Effects Of Various Metal Ions Research Articles

Purification and characterization of novel manganese peroxidase from Trichoderma parestonica and its bio-conversion study of toxic arylamine

Trichoderma parestonica is a novel source of manganese peroxidase (MnP), [E.C.1.11.1.13] enzyme. The following was the sequence in which T. parestonica induced the production of MnP in the liquid culture medium: rice husk, corncob, sawdust, coir-dust and dried luffa on days 3, 4, 5, 2 and 3, respectively. The molecular weight of the enzyme was found to be 45kDa, determined from SDS-PAGE analysis which was further confirmed by mass analysis and was found to be 51.9kDa. Enzyme characterization parameters like optimum pH, temperature, Km, Kcat and catalytic efficiency are 5, 20ᵒC, 0.014mM (for Mn2+) and 0.028mM (for H2O2), 23.9 S-1 and 1.7 μM-1 S-1 respectively. Effect of various metal ions, such as bivalent Copper(Cu), Cobalt(Co), Mercury(Hg), Nickel(Ni), Cadmium(Cd), trivalent Chromium(Cr), Iron(Fe), Tetra-valent cerium(Ce), Penta-valent vanadium(V) and hexavalent molybdenum(Mo) the enzyme-catalyzed steady-state velocity was studied. The inhibition constant and nature of inhibition by these metal ions were determined. In the current investigation, MnP catalytic activities demonstrated that it could effectively catalyze the oxidation of phenols, aromatic amines and wastewater treatment. This communication suggested potential pathways for aromatic amine bioconversion mediated by MnP

Purification and characterization of an alkali-organic solvent-stable laccase with dye decolorization capacity from newly isolated Lysinibacillus fusiformis W11.

A new Lysinibacillus fusiformis strain with abundant laccase activity was isolated from soil under forest rotted leaf and identified as L. fusiformis W11 based on its 16S rRNA gene sequence and physiological characteristics. The laccase LfuLac was purified and characterized. The optimum temperature and pH of LfuLac on guaiacol were 45 °C and pH 9, respectively. LfuLac kept 78%, 88%, 92%, 74%, and 47% of activity at pH 7-11, respectively, suggesting the alkali resistance of the enzyme. The effects of various metal ions on LfuLac showed that Cu2+, Mg2+, and Na+ were beneficial to laccase activity and 10 mM Cu2+ increased the activity of LfuLac to 216%. LfuLac showed about 90% activity at 5% organic solvents and more than 60% activity at 20%, indicating its resistance to organic solvents. In addition, LfuLac decolorized different kinds of dyes. This study enriched our knowledge about laccase from L. fusiformis W11 and its potential industrial applications.

Synergistic effect of electrolyte additives on the suppression of dendrite growth in a flowless membraneless Zn–Br2 battery

Aqueous ZnBr2 batteries (ZBBs), particularly in flowless and membraneless form (FLML-ZBB), offer a cost-effective and promising large-scale energy storage system. Unfortunately, these batteries suffer from unavoidable Zn dendrite growth and inherent cross-diffusion of volatile and corrosive soluble Br3− species, which can eventually lead to a short circuit and serious self-discharge of the battery, respectively. To address these challenges, a single chamber FLML-ZBB is demonstrated in which the synergic effect of various metal ions (Li+, Na+, K+, Mn2+, and Cr3+) suppresses Zn dendritic growth. In addition, the use of a bromine complexing agent (BCA), 1-ethylpyridinium bromide (1-EPBr), efficiently inhibits cross-diffusion and induces reversible complexation of Br2/Br3−. The experimental results reveal that a battery with both Mn2+ ions and 1-EPBr electrolyte additives shows stable operation and exhibits a high Coulombic efficiency of 98.5% over 700 charge/discharge cycles at a high current density of 20 mA cm−2 and a long cycle life (1600 cycles) of over 3200 h at 1 mA cm−2.

Characterization of a Novel One-Domain Halotolerant Laccase from Parageobacillus thermoglucosidasius and Its Application in Dye Decolorization.

Laccases are widespread multi-copper oxidases and generally classified into three-domain laccases and two-domain laccases. In this study, a novel laccase PthLac from Parageobacillus thermoglucosidasius harbored only one domain of Cu-oxidase_4 and showed no sequence relatedness or structure similarity to three-domain and two-domain laccases. PthLac was heterologously expressed in Escherichia coli, purified, and characterized. The optimum temperature and pH of PthLac on guaiacol were at 60 ℃ and pH 6, respectively. The effects of various metal ions on PthLac were analyzed. All the tested metal ions did not suppress the activity of PthLac, except for 10mM Cu2+, which increased the activity of PthLac to 316%, indicating that PthLac was activated by Cu2+. Meanwhile, PthLac kept 121% and 69% activity when incubated at concentrations of 2.5 and 3M NaCl for 9h, suggesting the long-term halotolerancy of this enzyme. In addition, PthLac showed resistance to the organic solvents and surfactants, and displayed dye decolorization capacity. This study enriched our knowledge about one-domain laccase and its potential industrial applications.

Study of influence of metal ions in the diagnosis of recombinant hepatitis B surface antigen (HBsAg) using ELISA technique

ObjectivesPreliminary diagnosis of hepatitis B is performed by measuring the levels of serological markers using enzyme-linked immunosorbent assay (ELISA). Errors in diagnosis of the disease using serum markers might occur due to the presence of metallic salts in diagnostic samples. The present study was designed to evaluate the effects of various metal ions on the diagnosis of recombinant hepatitis B surface antigen (HBsAg). MethodsA simple step sandwich ELISA technique was used to evaluate the effects of metal ions on the diagnosis of HBsAg. Further, the zeta potential, electrical conductivity and redox potential of solutions with proteins and metal salts were measured to evaluate the interaction of these metal ions with the protein. ResultsThe results showed that recombinant HBsAg significantly interacted with the tested metal ions, with lithium ion showing the highest interaction, followed by barium, aluminium, and nickel as the percent decrease in the free HBsAg concentration in presence of these metal ions were found to be 95.5, 94.1, 93.5, and 91.6%, respectively. Zeta potential of the protein in reaction mixture also changed considerably and marked decrease in the concentration of free HBsAg was observed. This decrease in the concentration of free HBsAg resulted into error in the ELISA test which was negative in presence of these metal ions due to strong interaction with the HBsAg protein. ConclusionsThe findings of this study clearly demonstrated that the presence of metal ions in pathological samples can result in interaction with the antigen leading to possible diagnostic errors.

Isolation, morphological identification, and xylanase characteristics of anaerobic gut fungi Neocallimastix from Anatolian wild goat.

This study shows the morphological identification of anaerobic fungal strains isolated from fecal samples of goats inhabiting Turkey and the effects of various metal ions and chemicals on extracellular xylanase production. Three different anaerobic gut fungi isolated from wild goats in Turkey were identified as Neocallimastix spp. xylanase, cellulase, and lichenase production were tested in culture supernatants, and the maximum-specific activities were found as 560.42 ± 9.39, 159.70 ± 3.88, and 157.36 ± 3.83 (μmol/min/mg protein), respectively. While the optimum temperature range of exo-xylanases was found as 40-50°C, their optimum pH range was determined as 6.0-6.5. Xylanase activity decreased in metal ions and other chemical reactants based on dose. The metal ion that significantly inhibited xylanase activity was Fe+3 . It was found that the ferric ions inhibited xylanase activity in all three anaerobic gut fungi by 30%-90% depending on molarity. On the contrary, the 1 mM concentrations of the Mn+2 , Ba+2 , Co+2 , Cu+2 , Sn+2 , and Mg+2 metal ions and the ethylenediaminetetraacetic acid and β-mercaptoethanol reagents had a positive effect at rates in the range of 3%-92%. In conclusion, these findings demonstrate that anaerobic gut fungus has very stable fibrolytic enzymes that need to be separated, as well and the existence of a unique resource for industrial applications.

Purification and biochemical characterization of catalase that confers protection against hydrogen peroxide induced by stressful desert environment: the Camelus Dromedarius kidney catalase

Camel is continually exposed to stressful desert environment that enhances generation of reactive oxygen species, including hydrogen peroxide (H2O2). Catalase plays an important role in detoxification of H2O2. A highly active catalase from camel kidney was purified to homogeneity, with a specific activity of 1,774,392 U/mg protein, using ion exchange and metal chelate affinity chromatography. The molecular weight of the enzyme was 268 kDa consisting of four identical subunits of 63 kDa. The enzyme showed higher optimum temperature (45 °C) and higher activation energy (4.37 kJ mol−1). The thermodynamic parameters, ΔH, ΔG and ΔS, were determined. The effect of various metal ions and chemicals on enzyme activity was investigated. Km , Vmax , kcat and kcat /Km values for H2O2 were found to be 46 mM, 10,715,045 U/mg, 48,265,968 s−1 and 2,966,562 s−1 mM−1, respectively. Camel kidney catalase displayed higher affinity efficiency for H2O2 and can protect reduced glutathione (GSH) from oxidation by H2O2. Sodium azide was found to be a noncompetitive inhibitor of enzyme with Ki and IC50 of 17.88 µM and 20.94 µM, respectively. Camel catalase showed unique biochemical properties. Interestingly, camel catalase can protect molecules (GSH) and organ functions (kidney) from the toxic effects of H2O2 induced by stressful desert environment.

Influence of monovalent and divalent cations on monazite flotation

Understanding of flotation mechanism of monazite with oleate collector in saline water is essential for the rare earth mining industry. This work investigates the flotation performance of monazite in the presence of different ions such as Ca2+, Mg2+, Na+, and K+. The research integrates flotation experiments with colloid and surface characterizations using zeta potential, XPS and UV adsorption. The results showed that the presence of divalent metal ions (Mg2+, Ca2+) was highly detrimental to monazite flotation while the presence of monovalent metal ions (K+ and Na+) affected monazite flotation slightly only. The reason is that oleate formed insoluble products with divalent metal ions while the opposite is true in the case of monovalent metal ions. Therefore, the amount of soluble oleate ions in the presence of divalent ions was significantly reduced, resulting in less adsorption of oleate ions on monazite particles and thus reduced monazite recovery. The reduced adsorption of oleate on monazite was confirmed using XPS and UV adsorption experiments. This work explains the detrimental effect of various metal ions in saline water on monazite flotation.

Protease from jute endophyte Micrococcus luteus MBL-Bac7 functions as a potential bating agent for the leather industry

Ever since the discovery of jute endophytes, testing their potential for commercial uses has been a matter of interest. Considering the same, jute endophyte Micrococcus luteus MBL-Bac7, capable of producing extracellular proteases, was selected for in vitro and in silico analysis to assess its role as a bating agent required in rawhide processing. The presence of extracellular protease was confirmed from the plate assay. As the enzyme is tested for commercial use, the effect of various metal ions and reaction conditions (pH, temperature) have been optimized. The protease activity appears to be retained even at 85°C. It also showed significant activity in a wide range of pH (pH 3.0-8.5). Metal ion Mn2+ increased the protease activity significantly, but Fe2+, Zn2+, and Co2+ ions showed the opposite effect. Molecular identification of the protease was done from the whole genome sequence data. Using PSORTb v.3.0.2, SecretomeP-1.0, TMHMM-2.0, and protein molecular weight software, the physicochemical properties of the protease were predicted. The isolated protease shared a strong evolutionary link with Micrococcus species' S8 family serine peptidase. Finally, in the bating of cowhide, effects similar to that of commercial agents were obtained during finger prick, lastometer, and tensile tests. The findings of this study corroborate the possibility of using this protease as a potential bating agent. However, further studies are necessary to reduce the production cost for higher yield and commercialization. J. Bangladesh Acad. Sci. 46(1); 31-43: June 2022

Synergistic effect of various metal ions on the mechanical, thixotropic, self-healing, swelling and water retention properties of bimetallic hydrogels of alginate

We demonstrate ultrafast gelation between alginate (Alg) and a series of metal ions Fe3+, Cu2+, Co2+, Ni2+, and Mn2+ in their single and binary mixtures. The hydrogels exhibit remarkable mechanical properties, thixotropy, and self-healing ability. The mixing of Fe3+ or Cu2+ metal ions to the Alg hydrogels formed by the Mn2+, Co2+ and Ni2+ metal ions that possess lower mechanical strength and higher stretchability were observed to enhance their mechanical strength, reduce the stretchability and decrease the water retention ability. These properties of divalent metal ion alginate hydrogels involving Co2+, Ni2+, and Mn2+ are significantly improved in their mixed state. The self-healing ability is not seen in Fe-Alg and all the bimetallic hydrogels containing Fe3+ as one of the metal ions. However, all the other monometallic or bimetallic hydrogels show excellent self-healing ability. Bimetallic hydrogels containing Fe3+ or Cu2+ as one of the components do not show the swelling property. But the bimetallic hydrogels containing Mn2+, Co2+, and Ni2+ not only show high swelling property but also high water retention ability. The synthesized hydrogels were observed to be multi-stimuli-responsive and depict sol-gel transition in response to EDTA and applied shear strain. Both single, as well as mixed metals, could be removed from hydrogel by changing the pH indicating their reusability and their prospective use in wastewater treatment. The hydrogels also possess good processability and excellent moldability which form rigid 3D shapes. This study highlights the importance of using the mixed metal ions for tunning the rheological, thermal, swelling, self-healing, and water retention properties of the alginate hydrogels.

Effects of various metal ions on the growth of some phytopathogenic and biological control fungi

Effects of various metal ions on the growth of some phytopathogenic and biological control fungi

Production and Optimization of Cellulase Activity of Thermomonospora Viridis Isolated From Rice Straw

A highly cellulolytic actinomycete SR1 was locally isolated from rice straw and provisionally identified as Thermomonospora viridis. Optimum pH, temperature, carbon and nitrogen sources for its cellulase production were 6.5, 35°C, Carboxymethyl cellulase (CMC) and yeast extract, respectively whereas those of cellulase activity were 7.5, 40°C, CMC and peptone respectively. The effects of various metal ions and different reductant and inhibitors on its cellulase activity were investigated. Univalent Ag+ was found to decrease the enzyme activity whereas increased by bivalent Mg2+. Ethylene diamine tetraacetic acid (EDTA) caused remarkable decrease of cellulase activity but β-Mercaptoethanol stimulated its cellulase activity.
 Bangladesh J. Bot. 50(2): 395-404, 2021 (June)

Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa

ABSTRACT Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium Paenibacillus polymyxa. We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg2+ concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the P. polymyxa pistol ribozyme – an aspect that has not been previously analysed for this ribozyme class. We determined that the P. polymyxa pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from P. polymyxa. We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.

Electron configurations at 3d orbital of metal ion determining charge transition process in memory devices

Functional polymeric materials with electrical bi-stable states possess significant potential for high-density data storage due to their nanoscale memory site, three-dimensional-stacking ability and intrinsic flexibility. Aromatic polyimides bearing donor-acceptor (D-A) skeleton could form charge transfer complex (CTC) under electrical field, leading to their feasibility as memory materials. Three novel porphyrinated polyimides DATPP-DSDA, Zn-DATPP-DSDA and Mn-DATPP-DSDA were designed and synthesized for information memory applications. Metal ions with different electron configurations at 3d orbital have a determining influence on memory behaviors of polyimides: nonvolatile write-once-read-many-times memory (WORM) for DATPP-DSDA, volatile static random access memory (SRAM) for Zn-DATPP-DSDA, but no memory performance for Mn-DATPP-DSDA. By comparing the contribution of orbital transition and hole-electron distribution of charge-transfer excited states, roles of metal ions in regulating memory types were discussed. Molecular simulation results indicate that the Zn ion could play a bridge role in paving the route for excited electrons from a D to an A, while a trap role for the Mn ion in hindering this process. This study proves the feasibility of the strategy for modulating the memory behaviors of porphyrinated polyimides by varying the central metal ion and provides the exact effects of various metal ions on regulating charge transfer processes.

The nature of electronic and carrier transport properties of M-(quinolate)2 complexes (M = Be, Mg, Ca, and Sr): A computational study

A series of metal-quinolate (MQ 2 ) materials (M = Be 2+ , Mg 2+ , Ca 2+ , and Sr 2+ ) were theoretically investigated to understand their electronic and charge transport properties as an application of electron injection layer (EIL) and additive material to electron transporting layer (ETL). It was found that the Be 2+ , Mg 2+ , and Ca 2+ are tetragonally coordinated to the quinolate ligands whereas the Sr 2+ is planarly coordinated. The analyses of electronic properties indicated that the stable lowest unoccupied molecular orbital (LUMO) energy level of these materials relative to LiQ is expected to improve the electron injection from cathode and enhance the electron transport in ETL. In perspective of electrochemical reactivity, the chemical hardness values of MQ 2 materials showed that hole and electron carriers can be easily facilitated in order of BeQ 2 < MgQ 2 < CaQ 2 < SrQ 2 . In particular, the results of charge carrier transport properties clearly showed that k e of SrQ 2 is 64% higher than that of LiQ, which is an advantage for both EIL and ETL additive. Comprehensive analyses showed that SrQ2 is superior to LiQ as a replacement in perspective of electron injection and transport properties. • The effect of various metal ions on electronic properties of multiligand metal quinolates was investigated by DFT simulation. • The electronic and electrical properties of SrQ 2 were found to be superior to those of LiQ. • SrQ 2 can be potentially utilized as electron injection layer and/or additive material to electron transport layer.

Effect of various metal ions on gypsum precipitation

ABSTRACT The effect of various metal ions, namely Mn2+, Al3+, Pb2+, Cr3+, and Zn2+, on the precipitation of gypsum was investigated in the present study. The structure, functional groups, morphology, and size of gypsum crystals precipitated with additives using the batch method were evaluated experimentally through X-ray diffraction (XRD), Raman spectroscopy, thermogravimetric analysis (TGA)-Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and particle size and morphological analysis. The results indicate that Pb2+ and Zn2+ ions, in particular, exerted significant effects on gypsum precipitation. When Al3+ ions were the additive, only needle-like gypsum formed, whereas compact plate-like gypsum was observed in the presence of Pb2+ ions. The addition of Zn2+ ions resulted in gypsum particles of larger sizes with agglomerated shapes, simultaneously achieving an increased filtration rate of the suspensions from 5.88×10−7–2.44 × 10−6 m3/s. A detailed analysis of the crystal shape showed that while the elongation values decreased, the circularity values increased as compared with the gypsum precipitated in pure medium. These results show how metal ions can be used to effectively modify gypsum size, shape, and crystal morphology.

Enhancement of Thermal Resistance by Metal Ions in Thermotolerant Zymomonas mobilis TISTR 548.

The thermal resistance of fermenting microbes is a key characteristic of stable fermentation at high temperatures. Therefore, the effects of various metal ions on the growth of Zymomonas mobilis TISTR 548, a thermotolerant ethanologenic bacterium, at a critical high temperature (CHT) were examined. Addition of Mg2+ and K+ increased CHT by 1°C, but the effects of the addition of Mn2+, Ni2+, Co2+, Al3+, Fe3+, and Zn2+ on CHT were negligible. To understand the physiological functions associated with the addition of Mg2+ or K+, cell morphology, intracellular reactive oxygen species (ROS) level, and ethanol productivity were investigated at 39°C (i.e., above CHT). Cell elongation was repressed by Mg2+, but not by K+. Addition of both metals reduced intracellular ROS level, with only K+ showing the highest reduction strength, followed by both metals and only Mg2+. Additionally, ethanol productivity was recovered with the addition of both metals. Moreover, the addition of Mg2+ or K+ at a non-permissive temperature in 26 thermosensitive, single gene-disrupted mutants of Z. mobilis TISTR 548 revealed that several mutants showed metal ion-specific growth improvement. Remarkably, K+ repressed growth of two mutants. These results suggest that K+ and Mg2+ enhance cell growth at CHT via different mechanisms, which involve the maintenance of low intracellular ROS levels.

Synthesis of Microwave-Assisted Fluorescence Carbon Quantum Dots Using Roasted-Chickpeas and its Applications for Sensitive and Selective Detection of Fe3+ Ions.

A simple method for the green synthesis of fluorescent carbon quantum dots (CQDs) has been developed by using roasted chickpea as carbon source in one-step without using any chemical. Interestingly, not only the carbon source of CQDs and the whole synthesis procedure are environmentally friendly, but also the synthesized CQDs have shown many advantageous properties such as high fluorescence intensity, excellent photostability, and good water solubility. CQDs which were firstly synthesized from roasted-chickpeas by a microwave-assisted pyrolysis have been characterized using UV-vis absorption spectroscopy, fluorescence spectroscopy, fourier transform infrared spectroscopy (FTIR) spectroscopy, X-ray diffraction (XRD) technique and transmission electron microscopy (TEM). In addition, the details of the structure have been revealed by the electron diffraction (SAED; selected-area electron diffraction) method based on the TEM images. The synthesized CQDs emits blue fluorescence under UV light (at 365nm). A microwave oven (350 watts) was used to prepare CQDs in 120s. The effect of various metal ions on the fluorescence intensity of CQDs was investigated in order to determine its utality in the detection of metal ions. It was determined that 1.38mg/L of Fe3+ ions quenched the fluorescence intensity of the CQDs by 65%. The linear range is observed between 11.25 and 37.50μM with limit of detection (LOD) and limit of quantification (LOQ) of 2.74μM and 8.22μM, respectively. CQDs which are highly selective and sensitive for Fe3+ ions was used to determine Fe3+ ions in certified reference material (CRM-SA-C). Fe3+ ions concentration was determined with precision values of more than 95% as intra-day and inter-day relative standard deviation (RSD%) is 5 at room temperature.

Artificial Cells Capable of Long-Lived Protein Synthesis by Using Aptamer Grafted Polymer Hydrogel.

Herein we report a new type of artificial cells capable of long-term protein expression and regulation. We constructed the artificial cells by grafting anti-His-tag aptamer into the polymer backbone of the hydrogel particles, and then immobilizing the His-tagged proteinaceous factors of the transcription and translation system into the hydrogel particles. Long-term protein expression for at least 16 days was achieved by continuously flowing feeding buffer through the artificial cells. The effect of various metal ions on the protein expression in the artificial cells was investigated. Utilizing the lac operator-repressor system, we could regulate the expression level of eGFP in the artificial cells by controlling the β-D-1-thiogalatopyranoside (IPTG) concentration in the feeding buffer. The artificial cells based on the aptamer grafted hydrogel provide a useful platform for gene circuit engineering, metabolic engineering, drug delivery, and biosensors.

Characterization of a cold-active, detergent-stable metallopeptidase purified from Bacillus sp. S1DI 10 using Response Surface Methodology

The colder regions of Earth are inhabited by cold-adapted microorganisms designated as psychrophiles that are known to produce cold-active enzymes, such as peptidases, chaperones, lipases, cellulases, and phosphatases. These types of enzymes are a major part of the market of industrial enzymes. Bacteria isolated from water samples collected from the Chamba region in the Himalayas were screened for peptidase production using skim milk agar plates. Among the peptidase-producing bacteria isolated, 20% of the isolates exhibited fast growth and maximum zones of clearance, and thus, were used for further studies. The 16S rDNA sequence analysis of isolate S1DI 10 identified it as a Bacillus sp. The peptidase was cloned in pET28a vector and expressed in Escherichia coli BL21(DE3) and the His-tagged recombinant protein was purified using Ni-NTA column. The purified peptidase of SIDI 10 was found to be an alkaline, cold-active peptidase with optimal enzyme activity at 10°C and pH 8. An approach of one variable at a time was used to further study the effect of various metal ions, organic solvents and detergents on the peptidase enzyme. The peptidase activity was enhanced in the presence of Fe2+ and Mn2+ (metal ions), hexane (organic solvent), SDS- sodium dodecyl sulfate (anionic detergent) and Tween 80 (nonionic detergent). Response surface methodology (RSM) was used to determine the cumulative effect of these five variables. A 25 full factorial central composite design was applied for the five independent variables to determine the optimal combinations of these constituents at the maximum peptidase activity.