Ammonium Sulfate Fractionation Research Articles

Significant Hydrolysis of Wheat Gliadin by Bacillus tequilensis (10bT/HQ223107): a Pilot Study.

Peptidase therapy is suggested to be effective to minimize gliadin toxicity in celiac disease (CD). Hence, present study deals with gliadin-hydrolysing peptidases. The efficient peptidase from the Bacillus tequilensis was purified using ammonium sulfate fractionation and preparative electrophoresis. Analysis of in-solution and in-gel hydrolysis of gliadin using one and two-dimensional SDS-PAGE revealed nearly complete hydrolysis of gliadin peptides after 180min of incubation with B. tequilensis protease. Purified peptidase was found to be stable at acidic (pH 3.5) to neutral (pH 7.2) pH range. The molecular mass and isoelectric point of the peptidase were observed around 29kDa and 5.2, respectively. The internal protein sequence obtained through mass spectrometric analysis suggested that peptidase might belong to peptidase S9 family known for prolyl-specific peptidases. This study recommends the possible applicability of this peptidase for elimination of immunotoxic gliadin peptides and may prove useful in CD treatment.

Purification and antioxidant activity of phycocyanin from Synechococcus sp. R42DM isolated from industrially polluted site

The cyanobacterium Synechococcus sp. R42DM, isolated from an industrially polluted site Vatva, Gujarat, India was recognized to produce phycocyanin (PC) as major phycobiliprotein. In present study, the combinatorial approach of chemical and physical methods i.e. Triton-X 100 treatment and ultra-sonication was designed for extraction of PC. From cell extract, the intact and functional-PC was purified up to purity 4.03 by ammonium sulphate fractionation and ion-exchange chromatography. The PC displayed considerable in vitro antioxidant and radical-scavenging activity. This PC was further noticed to scavenge intracellular-ROS and to increase tolerance against thermal and oxidative stress in Caenorhabditis elegans. Moreover, the PC was noticed to improve the physiological behaviour and longevity of C. elegans. In addition, the PC showed remarkable stability under physico-chemical stressors, which is desirable for their use in biomedical applications. In conclusion, present paper added up evidence in support of the prospective use of PC as an antioxidant nutraceutical.

Antibacterial activity of fusion from biosynthesized acidocin/silver nanoparticles and its application for eggshell decontamination.

Bacteriocins from lactic acid bacteria (LAB) are useful to control the persistent development of pathogenic microorganisms in food and medicine fields. The bacteriocin acidocin was extracted from Lactobacillus acidophilus M1 that was isolated from fermented milk, purified using ammonium sulphate fractionation, and gel filtration column chromatography using Sephadex matrix and applied as a potential antibacterial agent. The molecular weight of the purified acidocin was estimated using SDS-PAGE to be 6.6 kDa. The acidocin was compared with silver nanoparticles (SNPs), biosynthesized by Aspergillus brasiliensis (niger) ATCC 16404, against two bacterial strains Bacillus cereus ATCC 14579 and Staphylococcus aureus ATCC 25923. Both acidocin and SNPs showed significance antibacterial effects using disc and well diffusion methods; the maximum antibacterial activity was proved against B. cereus from acidocin/SNPs composite using a ratio of 1/1 from each agent. The application of acidocin/SNPs composite as immersion solution, for disinfecting chicken eggshells, resulted in remarkable reduction in microbial load on the shells of 5.53 log10 CFU/eggshell. Results could provide an eco-friendly approach for potential antimicrobial composites to be used in food preservation and other health protection researches.

Purification of thermo and acid tolerant extracellular phytase from a new soil isolate of Amycolatopsis vancoresmycina S-12

Abstract Amycolatopsis vancoresmycina S-12, a member of soil Actinomycetes, was explored regarding its extracellular phytase. The enzyme was recovered from the culture broth by ammonium sulfate fractionation and purified to near homogeneity following dialysis, DEAE Sepharose and Sphadex G-100 column. The enzyme was purified 5.51 fold with a final yield of 14.9%. It was characterized by SDS PAGE, Zymogram, Native PAGE, 2D PAGE as monomeric protein of 53 kDa. and p I 5.4. Peptide mass fingerprinting of MALDI-TOF MS result indicates its phytase nature. Optimum enzyme activity was at 37 °C and at pH 5.4. Optimum stability was at 60 °C and pH 8.0. The enzyme activity is stimulated in presence of Mg 2+ . The plant growth promotion experiment shows a significant effect, with an increase of 10% root and 25% of shoot length, resulting in a 2.2 fold increase in biomass.

Hyper-Productivity, Characterization, and Exploitation of a Cellulase Complex from a Novel Isolate of Aspergillus tubingenesis S2 using Lignocellulose-based Material

The hyper-production potential of a cellulase complex from a local strain of Aspergillus tubingensis S2, indigenously isolated from rotten tomato, was investigated. A total of nine fungal species of Aspergillus and Trichoderma were isolated and confirmed through triple-phase screening via 18S ribosomal DNA sequencing and construction of a phylogenetic tree. Congo red testing and the zone of clearance method were used to confirm the cellulase production from A. tubingenesis S2 isolate. A. tubingenesis S2 revealed maximum cellulase production (78 µg/mL/min) and was selected for further study. The optimum fermentative conditions, including the incubation period, pH, and temperature values, were determined to be 96 h, pH 4.8, and 40 °C, respectively, for obtaining the cellulase activity of 86.4±2.1 µg/mL/min. The cellulase was 5.14-fold purified by ammonium sulfate fractionation and gel permeation chromatography. Characterization revealed that maximum activity (130.5 µg/mL/min and 133.5 µg/mL/min) was achieved at 4.5 pH and 40 °C, respectively. A monomeric protein with an apparent molecular weight of 76 kDa was evident after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cellulase revealed maximal activity with 40-mesh size corn stover as compared with 20-mesh size corn stover and 80-mesh size corn stover after 36 h of incubation at 40 °C.

Isolation of recombinant human untagged glyceraldehyde-3-phosphate dehydrogenase from E. coli producer strain

The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass). The specific activity of the freshly purified hGAPDH constitutes 117 ± 5 μmol NADH/min per mg protein (pH 9.0, 22 °C), which is close to the specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase determined under the same conditions and several times exceeds the specific activity of his-tagged GAPDH preparations. The high enzymatic activity suggests that the recombinant enzyme retains its native structure. The described procedure may be useful for researchers who need a preparation of native hGAPDH without admixture of misfolded forms for their investigations.

Chitosan-immobilized pectinolytics with novel catalytic features and fruit juice clarification potentialities

Biological macromolecules are primarily composed of complex polysaccharides that strengthen microbial growth for the production of industrially relevant enzymes. The presence of polysaccharides in the form of the disrupted cell wall and cell materials are among major challenges in the fruit juice industry. The breakdown of such biological macromolecules including cellulose and pectin is vital for the juices processing. In this background, pectinolytic enzymes including polygalacturonase (PG), pectin lyase (PL), and pectin methylesterase (PME) were isolated from Aspergillus ornatus, statistically optimized and purified via ammonium sulfate fractionation (ASF), dialysis, and Sephadex G-100 gel permeation chromatography. After passing through Sephadex G-100 column, PG, PL, and PME were 2.60-fold, 3.30-fold, and 4.52-fold purified with specific activities of 475.2U/mg, 557.1U/mg, and 205.7U/mg. The active PG, PL, and PME, each separately, were surface immobilized using various concentrations of chitosan and dextran polyaldehyde as a macromolecular crosslinking agent. Prior to exploit for juice clarification purposes, various parameters including pH, thermal and Michaelis-Menten kinetic constants of purified and chitosan-immobilized fractions were investigated. A considerable improvement in the pH and thermal profiles was recorded after immobilization. However, the negligible difference between the Km and Vmax values of purified free and chitosan-immobilized fractions revealed that the conformational flexibility of pectinolytics was retained as such. A significant color and turbidity reductions were recorded after 60min treatment with CTS-PG, followed by CTS-PME, and CTS-PL. It can be concluded that the clarification of apples, mango, peach, and apricot juices was greatly affected by CTS-PG, CTS-PME, and CTS-PL treatments rendering them as potential candidatures for food industry applications.

Purification, characterization and retting of Crotolaria juncea fibres by an alkaline pectin lyase from Fusarium oxysporum MTCC 1755

Using solid-state fermentation, production of an industrially important pectin lyase from a fungal strain Fusarium oxysporum MTCC 1755 was attempted, which was further subjected to purification and characterization. The enzyme was purified by three steps, namely ammonium sulfate fractionation, cation-exchange chromatography on CM cellulose followed by gel filtration chromatography using Sephadex G-100 column. A 16-fold purification with 31.2% yield and 3.2 U/mg specific activity was achieved. The optimum pH of the purified enzyme was 9.0 and stability ranged from pH 5.0–7.0 for 24 h. Optimum temperature of purified enzyme was found to be 40 °C while temperature stability ranged from 10 to 50 °C for 30 min. The Km and kcat of the enzyme was 1.75 mg/ml and 83.3 s−1, respectively. The purified enzyme was found to be highly stimulated by Ca2+ ions while sugars like mannitol and sorbitol, and salts like NaCl and CaCl2 enhanced the thermostability. The purified pectin lyase was found suitable for retting of Crotolaria juncea fiber.

Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262.

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu2+, Mg2+, and Fe2+. The enzyme showed a fibrinolytic activity of 22.53U/mL at 40°C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

Effect of Physical and Chemical Parameters on the Activity of Purified Phosphatase Enzyme Produced by Bacillus cereus

Phosphorus is one of the most important nutrients for plant growth and development. This study aimed to survey the optimum condition for phosphatase enzyme production, purified and studied its properties. Phosphatase enzyme activity was determined by end point method. Phosphatase enzyme was obtained from Bacillus cereus EME 66 isolate. The enzyme was purified and characterized, using a three-step purification procedure with 12.3-fold. The phosphatase enzyme was partially purified using ammonium sulfate fractionation followed by ultrafiltration. The acid phosphatase is a monomer protein purified gel filtration to 5.6 fold. Results showed that the optimum temperature for the purified enzyme activity was 60 °C and it was stable at temperatures below 60 °C. This enzyme was stable between pH 4.0-6.0, and the optimal pH activity was found to 5.0. The activity of the enzyme enhanced by heavy metals (Fe 3+ , Cu 2+ , Ca 2+ , Mg 2+ and K + ). The enzyme activity was strongly inhibited by heavy metals Zn 2+ . The present article reveals on enzymatic characterization of acid phosphatase enzyme.Â

Two isoforms of lipoxygenase from mature grains of pearl millet [Pennisetum glaucum (L.) R. Br.]: purification and physico-chemico-kinetic characterization.

This study describes the partial purification and characterization of lipoxygenase (LOX) from pearl millet mature grains of inbred HBL 0843-2. Two isoforms of LOX, i.e., LOX 1 and LOX 2, were purified using ammonium sulphate fractionation, gel filtration chromatography and ion exchange chromatography near homogeneity to 56 and 40 folds with yield of 28 and 24%, respectively. LOX 1 and LOX 2 having molecular masses of approximately 85 and 79kDa, respectively were purified. LOX 1 and LOX 2 exhibited maximum activity at pH 4.5 and 4.8, respectively at 25°C temperature. Both the isoforms, which showed thermostability up to 35°C when incubated for 30min, were stable at a pH range of 7-7.8. LOX 1 and LOX 2 had apparent Km value of 0.86 and 0.57µM, respectively. Ascorbic acid and vitamin E inhibited 66-78 and 61-69% activity of LOX 1 and LOX 2, respectively but Na+, Zn2+ and K+ strongly inhibited the activity of these isozymes. The present information about lipoxygenase enzyme might be valuable in drafting the strategies for its inactivation, which in turn can obstruct the LOX damaging effects on food products during processing and storage.

A cold active transglutaminase from Antarctic krill (Euphausia superba): Purification, characterization and application in the modification of cold-set gelatin gel

Transglutaminase (TGase), EC 2.3.2.13, was purified from whole Antarctic krill (Euphausia superba) using ammonium sulfate fractionation and DEAE-Sephacel chromatography. The purified enzyme had specific activity, purification fold and yield of 53.518U/mg, 10.272 and 10.992%, respectively. The molecular weight of the purified Antarctic krill TGase was estimated to be 78kDa using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for the activity of the purified TGase were pH 8.0–9.0 and 0–10°C, respectively. However, the TGase activity reduced to 50% at a higher temperature of 45°C. The cations Ca++ and Na+ activated the purified TGase activity optimally at levels of incorporation of 10mM and 1.8mM, respectively. Addition of TGase at 0.1U/mg increased the gel strength (p<0.05), setting temperature, setting time (p<0.05) and melting temperature (p<0.05) of cold-set gelatin gel.

English

Proteases constitute most important enzymes owing to their wide variety of functions and have immense applications in various industries viz., medical, pharmaceutical, biotechnology, leather, detergent, and food industries. Despite of their wide spread occurrence in various sources, microorganisms present remarkable potential for proteolytic enzymes production due to their extensive biochemical diversity and susceptibility to genetic manipulation. The present study was aimed at isolating alkaline protease producing fungal members from dairy form effluents, designing the process variables for maximizing the protease production and determining the fibrinolytic potential of the partially purified alkaline protease. To achieve the specified objectives, the dairy form effluent was processed for the isolation of proteolytic fungi using suitable microbiological medium. All the fungal isolates were screened for their protease producing ability and the isolate showing highest alkaline protease production was selected for further studies. Optimization of different fermentative variables like carbon, nitrogen sources, pH, temperature and incubation period were carried out to enhance enzyme production. Ammonium sulphate fractionation was employed to partially purify the enzyme following which its fibrinolytic potential was determined. Based on morphological and microscopic studies, the selected fungal isolate was identified as Aspergillus niger. Optimization studies using OVAT (one variable at a time) method revealed an enhanced protease production in the presence of fructose as additional carbon source and ammonium sulphate as nitrogen source. The optimum incubation period, temperature and pH for enzyme production by the selected fungal isolate was found to be 92 h, 50°C and 10, respectively. The partially purified alkaline protease was efficient in the removal of blood stains emphasizing its fibrinolytic ability. An alkaline protease producing Fungal sp. was screened and isolated from dairy form effluent and it was found to be efficient in the removal of blood stains proving its fibrinolytic potential. Enzymes produced from microorganisms that can survive under extremes of pH could be particularly useful for commercial applications under high alkaline conditions. Key words: Alkaline protease, dairy form effluent, optimization, Aspergillus niger, fibrinolytic potential.

Alkaline Protease Production Using Response Surface Methodology, Characterization and Industrial Exploitation of Alkaline Protease of Bacillus subtilis sp.

Bacterial proteases are considered potential candidates for industrial and biotechnological applications. In the present study, the influence of various parameters on an in-house isolated alkaline protease was investigated through central composite design from a well-known response surface methodology technique. A high level of alkaline protease productivity up to 8.9-folds was achieved from a novel strain of Bacillus subtilis isolated from local Tannery, Lahore Distt. (Pakistan). The purification profile revealed 8.48-fold increase to homogeneity by ammonium sulfate fractionation and Sephadex G-100-based gel filtration chromatography. The purified fraction was identified as a monomeric with apparent molecular weight of 15 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The catalytic characterization was performed with various substrates using Michaelis–Menten equation. The alkaline protease remained optimally active within a broader pH range of 8–12 having a half-life of 37.8 min at 87 °C, along with a maximum velocity (Vmax) of 200 IU/mL/min and 0.090 mg/mL Michaelis constants. Moreover, a complete de-staining and dehairing were recorded within the short incubation period. In conclusion, the data thus obtained in this study suggest a high potential for enzymatic treatment in an industrial setting.

Purification, Kinetic, and Thermodynamic Characteristics of an Exo-polygalacturonase from Penicillium notatum with Industrial Perspective.

An extracellular exo-polygalacturonase (exo-PG) produced by Penicillium notatum was purified (3.07-folds) by ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Two distinct isoforms of the enzyme, namely exo-PGI and exo-PGII, were identified during column purification with molecular weights of 85 and 20kDa, respectively, on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed its optimum activity at pH 6.0 and 50°C and was found to be stable in the slightly acidic pH (ranging from 4.5 to 6.0). Michaelis-Menten parameters, i.e., K m (app) and V max for pectin hydrolysis, were calculated to be 16.6mg/mL and 20μmol/mL/min, respectively. The enzyme followed biphasic deactivation kinetics. Phase I of the exo-PGI showed half-lives of 6.83 and 2.39min at 55 and 80°C, respectively, whereas phase II of the enzyme exhibited a half-life of 63.57 and 22.72min at 55 and 80°C, respectively. The activation energy for denaturation was 51.66 and 44.06kJ/mol for phase I and phase II of the exo-PGI, respectively. The enzyme activity was considerably enhanced by Mn2+, whereas exposure to a hydrophobic environment (urea and sodium azide solution) drastically suppressed the enzyme activity. Results suggest that exo-PGI might be considered as a potential candidate for various applications, particularly in the food and textile industries.

Purification and characterisation of soluble acid invertase from mango fruits

AbstractSoluble acid invertase (SAI) was purified from mango fruits (Mangifera indica L.) by ammonium sulphate fractionation and anion‐exchange chromatography (DEAE‐Sepharose Fast Flow). Molecular mass of the enzyme is 45 kDa estimated by SDS–PAGE. Dynamic light scattering analysis suggests the hydrodynamic radius of SAI distributes from 4 to 20 nm with a peak at 6.68 nm. Transmission electron microscopy shows that SAI is a globulin with diameter of 10–30 nm. Its optimal pH and temperature are 4.0 and 60 °C, respectively. The enzyme is not stable at high temperature (≥60 °C) or in alkaline (pH ≥ 8) environment. Using sucrose as substrate, its KM and Vmax are 25.55 mm and 1.002 mmol min−1 mL−1, respectively. Its circular dichroism spectrum shows a negative band at 220 nm and a positive band at 195 nm, suggesting a β‐sheet structure. The fluorescence spectra reflect that the tryptophan and tyrosine residues of SAI are partially exposed.

Characterization and thermal inactivation kinetics of highly thermostable ramie leaf β-amylase.

We characterized ramie leaf β-amylase, and determined its thermostability and kinetic parameters. The enzyme was purified 53-fold using ammonium sulfate fractionation (40-60% saturation), anion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Superdex-200. The purified enzyme was identified as β-amylase with molecular mass of 42kD. The enzyme displayed Km and kcat values for soluble potato starch of 1.1mg/mL and 7.8s-1, respectively. The enzyme had a temperature optimum of 65°C, and its activity at 70°C was 92% of that at the optimal temperature after a 15-min incubation. Furthermore, enzyme activity was stable during treatment at 55°C for 60min but was inactivated rapidly at >75°C. This thermal behavior indicates that ramie leaf β-amylase has excellent intermediate temperature-stable enzyme properties for the baking and bio-industries. Inactivation of the enzyme followed first-order kinetics in the range of 55-80°C. The enthalpy change of thermal inactivation (ΔH‡), ΔG‡, and ΔS‡ were 237.2kJ/mol, 107.7kJ/mol, and 0.39kJ/molK at 333K, respectively. The D-value at 65°C (=110min) and the z-value (=9.4°C) are given for food processing.

Detection of histidine-rich glycoprotein and fibrinogen with nickel-enzyme conjugates: Purification of rabbit HRG

Nickel-bound alkaline phosphatase and peroxidase enzymes were used to investigate nickel binding to plasma proteins. Rabbit plasma dilutions to 25,000 were positive by ELISA, while Western blot analysis showed a prominent reaction with histidine-rich glycoprotein (HRG)1 and lower reaction with fibrinogen (Fgn). To confirm their identities, purified HRG and Fgn were demonstrated to react with the nickel-bound enzymes by Western analysis. With disulfide bonds reduced, HRG and Fgn α-chain reactions were demonstrated. HRG reactions were shown in other species, including human, bovine, chicken and guinea pig, demonstrating general applicability of the detection method. To enhance the purification of rabbit HRG, ammonium sulfate fractionation, immobilized metal ion chromatography and ion-exchange chromatography were optimized. Purified HRG contained trace components larger than HRG that reacted with nickel-enzymes and also with an antibody to HRG by Western analysis, confirming the trace components are related to HRG. These results demonstrate the utility of nickel-enzymes together with antibodies to detect HRG.

Purification, characterization, and application of a thermostable dextranase from Talaromyces pinophilus.

Dextranase can hydrolyze dextran to low-molecular-weight polysaccharides, which have important medical applications. In the study, dextranase-producing strains were screened from various soil sources. The strain H6 was identified as Talaromyces pinophilus by a standard ITS rDNA analysis. Crude dextranase was purified by ammonium sulfate fractionation and Sepharose 6B chromatography, which resulted in a 6.69-fold increase in the specific activity and an 11.27% recovery. The enzyme was 58kDa, lower than most dextranase, with an optimum temperature of 45°C and an optimum pH of 6.0, and identified as an endodextranase. It was steady over a pH range from 3.0 to 10.0 and had reasonable thermal stability. The dextranase activity was increased by urea, which enhanced its activity to 115.35% and was conducive to clinical dextran production. Therefore, T. pinophilus H6 dextranase could show its superiority in practical applications.

Optimization and partial characterization of intracellular anticandidal protein from Aspergillus giganteus MTCC 8408 using taguchi DOE

ABSTRACT A new intracellular antifungal protein (afp) production with average molecular weight 24.3 kDa and yield of 0.65 ± 0.1 mg/gram dry cell weight (gdcw) of mycelia in submerged fermentation of Aspergillus giganteus MTCC 8408 was optimized. Taguchi's DOE (design of experiment) L27 orthogonal array (OA) was constructed using Qualitek-4 software with 8 most influensive factors namely, culture pH, temperature, slant age, inoculum volume, agitation and KH2PO4. Scanning electron microscopy (SEM) was used to correlate the effect of selected factors on fungal cell morphology and afp production. The crude protein purification was accomplished using pure ammonium sulfate fractionation followed by carboxymethyl cellulose (CMC) ion-exchange chromatography and sephadex G-100 gel filtration. The average molecular mass of the purified protein was figured by silver stained SDS (sodium dodecylsulphate)-PAGE (poly-acryl amide gel electrophoresis). In vitro antifungal susceptibility assay was profiled against Candida albicans NCIM 3471 and minimum inhibitory concentrations (MICs) were in the range 3 to 4 µg/ml. Characterization of protein was observed with FTIR (Fourier transform infrared spectroscopy) analysis. The optimal production condition for crude afp was obtained as follows: soluble starch: 20 g/l; Corn steep liquor (CSL, 2%) + proteose peptone (PP, 1%): 30 g/l; pH: 5.8; temperature: 25°C; slant age: 3 d; inoculum size: 5% (v/v); agitation: 180 rpm; KH2PO4: 0.1 g/l. The validation experiments using optimized conditions confirmed an improvement in afp production by 59.4% against the expected enhancement of afp production by 61.22%. The present statistical optimization study revealed an opportunity to promote economical design at the industrial level for future scale up of effective antifungal agent against opportunistic oral and vaginal infection.