Search results for: enzyme purification

Authors: Joseph A. Bentil, Anders Thygesen, Lene Langea, Moses Mensah, Anne Meyer

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The study investigated the saccharification potential of in-house enzymes produced from a white-rot basidiomycete strain, Polyporus ciliatus CBS 366.74. The in-house enzymes were produced by growing the fungus on mono and composite substrates of cocoa pod husk (CPH) and green seaweed (GS) (Ulva lactuca sp.) with and without the addition of 25mM ammonium nitrate at 4%w/v substrate concentration in submerged condition for 144 hours. The crude enzyme extracts preparations (CEE 1-5 and CEE 1-5+AN) obtained from the fungal cultivation process were sterile-filtered and used as enzyme sources for enzymatic hydrolysis of hydrothermally pretreated corn stover using a commercial cocktail enzyme, Cellic Ctec3, as benchmark. The hydrolysis was conducted at 50ᵒC with 50mM sodium acetate buffer, pH 5 based on enzyme dosages of 5 and 10 CMCase Units/g biomass at 1%w/v dry weight substrate concentration at time points of 6, 24, and 72 hours. The enzyme activity profile of the in-house enzymes varied among the growth substrates with the composite substrates (50-75% GS and AN inclusion), yielding better enzyme activities, especially endoglucanases (0.4-0.5U/mL), β-glucosidases (0.1-0.2 U/mL), and xylanases (3-10 U/mL). However, nitrogen supplementation had no significant effect on enzyme activities of crude extracts from 100% GS substituted substrates. From the enzymatic hydrolysis, it was observed that the in-house enzymes were capable of hydrolysing the pretreated corn stover at varying degrees; however, the saccharification yield was less than 10%. Consequently, theoretical glucose yield was ten times lower than Cellic Ctec3 at both dosage levels. There was no linear correlation between glucose yield and enzyme dosage for the in-house enzymes, unlike the benchmark enzyme. It is therefore recommended that the in-house enzymes are used to complement the dosage of commercial enzymes to reduce the cost of biomass saccharification.

Keywords: enzyme production, hydrolysis yield, feedstock, enzyme blend, Polyporus ciliatus

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Authors: T. Sadunishvili, L. Kutateladze, T. Urushadze, R. Khvedelidze, N. Zakariashvili, M. Jobava, G. Kvesitadze

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Multiple repeated soil-climatic zones in Georgia determines the diversity of microorganisms. Hundreds of microscopic fungi of different genera have been isolated from different ecological niches, including some extreme environments. Biosynthetic ability of microscopic fungi has been studied. Trichoderma ressei, representative of the Ascomycetes secrete cellulolytic and xylanolytic enzymes that act in synergy to hydrolyze polysaccharide polymers to glucose, xylose and arabinose, which can be fermented to biofuels. The other mesophilic strains producing cellulases are Allesheria terrestris, Chaetomium thermophile, Fusarium oxysporium, Piptoporus betulinus, Penicillium echinulatum, P. purpurogenum, Aspergillus niger, A. wentii, A. versicolor, A. fumigatus etc. In the majority of the cases the cellulases produced by strains of genus Aspergillus usually have high β-glucosidase activity and average endoglucanases levels (with some exceptions), whereas strains representing Trichoderma have high endo enzyme and low β-glucosidase, and hence has limited efficiency in cellulose hydrolysis. Six producers of stable cellulases and xylanases from mesophilic and thermophilic fungi have been selected. By optimization of submerged cultivation conditions, high activities of cellulases and xylanases were obtained. For enzymes purification, their sedimentation by organic solvents such as ethyl alcohol, acetone, isopropanol and by ammonium sulphate in different ratios have been carried out. Best results were obtained with precipitation by ethyl alcohol (1:3.5) and ammonium sulphate. The yields of enzyme according to cellulase activities were 80-85% in both cases. Cellulase activity of enzyme preparation obtained from the strain Trichoderma viride X 33 is 126 U/g, from the strain Penicillium canescence D 85–185U/g and from the strain Sporotrichum pulverulentum T 5-0 110 U/g. Cellulase activity of enzyme preparation obtained from the strain Aspergillus sp. Av10 is 120 U/g, xylanase activity of enzyme preparation obtained from the strain Aspergillus niger A 7-5–1155U/g and from the strain Aspergillus niger Aj 38-1250 U/g. Optimum pH and temperature of operation and thermostability, of the enzyme preparations, were established. The efficiency of hydrolyses of different agricultural residues by the microscopic fungi cellulases has been studied. The glucose yield from the residues as a result of enzymatic hydrolysis is highly determined by the ratio of enzyme to substrate, pH, temperature, and duration of the process. Hydrolysis efficiency was significantly increased as a result of different pretreatment of the residues by different methods. Acknowledgement: The Study was supported by the ISTC project G-2117, funded by Korea.

Keywords: cellulase, xylanase, microscopic fungi, enzymatic hydrolysis

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Authors: Chanchal Mewar, Shikha Gangil, Yashwant Parihar, Virendra Dhakar, Bharat Modhera

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Biodiesel was prepared from Karanja oil by three step methods: saponification, acidification and esterification. In first step, saponification was done in presence of methanol and KOH or NaOH with Karanja oil. During second step acidification, various acids such as H3PO4, HCl, H2SO4 were used as acid catalyst. In third step, esterification followed by purification was done with various silica sources as Ludox (colloidal silicate) and fumed silica gel. It was found that there was no significant change in density, kinematic viscosity, iodine number, acid value, saponification number, flash point, cloud point, pour point and cetane number after purification by these adsorbents. The objective of this research is the comparison among different adsorbents which were used for the purification of biodiesel. Ludox (colloidal silicate) and fumed silica gel were used as adsorbents for the removal of glycerin from biodiesel and evaluate the effectiveness of biodiesel purity. Furthermore, this study compared the results of distilled water washing also. It was observed that Ludox, fumed silica gel and distilled water produced yield about 93%, 91% and 83% respectively. Highest yield was obtained with Ludox at 100 oC temperature using H3PO4 as acid catalyst and NaOH as base catalyst with methanol, (3:1) alcohol to oil molar ratio in 90 min.

Keywords: biodiesel, three step method, purification, silica sources

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Authors: Wen Po Cheng, Chi Hua Fu, Ping Hung Chen, Ruey Fang Yu

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Recovering resources from water purification sludge (WPS) have been gradually stipulated in environmental protection laws and regulations in many nations. Hence, reusing the WPS is becoming an important topic, and recovering alum from WPS is one of the many practical alternatives. Most previous research efforts have been conducted on studying the amphoteric characteristic of aluminum hydroxide for investigating the optimum pH range to dissolve the Al(III) species from WPS, but it has been lack of reaction kinetics or mechanisms related discussion. Therefore, in this investigation, water purification sludge (WPS) solution was broken by ultrasound to make particle size of reactants smaller, specific surface area larger. According to the reaction kinetics, these phenomena let the dissolved aluminum salt quantity increased and the reaction rate go faster.

Keywords: aluminum, acidification, sludge, recovery

Procedia PDF Downloads 586

Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcuslactis, recombinant, phytase, poultry

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Authors: Ashish Shukla, K. P. Mishra, Pushplata Tripathi

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This paper investigates the production and optimization of β-galactosidase enzyme using synthetic medium by isolated wild strains (S1, S2) mutated strains (M1, M2) through SSF and SmF. Among the different cell disintegration methods used, the highest specific activity was obtained when the cells were permeabilized using isoamyl alcohol. Wet lab experiments were performed to investigate the effects of carbon and nitrogen substrates present in Vogel’s medium on β-galactosidase enzyme activity using S1, S2, and M1, M2 strains through SSF. SmF experiments were performed for effects of carbon and nitrogen sources in YLK2Mg medium on β-galactosidase enzyme activity using S1, S2 and M1, M2 strains. Effect of pH on β-galactosidase enzyme production was also done using S1, S2, and M1, M2 strains. Results were found to be very appreciable in all the cases.

Keywords: β-galactosidase, cell disintegration, permeabilized, SSF, SmF

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Authors: Nuha Mansour Alhazmi

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Replacement of petro-based plastics by a biodegradable plastic are vastly growing process. Poly-β-hydroxybutyrate (PHB) is a biodegradable biopolymer, synthesized by some bacterial genera. The objective of the current study is to explore the ability of some fungi to biodegrade PHB. The degradation of (PHB) was detected in Petri dish by the formation of a clear zone around the fungal colonies due to the production of depolymerase enzyme which has an interesting role in the PHB degradation process. Among 10 tested fungi, the most active PHB biodegraded fungi were identified as Trichoderma asperellum using morphological and molecular characters. The highest PHB degradation was at 25°C, pH 7.5 after 7 days of incubation for the tested fungi. Finally, the depolymerase enzyme was isolated, purified using column chromatography and characterized. In conclusion, PHB can be biodegraded in solid and liquid medium using depolymerase enzyme from T. asperellum.

Keywords: degradation, depolymerase enzyme, PHB, Trichoderma asperellum

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Authors: Evgeniya Y. Yuzbasheva, Tigran V. Yuzbashev, Natalia I. Perkovskaya, Elizaveta B. Mostova

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Cell-surface display of lipase is of great interest as it has many applications in the field of biotechnology owing to its unique advantages: simplified product purification, and cost-effective downstream processing. One promising area of application for whole-cell biocatalysts with surface displayed lipase is biodiesel synthesis. Biodiesel is biodegradable, renewable, and nontoxic alternative fuel for diesel engines. Although the alkaline catalysis method has been widely used for biodiesel production, it has a number of limitations, such as rigorous feedstock specifications, complicated downstream processes, including removal of inorganic salts from the product, recovery of the salt-containing by-product glycerol, and treatment of alkaline wastewater. Enzymatic synthesis of biodiesel can overcome these drawbacks. In this study, Lip2p lipase was displayed on Yarrowia lipolytica cells via C- and N-terminal fusion variant. The active site of lipase is located near the C-terminus, therefore to prevent the activity loosing the insertion of glycine-serine linker between Lip2p and C-domains was performed. The hydrolytic activity of the displayed lipase reached 12,000–18,000 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. In case of C-terminal fusion variant, the leakage was occurred due to the proteolytic cleavage within the linker peptide. In case of N-terminal fusion variant, the leaking enzyme was presented as three proteins, one of which corresponded to the whole hybrid protein. The calculated number of recombinant enzyme displayed on the cell surface is approximately 6–9 × 105 molecules per cell, which is close to the theoretical maximum (2 × 106 molecules/cell). Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, cell-bound lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. It retained 74% of original activity at 60°C for 5 min of incubation, and 83% of original activity after incubation at 50°C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1–% and 71.0–% methyl esters after 33–h and 45–h reactions, respectively.

Keywords: biodiesel, cell-surface display, lipase, whole-cell biocatalyst

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Authors: Jamal Abd Awadallak, Thiago Olinek Reinehr, Eduardo Raizer, Deise Molinari, Edson Antonio, Camila da Silva da Silva

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The hydrolysis of soy oil catalyzed by 1,3-specific enzyme (Lecitase Ultra) in a well-stirred bioreactor was studied. Two forms of applications of the ultrasound were evaluated aiming to increase reaction rates, wherein the use of probe ultrasound associated with the use of surfactant to pre-emulsify the substrate showed the best results. Two different reaction periods were found: the first where the ultrasound has great influence on reaction rates, and the second where ultrasound influence is minimal. Studies on the time of pre-emulsification, surfactant concentration and enzyme concentration showed that the initial rate of hydrolysis depends on the interfacial area between the oil phase and the aqueous phase containing the enzyme.

Keywords: specific enzyme, free fatty acids, Hydrolysis, lecitase ultra, ultrasound

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Authors: Misha Ali, Qayyum Husain, Nida Alam, Masood Ahmad

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Improving the activity and stability of the enzyme is an important aspect in bioremediation processes. Immobilization of enzyme is an efficient approach to amend the properties of biocatalyst required during wastewater treatment. The present study was done to immobilize partially purified ginger peroxidase on amino functionalized silica coated titanium dioxide nanocomposite. Interestingly there was an enhancement in enzyme activity after immobilization on nanosupport which was evident from effectiveness factor (η) value of 1.76. Immobilized enzyme was characterized by transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. There was a 4 fold increase in Vmax of immobilized peroxidase as compared to free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process. Our study shows that this bio-remediating system has remarkable potential for treatment of aromatic pollutants present in wastewater.

Keywords: acid yellow 42, decolorization, ginger peroxidase, immobilization

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Authors: Suphalucksana, Wichai, Sangsoponjit Settasit, Soytong Kasem

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A study on the efficiency for enzyme production of fungi isolated from stomach of buffalo was conducted. The fungi were collected from 4 parts of stomach as rumen, reticulum, omasum and abomasums. The objective to study the efficiency of fungi from stomach of buffalo had effected to produced enzyme and to selected fungi for their ability to produced enzyme cellulase, hemicellulase and ligninase. Results shown that the fungi isolated from rumen were: Eupenicillium sp. (B-RU-01-1), Eupenicillium sp. (B-RU-02-3G), Rhyzopus stolonifer (B-RU-01-4) and Trichoderma sp. (B-RU-01-2). From the reticulum, Aspergillus glaucus (B-RET-02-3), Aspergillus orchraceus (B-RET-02-2) and Penicillium sp. (B-RET-02-4) were found. In the omasum Aspergillus fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4) and Rhizopus stolonifer (B-OMA-02-3) were isolated and in the abomasums Aspergillus flavas (B-ABO-02-3), Aspergillus fumigatus (B-ABO-02-1), Aspergillus niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4) and Mucor sp. (B-ABO-02-4G). Results of enzyme analysis revealed that cellulase was produced by isolated: Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Penicillium sp. (B-RET-02-4), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1), Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4). Hemicellulase was produced Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Rhizopus stolonifer (B-RU-01-4), Trichoderma sp. (B-RU-01-2), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Penicillium sp. (B-RET-02-4), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA -01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1) Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4), Mucor sp. (B-ABO-02-4G). For the enzyme ligninase, two isolates were found to produced this enzyme namely : Trichoderma sp. (B-RU-01-2) and Mucor sp. (B-ABO-02-4G).

Keywords: enzyme production from fungi, enzyme production, fungi, agricultural technology

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Authors: Sibashish Baksi, Ujjaini Sarkar, Sudeshna Saha

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In the present study, hydrolysis of Pinus roxburghi wood powder was carried out with Viscozyme, and kinetics of the hydrolysis has been investigated. Finely ground sawdust is submerged into 2% aqueous peroxide solution (pH=11.5) and pretreated through autoclaving, probe sonication, and alkaline peroxide pretreatment. Afterward, the pretreated material is subjected to hydrolysis. A chain of experiments was executed with delignified biomass (50 g/l) and varying enzyme concentrations (24.2–60.5 g/l). In the present study, 14.32 g/l of glucose, along with 7.35 g/l of xylose, have been recovered with a viscozyme concentration of 48.8 g/l and the same condition was treated as optimum condition. Additionally, thermal deactivation of viscozyme has been investigated and found to be gradually decreasing with escalated enzyme loading from 48.4 g/l (dissociation constant= 0.05 h⁻¹) to 60.5 g/l (dissociation constant= 0.02 h⁻¹). The hydrolysis reaction is a pseudo first-order reaction, and therefore, the rate of the hydrolysis can be expressed as a fractal-like kinetic equation that communicates between the product concentration and hydrolytic time t. It is seen that the value of rate constant (K) increases from 0.008 to 0.017 with augmented enzyme concentration from 24.2 g/l to 60.5 g/l. Greater value of K is associated with stronger enzyme binding capacity of the substrate mass. However, escalated concentration of supplied enzyme ensures improved interaction with more substrate molecules resulting in an enhanced de-polymerization of the polymeric sugar chains per unit time which eventually modifies the physiochemical structure of biomass. All fractal dimensions are in between 0 and 1. Lower the value of fractal dimension, more easily the biomass get hydrolyzed. It can be seen that with increased enzyme concentration from 24.2 g/l to 48.4 g/l, the values of fractal dimension go down from 0.1 to 0.044. This indicates that the presence of more enzyme molecules can more easily hydrolyze the substrate. However, an increased value has been observed with a further increment of enzyme concentration to 60.5g/l because of diffusional limitation. It is evident that the hydrolysis reaction system is a heterogeneous organization, and the product formation rate depends strongly on the enzyme diffusion resistances caused by the rate-limiting structures of the substrate-enzyme complex. Value of the rate constant increases from 1.061 to 2.610 with escalated enzyme concentration from 24.2 to 48.4 g/l. As the rate constant is proportional to Fick’s diffusion coefficient, it can be assumed that with a higher concentration of enzyme, a larger amount of enzyme mass dM diffuses into the substrate through the surface dF per unit time dt. Therefore, a higher rate constant value is associated with a faster diffusion of enzyme into the substrate. Regression analysis of time curves with various enzyme concentrations shows that diffusion resistant constant increases from 0.3 to 0.51 for the first two enzyme concentrations and again decreases with enzyme concentration of 60.5 g/l. During diffusion in a differential scale, the enzyme also experiences a greater resistance during diffusion of larger dM through dF in dt.

Keywords: viscozyme, glucose, fractal kinetics, thermal deactivation

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Authors: K. Eryilmaz, G. Mercanoglu

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Aim: Purification of the final product, which is the last step in the synthesis of lanthanide-labeled peptide compounds, can be accomplished by different methods. Among these methods, the two most commonly used methods are C18 solid phase extraction (SPE) and weak cation exchanger cartridge elution. SPE C18 solid phase extraction method yields high purity final product, while elution from the weak cation exchanger cartridge is pH dependent and ineffective in removing colloidal impurities. The aim of this work is to develop an additional purification method for the lanthanide-labeled peptide compound in cases where the desired radionuclidic and radiochemical purity of the final product can not be achieved because of pH problem or colloidal impurity. Material and Methods: For colloidal impurity formation, 3 mL of water for injection (WFI) was added to 30 mCi of 177LuCl3 solution and allowed to stand for 1 day. 177Lu-DOTATATE was synthesized using EZAG ML-EAZY module (10 mCi/mL). After synthesis, the final product was mixed with the colloidal impurity solution (total volume:13 mL, total activity: 40 mCi). The resulting mixture was trapped in SPE-C18 cartridge. The cartridge was washed with 10 ml saline to remove impurities to the waste vial. The product trapped in the cartridge was eluted with 2 ml of 50% ethanol and collected to the final product vial via passing through a 0.22μm filter. The final product was diluted with 10 mL of saline. Radiochemical purity before and after purification was analysed by HPLC method. (column: ACE C18-100A. 3µm. 150 x 3.0mm, mobile phase: Water-Acetonitrile-Trifluoro acetic acid (75:25:1), flow rate: 0.6 mL/min). Results: UV and radioactivity detector results in HPLC analysis showed that colloidal impurities were completely removed from the 177Lu-DOTATATE/ colloidal impurity mixture by purification method. Conclusion: The improved purification method can be used as an additional method to remove impurities that may result from the lanthanide-peptide synthesis in which the weak cation exchange purification technique is used as the last step. The purification of the final product and the GMP compliance (the final aseptic filtration and the sterile disposable system components) are two major advantages.

Keywords: lanthanide, peptide, labeling, purification, radionuclide, radiopharmaceutical, synthesis

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Authors: Sandeep Kaur

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Phycoerythrin (PE) from the mesophilic filamentous cyanobacterium Nostoc piscinale PUPCCC 405.17, a good producer of phycobiliproteins, has been characterized in terms of their unit assembly and stability. The phycoerythrin was extracted by freeze-thawing the cells in water, concentrated by ammonium sulphate fractionation and purified by anion exchange chromatography. The purification process resulted in 2.90 fold increase in phycoerythrin purity reaching to 1.54. Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis of purified PE demonstrated three protein bands of 14.3, 27.54 and 39.81 kDa. The native PE also showed one band of 125.87 kDa, assumed to be a dimer (αβ)2γ based on results of non-denaturing PAGE. Lyophilized powder PE was more stable compared to phycoerythrin in the solution. The half-life of dry PE is 80 days when stored at 4 °C under dark. The phycoerythrin from this organism has potential applications in food as natural colour and as a fluorescent marker.

Keywords: characterization, Nostoc piscinale, phycoerythrin, purification

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Authors: Shweta Sinha, Mukesh Doble, Manju S. L.

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Pyrazole scaffold is an important group of compound in heterocyclic chemistry and is found to possess numerous uses in chemistry. Pyrazole derivatives are also known to possess important biological activities including antitumor, antimicrobial, antiviral, antifungal, anticancer and anti-inflammatory. Inflammation is associated with pain, allergy and asthma. Leukotrienes are mediators of various inflammatory and allergic disorders. 5-Lipoxygenase (5-LOX) is an important enzyme involved in the biosynthesis of leukotrienes and metabolism of arachidonic acid (AA) and thus targeted for anti-inflammation. In vitro inhibitory activity of pyrazol-3-yl thiazole 4-carboxylic acid derivatives is tested against enzyme 5-LOX. Most of these compounds exhibit good inhibitory activity against this enzyme. Binding mode study of these compounds is determined by computational tool. Further experiments are being done to understand the mechanism of action of these compounds in inhibiting this enzyme. To conclude, these compounds appear to be a promising target in drug design against 5-LOX.

Keywords: inflammation, inhibition, 5-lipoxygenase, pyrazole

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Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh

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Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

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Authors: Leila Mosafa, Majid Moghadam, Mohammad Shahedi

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In this work, pectinase was immobilized on the surface of silica-coated magnetite nanoparticles via covalent attachment. The magnetite-immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electron microscopy and vibrating sample magnetometry techniques. Response surface methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30°C and pH 5.5 with 42.97 µl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm2s-1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.

Keywords: magnetite nanoparticles, pectinase enzyme, immobilization, juice clarification, enzyme activity

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Authors: Debajyoti Bose

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Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Chitosan is a positively charged natural biodegradable and biocompatible polymer. It is a linear polysaccharide consisting of β-1,4 linked monomers of glucosamine and N-acetylglucosamine. Chitosan can be mainly obtained from fungal sources during large fermentation process. In this study,three different fungal strains Aspergillus niger NCIM 1045, Aspergillus oryzae NCIM 645 and Mucor indicus MTCC 3318 were used for the production of chitosan. The growth mediums were optimized for maximum fungal production. The produced chitosan was characterized by determining degree of deacetylation. Chitosan possesses one reactive amino at the C-2 position of the glucosamine residue, and these amines confer important functional properties to chitosan which can be exploited for biofabrication to generate various chemically modified derivatives and explore their potential for pharmaceutical field. Chitosan nanoparticles were prepared by ionic cross-linking with tripolyphosphate (TPP). The major effect on encapsulation and release of protein (e.g. enzyme diastase) in chitosan-TPP nanoparticles was investigated in order to control the loading and release efficiency. It was noted that the chitosan loading and releasing efficiency as a nanocapsule, obtained from different fungal sources was almost near to initial enzyme activity(12026 U/ml) with a negligible loss. This signify, chitosan can be used as a polymeric drug as well as active component or protein carrier material in dosage by design due to its appealing properties such as biocompatibility, biodegradability, low toxicity and relatively low production cost from abundant natural sources. Based upon these initial experiments, studies were also carried out on modification of chitosan based nanocapsules incorporated with physiologically important enzymes and nutraceuticals for target delivery.

Keywords: fungi, chitosan, enzyme, nanocapsule

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Authors: N. Zanganeh, M. Zabet

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Lactulose is a synthetic disaccharide which has remarkable applications in food and pharmaceutical fields. Lactulose is not found in nature and it is produced by isomerization reaction of lactose in an alkaline environment. It should be noted that this reaction has a very low yield since significant amount of lactose stays un-reacted in the system. Basically, purification of lactulose is difficult and costly. Previous studies have revealed that solubility of lactose and lactulose are significantly different in ethanol. Considering the fact that solubility is also affected by temperature itself, we investigated the effect of ethanol and temperature on separation process of lactose from the syrup containing lactose and lactulose. For this purpose, a saturated solution containing lactulose and lactose was made at three different temperatures; 25⁰C (room temperature), 31⁰C, and 37⁰C first.  Five samples containing 2g saturated solution was taken and then 2g, 3g, 4g, 5g, and 6g ethanol separately was added to the sampling tubes. Sampling tubes were kept at respective temperatures afterward. The concentration of lactose and lactulose after separation process measured and analyzed by High Performance Liquid Chromatography (HPLC). Results showed that ethanol has such a greater impact than operating temperature on purification process. Also, it was observed that the maximum rate of separation occurred at initial amount of added ethanol.

Keywords: lactulose, lactose, purification, solubility

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Authors: Samira Rostom, Robert Symonds, Robin W. Hughes

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Hydrogen is considered as one of the most important clean and renewable energy carriers for a sustainable energy future. However, its efficient and cost-effective purification remains challenging. This paper presents the potential of using metal–organic frameworks (MOFs) in combination with pressure swing adsorption (PSA) technology for syngas based H2 purification. PSA process analysis is done considering high pressure and elevated temperature process conditions, it reduces the demand for off-gas recycle to the fuel reactor and simultaneously permits higher desorption pressure, thereby reducing the parasitic load on the hydrogen compressor. The elevated pressure and temperature adsorption we present here is beneficial to minimizing overall process heating and cooling demand compared to existing processes. Here, we report the comparative performance of zeolite-5A, Cu-BTC, and the mix of zeolite-5A/Cu-BTC for H2 purification from syngas typical of those exiting water-gas-shift reactors. The MOFs were synthesized hydrothermally and then mixed systematically at different weight ratios to find the optimum composition based on the adsorption performance. The formation of different compounds were characterized by XRD, N2 adsorption and desorption, SEM, FT-IR, TG, and water vapor adsorption technologies. Single-component adsorption isotherms of CO2, CO, CH4, N2, and H2 over single materials and composites were measured at elevated pressures and different temperatures to determine their equilibrium adsorption capacity. The examination of the stability and regeneration performance of metal–organic frameworks was carried out using a gravimetric system at temperature ranges of 25-150℃ for a pressure range of 0-30 bar. The studies of adsorption/desorption on the MOFs showed selective adsorption of CO2, CH4, CO, and N2 over H2. Overall, the findings of this study suggest that the Ni-MOF-74/Cu-BTC composites are promising candidates for industrial H2 purification processes.

Keywords: MOF, H2 purification, high T, PSA

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Authors: N. Bouzouita, A. Snoussi , H. Ben Haj Koubaier, I. Essaidi, M. M. Chaabouni, S. Zgoulli, P. Thonart

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Color is one of the most important quality attributes for the food industry. Grape marc, a complex lignocellulosic material is one of the most abundant and worth less byproduct, generated after the pressing process. The development of the process of purification by micro filtration, ultra filtration, nano filtration and drying by atomization of the anthocyanins of Tunisian origin is the aim of this work. Locust bean gum is the ground endosperm of the seeds of carob fruit; owing to its remarkable water-binding properties, it is widely used to improve the texture of food and largely employed in food industry. The purification of LGB causes drastically reduced ash and proteins contents but important increase for galactomannan.

Keywords: Carob, food additives, grape pomace, locust bean gum, natural colorant, nano filtration, thickener, ultra filtration

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Authors: S. Md. Shaarani, J. Md. Jahim, R. A. Rahman, R. Md. Illias

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Modern developments in biotechnology have paved the way for extensive use of biocatalysis in industries. Although it offers immense potential, industrial application is usually hampered by lack of operational stability, difficulty in recovery as well as limited re-use of the enzyme. These drawbacks, however, can be overcome by immobilization. Cross-linked enzyme aggregates (CLEAs), a versatile carrier-free immobilization technique is one that is currently capturing global interest. This approach involves precipitating soluble enzyme with an appropriate precipitant and subsequent crosslinking by a crosslinking reagent. Without ineffective carriers, CLEAs offer high enzymatic activity, stability and reduced production cost. This study demonstrated successful CLEA synthesis of recombinant xylanase from Trichoderma reesei using ethanol as aggregating agent and glutaraldehyde (2% (v/v); 100 mM) as crosslinker. Effects of additives including proteic feeder such as bovine serum albumin (BSA) and poly-L-Lysine were investigated to reveal its significance in enhancing the performance of enzyme. Addition of 0.1 mg BSA/U xylanase showed considerable increment in CLEA development with approximately 50% retained activity.

Keywords: cross-linked, immobilization, recombinant, xylanase

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Authors: Ashish Kumar Singh, Mehak Gulati, Neelam Verma

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Arginine majorly acts as a substrate for the enzyme nitric oxide synthase (NOS) for the production of nitric oxide, a strong vasodilator. Current study demonstrated a novel amperometric approach for estimation of arginine using nitric oxide synthase. The enzyme was co-immobilized in carbon paste electrode with NADP+, FAD and BH4 as cofactors. The detection principle of the biosensor is enzyme NOS catalyzes the conversion of arginine into nitric oxide. The developed biosensor could able to detect up to 10-9M of arginine. The oxidation peak of NO was observed at 0.65V. The developed arginine biosensor was used to monitor arginine content in fruit juices.

Keywords: arginine, biosensor, carbon paste elctrode, nitric oxide

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Authors: Iranga Weerakkody, Palitha Sri Geegana Arachchige, Dasith Tilakaratna

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The aim of this research is to study how folk wisdom can be applied to assist in the process of purification of water. This is qualitative research, and by random sampling, it is focused on to the dry zone of Sri Lanka. The research limitation has been set to the use of Ingini seeds (Strychnos potatorum) to purify water. Here the research is based on connecting traditional knowledge regarding water purification using Ingini seeds to modern times and the advantages and disadvantages of using Ingini seeds to purify water sources. Ingini seeds have been used among villagers of the dry zone to purify water for a long time by methods such as planting Ingini plants around water sources and depositing seeds covered with a cotton cloth inside wells. Crushed Ingini seeds have been put into clay water pots to reduce the hardness of water, as well as the number of impurities present in the water. This shows that Ingini seeds have a property that is successful in precipitating dissolved impurities in water. Ingini seeds are also used to precipitate solid impurities in herbal wine. The advantages of using Ingini seeds are that it can be obtained naturally from the ecology without an additional cost and that it is completely organic forest produce. Another specialty is that in practices, it is used to treat kidney stones and other water-related diseases affecting the kidneys.

Keywords: folklife, Ingini seeds, Strychnos potatorum, organic forest produce, water purification

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Authors: Ziqu Ouyang, Kun Su

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A novel efficient and clean coal combustion system, namely the purification-combustion system, was designed by the Institute of Engineering Thermal Physics, Chinese Academy of Science, in 2022. Among them, the purification system was composed of a mesothermal activating unit and a hyperthermal reductive unit, and the combustion system was composed of a mild combustion system. In the purification-combustion system, the deep in-situ removal of coal-N could be realized by matching the temperature and atmosphere in each unit, and thus the NOx emission was controlled effectively. To acquire the methods for realizing the efficient and clean coal combustion, this study investigated the effect of the activating temperature (including 822 °C, 858 °C, 933 °C, 991 °C), which was the key factor affecting the system operation, on combustion and NOx emission characteristics of pulverized coal in a 30 kW purification-combustion test bench. The research result turned out that the activating temperature affected the combustion and NOx emission characteristics significantly. As the activating temperature increased, the temperature increased first and then decreased in the mild combustion unit, and the temperature change in the lower part was much higher than that in the upper part. Moreover, the main combustion region was always located at the top of the unit under different activating temperatures, and the combustion intensity along the unit was weakened gradually. Increasing the activating temperature excessively could destroy the reductive atmosphere early in the upper part of the unit, which wasn’t conducive to the full removal of coal-N in the reductive coal char. As the activating temperature increased, the combustion efficiency increased first and then decreased, while the NOx emission decreased first and then increased, illustrating that increasing the activating temperature properly promoted the efficient and clean coal combustion, but there was a limit to its growth. In this study, the optimal activating temperature was 858 °C. Hence, this research illustrated that increasing the activating temperature properly could realize the mutual matching of improving the combustion efficiency and reducing the NOx emission, and thus guaranteed the clean and efficient coal combustion well.

Keywords: activating temperature, combustion characteristics, nox emission, purification-combustion system

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Authors: Kayla Youhanaie, Kenneth Dott, Greg Gillis-Smith

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Access to clean water is a global challenge that affects nearly one-third of the world’s population. A lack of safe drinking water negatively affects a person’s health, safety, and economic status. However, many regions of the world that face this clean water challenge also have high solar energy potential. To address this worldwide issue and utilize available resources, a solar-powered water purification device was developed that could be implemented in communities around the world that lack access to potable water. The device uses ozone to destroy water-borne pathogens and sand filtration to filter out particulates from the water. To select the best method for this application, a quantitative energy efficiency comparison of three water purification methods was conducted: heat, UV light, and ozone. After constructing an initial prototype, the efficacy of the device was tested using agar petri dishes to test for bacteria growth in treated water samples at various time intervals after applying the device to contaminated water. The results demonstrated that the water purification device successfully removed all bacteria and particulates from the water within three minutes, making it safe for human consumption. These results, as well as the proposed design that utilizes widely available resources in target communities, suggest that the device is a sustainable solution to address the global water crisis and could improve the quality of life for millions of people worldwide.

Keywords: clean water, solar powered water purification, ozonation, sand filtration, global water crisis

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Authors: Saba Pajuhan, Afshin Farahbakhsh, S. M. M. Dastgheib

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Biosurfactants and bioemulsifiers are a structurally diverse group of surface-active molecules synthesized by microorganisms, they are amphipathic molecules which reduce surface and interfacial tensions and widely used in pharmaceutical, cosmetic, food and petroleum industries. In this paper, several methods of bioemulsifer synthesis and purification by Bacillus licheniformis strains (namely ACO1, PTCC 1595 and ACO4) were investigated. Strains were grown in nutrient broth with different conditions in order to get maximum production of bioemulsifer. The purification of bio emulsifier and the quality evaluation of the product was done by adding sulfuric acid (H₂SO₄) (98%), Ethanol or HCl to the solution followed by centrifuging. To determine the optimal conditions yielding the highest bioemulsifier production, the effect of various carbon and nitrogen sources, temperature, NaCl concentration, pH, O₂ levels, incubation time are indispensable and all of them were highly effective in bioemulsifiers production.

Keywords: biosurfactant, bioemulsifier, purification, surface tension, interfacial tension

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Authors: Zohreh Bayat, Dariush Minai-Tehrani

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Lipases constitute one of the most important groups of industrial enzymes that catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids. Muscarinic antagonist relieves smooth muscle spasm of the gastrointestinal tract and effect on the cardiovascular system. In this research, the effect of a muscarinic antagonist on the lipase activity of Pseudomonas aeruginosa was studied. Lineweaver–Burk plot showed that the drug inhibited the enzyme by competitive inhibition. The IC50 value (60 uM) and Ki (30 uM) of the drug revealed the drug bound to the enzyme with high affinity. Determination of enzyme activity in various pH and temperature showed that the maximum activity of lipase was at pH 8 and 60°C both in presence and absence of the drug.

Keywords: bacteria, inhibition, kinetics, lipase

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Authors: Houmame Benbouali

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The risks, in general, exist in any project; one can hardly carry out a project without taking risks. The hydraulic works are rather complex projects in their design, realization and exploitation, and are often subjected at the multiple risks being able to influence with their good performance, and can have an negative impact on their environment. The present study was carried out to quote the impacts caused by purification plant STEP Chlef on the environment, it aims has studies the environmental impacts during construction and when designing this STEP, it is divided into two parts: The first part results from a research task bibliographer which contain three chapters (-cleansing of water worn-general information on water worn-proceed of purification of waste water). The second part is an experimental part which is divided into four chapters (detailed state initial-description of the station of purification-evaluation of the impacts of the project analyzes measurements and recommendations).

Keywords: treatment plant, waste water, waste water treatment, environmental impact

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Authors: Justina I. R. Udotong, Essien U. Essien

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Diagnostic enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined as indices of heavy metal pollution in Tilapia guinensis. Three different sets of fishes treated with lead (Pb), iron (Fe) and copper (Cu) were used for the study while a fourth group with no heavy metal served as a control. Fishes in each of the groups were exposed to 2.65 mg/l of Pb, 0.85 mg/l of Fe and 0.35 mg/l of Cu in aerated aquaria for 96 hours. Tissue fractionation of the liver tissues was carried out and the three diagnostic enzymes (AST, ALT, and ALP) were estimated. Serum levels of the same diagnostic enzymes were also measured. The mean values of the serum enzyme activity for ALP in each experimental group were 19.5±1.62, 29.67±2.17 and 1.15±0.27 IU/L for Pb, Fe and Cu groups compared with 9.99±1.34 IU/L enzyme activity in the control. This result showed that Pb and Fe caused increased release of the enzyme into the blood circulation indicating increased tissue damage while Cu caused a reduction in the serum level as compared with the level in the control group. The mean values of enzyme activity obtained in the liver were 102.14±6.12, 140.17±2.06 and 168.23±3.52 IU/L for Pb, Fe and Cu groups, respectively compared to 91.20±9.42 IU/L enzyme activity for the control group. The serum and liver AST and ALT activities obtained in Pb, Fe, Cu and control groups are reported. It was generally noted that the presence of the heavy metal caused liver tissues damage and consequent increased level of the diagnostic enzymes in the serum.

Keywords: diagnostic enzymes, enzyme activity, heavy metals, tissues investigations

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