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals. RESULTS: Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24-62%) and ß-glucosidase (22-76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and ß-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5-4.5 g l-1. CONCLUSION: Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

Comparative secretomic analysis of lignocellulose degradation by Lentinula edodes grown on microcrystalline cellulose, lignosulfonate and glucose.

Lentinula edodes has the potential to degrade woody and nonwoody lignocellulosic biomass. However, the mechanism of lignocellulose degradation by L. edodes is unclear. The aim of this work is to explore the profiling of soluble secreted proteins involved in lignocellulose degradation in L. edodes. For that, we compared the secretomes of L. edodes grown on microcrystalline cellulose, cellulose with lignosulfonate and glucose. Based on nanoliquid chromatography coupled with tandem mass spectrometry of whole-protein hydrolysate, 230 proteins were identified. Label-free proteomic analysis showed that the most abundant carbohydrate-active enzymes involved in polysaccharide hydrolysis were endo-ß-1,4-glucanase, α-galactosidase, polygalacturonase and glucoamylase in both cellulosic secretomes. In contrast, enzymes involved in lignin degradation were most abundant in glucose culture, with laccase 1 being the predominant protein (13.13%). When the cellulose and cellulose with lignosulfonate secretomes were compared, the abundance of cellulases and hemicellulases was higher in cellulose with lignosulfonate cultures, which was confirmed by enzyme activity assays. In addition, qRT-PCR analysis demonstrated that the expression levels of genes encoding cellulases and hemicellulases were significantly increased (by 32.2- to 1166.7-fold) when L. edodes was grown in cellulose with lignosulfonate medium. BIOLOGICAL SIGNIFICANCE: In this article, the secretomes of L. edodes grown on three different carbon sources were compared. The presented results revealed the profiling of extracellular enzymes involved in lignocellulose degradation, which is helpful to further explore the mechanism of biomass bioconversion by L. edodes.

Electrochemical Assay for a Total Cellulase Activity with Improved Sensitivity.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activities. Here, we report a simple and yet efficient electrochemical assay for the total activity of cellulase, a hydrolytic enzyme widely used in food and textiles industries, and for production of bioethanol. The assay exploits the difference in electrochemical signals from a soluble redox indicator, ferricyanide, on nitrocellulose films treated by cellulases. Ferricyanide electrochemistry is totally inhibited on graphite electrodes modified with an insulating nitrocellulose film and is evoked after the cellulase treatment. Ferricyanide voltammetric responses correlate with the increased permeability of the films and electrochemically active surface area of electrodes becoming accessible to the ferricyanide reaction after nitrocellulose digestion by cellulase. Trichoderma and Aspergillus niger cellulases activities were determined in a 5 min assay with a sensitivity of 10-8 U per assay, being 103-104-fold more sensitive than the standard commercially available optical assays. That makes the developed electrochemical approach the most prospective cost-effective alternative both for research and automated industrial applications.

Chromatographic Study of Azintamide in Bulk Powder and in Pharmaceutical Formulation in the Presence of Its Degradation Form.

Two specific, sensitive, and precise stability-indicating chromatographic methods were developed, optimized, and validated for the determination of Azintamide (AZ) in the presence of its degradation product. The first method was TLC combined with the densitometric determination of the separated bands. Separation was achieved using silica gel 60 F254 TLC plates and chloroform-acetone-glacial acetic acid (7.5 + 2.1 + 0.4, v/v/v) as the developing system. Good correlations were obtained between the integrated peak area of the studied drug and its corresponding concentrations in the linearity range. The second method used HPLC with UV diode-array detection, in which the proposed method was applied for the quantitative determination of AZ in the presence of its acidic degradation product and the quantitative determination of the acid-induced degradation product of AZ (AZ Deg) using pentoxifylline as the internal standard. The proposed components were separated on a reversed-phase C18 analytical column using acetonitrile-water (50 + 50, v/v). The flow rate was maintained at 0.55 mL/min and the detection wavelength was 260 nm. Linear regressions were obtained in the range of 1-30 and 0.3-16 µg/mL for AZ and AZ Deg, respectively. Different parameters affecting the suggested methods were optimized for maximum separation of the cited components. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and successfully applied for the determination of AZ in its pure powder form and in its pharmaceutical formulation. Both methods were also statistically compared with the reported method with no significant difference in performance observed.