Gel Entrapment Research Articles

Entrapment in HydrIL gels: Hydro-Ionic Liquid polymer gels for enzyme immobilization

Enzyme entrapment in hydro-ionic liquid gels (HydrIL gels) was explored as an enzyme immobilization strategy. The liquid phase of the gels was a mixture of buffer and tributyl methyl phosphonium alkanesulfonate [P4441][RSO3] ionic liquids. The ionic liquid modifies the liquid phase creating a more “organic” hydrophobic microenvironment around the enzyme. In the esterification of a secondary alcohol by Candida antarctica Lipase B, a 20-fold enhancement in final substrate conversion was achieved using an HydrIL gel compared to a standard poly(acrylamide) hydrogel. Co-polymerization of the poly(acrylamide) matrix with acrylate monomers led to further increases in reaction rate. A CalB HydrIL gel was reused over 10 reaction cycles, achieving a consistent performance from the fifth cycle. IL leaching became undetectable from the third reaction cycle.

Preparation of Idebenone as a Thermosetting Nasal Gel for Better Bioavailability and Histopathological Effect

Background Idebenone is an extensively metabolized drug with poor water solubility that is used to treat Leiber’s hereditary optical neuropathy. Objective This study aims to prepare idebenone nanoemulsion as a poloxamer-based nasal gel to overcome the extensive rate of hepatic metabolism for better bioavailability and lower histopathological effect on the nasal mucosa. Methods The formulation strategy was based on eliciting mutual concentration reduction between the nanoemulsion and the carrier gel by setting their gelation temperature between 30-32°C to overcome the mucociliary dose washout. The o/w nanoemulsions rely on cremophor EL and transcutol as an emulsifying system to stabilize idebenone-loaded lemongrass oil. The spontaneous emulsification method was used to prepare nanoemulsions that were characterized by zeta sizer while the thermosensitive hydrogels were prepared using the cold method. In-vitro dissolution test and ex-vivo permeation study through excised sheep nasal mucosa were performed to evaluate the enhanced permeation ratio, rate of permeation, and permeation coefficient. The histopathological effect of direct application on sheep nasal mucosa was studied using optical microscopy to evaluate cellular toxicity. Results The formula prepared from NE1 with poloxamer 407: poloxamer188 in concentrations 10:3% w/w respectively showed almost complete drug release in 120 minutes due to complete polymers blend erosion. Furthermore, thermosensitive nano-emulgel at a temperature of gelation 31.8°C was obtained at much lower concentrations of poloxamer 407 (10%) compared to previous studies. Nanoemulsions retained their globular size below 100nm due to further gel entrapment stabilization. Conclusions Drug permeation through excised sheep nasal mucosa elicited an increase in enhanced permeation ratio to 20.3 times and other flux kinetics parameters compared to those of IDB oil dispersion. Direct cellular toxicity showed a minor inflammatory response characterized by serous infiltration of inflammatory cells and edema. In contrast, most of the epithelial cells retained their histological characteristics compared to control slides. Received: Mar. 2023 Accepted: Aug 2023 Published: Oct.2023

Fabrication of 1,4-alpha-D-glucan glucanohydrolase holding Gel-Scaffolds using Agar-Agar, a natural polysaccharide and Polyacrylamide, a synthetic organic polymer for continuous liquefaction of starch

1,4-alpha-D-glucan glucanohydrolase is among the most widely used commercial hydrolytic enzymes acting randomly on the glycosidic linkages of starch resulting in its saccharification and liquefaction. Its applicability in different industries can be improved by enhancing its stability and reusability. Therefore, in the present study attempts have been made to enhance the industrial applicability of 1,4-alpha-D-glucan glucanohydrolase from Bacillus subtilis KIBGE-HAR by adapting immobilization technology. The study developed mechanically stable, enzyme containing gel-frameworks using two support matrices including agar-agar, a natural polysaccharide and polyacrylamide gel, a synthetic organic polymer. These catalytic gel-scaffolds were compared with each other in terms of kinetics and stability of entrapped 1,4-α-D-glucan glucanohydrolase. In case of polyacrylamide gel, Km value for immobilized enzyme increased to 7.95 mg/mL, while immobilization in agar-agar resulted in decreased Km value i.e 0.277 mg/mL as compared to free enzyme. It was found that immobilized enzyme showed maximum activity at 70 °C in both the supports as compared to free enzyme having maximum activity at 60 °C. Immobilized 1,4-α-D-glucan glucanohydrolase exhibited no change in optimal pH 7.0 before and after entrapment in polyacrylamide gel and agar-agar. The enzyme containing gel-scaffold was found suitable for repeated batches of starch liquefaction in industrial processes. Agar-agar entrapped 1,4-α-D-glucanglucanohydrolase was capable to degrade starch up to seven repeated operational cycles whereas polyacrylamide entrapped enzyme conserved its activity up to sixth operational cycle.

A review on distribution, properties, genetic organization, immobilisation and applications of urease

Urease, a nickel-containing metalloenzyme is getting remarkable attention due to a diverse range of applications for mankind. Urease plays a magnificent role in various field like agriculture, analytical, geological phenomena, beverage industry and is an important diagnostic tool. Urease is mainly present in bacteria, fungi, plants and invertebrates and its manifestation in specific genera may open new vistas for its taxonomic position. Various qualitative and quantitative assays are also reported for the estimation of urease enzyme. Urease based biosensors utilizing green synthesis on nanoparticles are also trending. Recently developed inhibitors against urease were discussed in the review. Inhibitory mechanisms involving the structural similarity of the substrate through modification or derivatization can also help in rational drug design by two possible competitive ways either by mimicking monodentate urea binding or binding as a tetrahedral intermediate. Immobilisation of urease through gel entrapment, using non-covalent and covalent protein tags, cross linkage, covalent bonding, using composite films, Teflon, co-precipitation and coating on nanoparticles is also reported. This review also comprised of various application of urease including enhancement of fertility in the soil, cell to cell organization, protection to predators, treatment of various bladder related diseases and infections, analysis of urea and heavy metal ions, biocementation, pollution control by bioleaching of heavy metals and making beverages urea and ethyl carbamate free. As researchers have a keen interest in urease enzyme at present, most of its aspects were incorporated in the article to make it helpful to the scientific community for further research related to the development of new inhibitors and add on applications of urease for the upliftment of the human as well as environment.

Enhancement of the Catalytic Performance and Operational Stability of Sol-Gel-Entrapped Cellulase by Tailoring the Matrix Structure and Properties.

Commercial cellulase Cellic CTec2 was immobilized by the entrapment technique in sol–gel matrices, and sol–gel entrapment with deposition onto magnetic nanoparticles, using binary or ternary systems of silane precursors with alkyl- or aryl-trimethoxysilanes, at different molar ratios. Appropriate tailoring of the sol–gel matrix allowed for the enhancement of the catalytic efficiency of the cellulase biocatalyst, which was then evaluated in the hydrolysis reaction of Avicel microcrystalline cellulose. A correlation between the catalytic activity with the properties of the sol–gel matrix of the nanobiocatalysts was observed using several characterization methods: scanning electron microscopy (SEM), fluorescence microscopy (FM), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA/DTA). The homogeneous distribution of the enzymes in the sol–gel matrix and the mass loss profile as a function of temperature were highlighted. The influence of temperature and pH of the reaction medium on the catalytic performance of the nanobiocatalysts as well as the operational stability under optimized reaction conditions were also investigated; the immobilized biocatalysts proved their superiority in comparison to the native cellulase. The magnetic cellulase biocatalyst with the highest efficiency was reused in seven successive batch hydrolysis cycles of microcrystalline cellulose with remanent activity values that were over 40%, thus we obtained promising results for scaling-up the process.

Effect of phosphorylated polyvinyl alcohol matrix size of cell entrapment on partial nitrification of ammonia in wastewater

ABSTRACT Partial nitrification is known as first and critical step for autotrophic nitrogen removal in high strength nitrogenous wastewater. Phosphorylated polyvinyl alcohol gel entrapment was used for suppressing oxygen to nitrite-oxidizing bacteria (NOB) in the gel matrix. The study investigated the effect of the size of gel matrix on partial nitrification. Results show that ammonia-oxidizing bacteria (AOB) proportion in the inoculum rather than the size of gel matrix governed ammonia oxidation. Nitrite oxidation depended on the size of gel matrix not the relative proportions of NOB and AOB in the inoculum. Larger size of gel matrix lead to less in situ oxygen penetration and available for NOB resulting in higher nitrite accumulation. This finding gains a better understanding of using suitable inoculum to control partial nitrification that is beneficial for the preparation of anaerobic ammonium oxidation-suited effluent.

Rapid cultivation of anammox sludge based on Ca-alginate cell beads.

Current gel entrapment technology has certain advantages for the enrichment of anammox sludge. In this study, the optimal preparation conditions and cultivation equipment of Ca-alginate cell beads for the culturing anammox sludge were proposed. The preparation parameters of the Ca-alginate cell beads were as follows: 3% sodium alginate, 4% CaCl2, VSA:Vcell = 1:1, a drop height of 9 cm, stirring speed of 300 rpm, and cross-linking time of 24 h. The prepared cell beads were regular spheres with a uniform size and hard texture. Throughout the 9 days of cultivation, the number of anammox bacteria in the Ca-alginate cell beads was 4.3 times that of the initial sludge, and the color of the cell beads changed from yellowish-brown to reddish-brown. Scanning electron microscopy (SEM) analysis showed that the SA gel beads had a good microporous structure. The fluorescence in situ hybridization (FISH) results illustrated that the bacteria were mostly dispersed inside the Ca-alginate cell beads. Additionally, the qPCR results implied that only a relatively small amount of anammox biomass (2.74×106 copies/gel-bead) was required to quickly start the anammox process. The anammox bacteria in the Ca-alginate cell beads grew with a fast growth rate in a short period and exhibited high activity due to diffusion limitations. In addition, the anammox bacteria cultivated in the Ca-alginate cell beads could adapt to the increase in substrate concentration in a short period. The optimal incubation time of this gel entrapment method for anammox sludge was no more than 17 days under the experimental conditions of this work. Therefore, this simple and practicable gel entrapment method may serve as a suitable pre-culture means for the rapid enrichment of anammox bacteria.

Long-Term Limitation Effects of Se(VI), Zn(II), and Ni(II) on Start-Up of the Anammox Process Using Gel Carrier.

Anaerobic ammonia oxidation (anammox) bacteria are inhibited by heavy metals at high concentrations but require trace amounts of some heavy-metal elements for growth and activity maintenance. The present study evaluates the long-term limitation effects of Se(VI), Zn(II), and Ni(II) on the start-up period of an anammox reactor. To strictly limit the levels of heavy metals in the reactor, all tests used ultrapure water as the influent synthetic wastewater and all reactors were installed in a clean booth. The anammox biomass was maintained through the gel entrapment technique. In the absence of Se(VI) and Ni(II), the anammox reactor start-up was 18.9 kg-N (m3-carrier d)−1 (nitrogen conversion rate (NCR) per gel carriers), indicating that Se(VI) and Ni(II) are not required or need not be continuously added to maintain the anammox process. Under Zn(II) limitation, the anammox process failed to start-up and the NCR tended to decrease rapidly. After readdition of 0.005 mg L−1 of Zn(II), the NCR did not decline further and instead partially recovered at a very slow rate. The NCR was completely recovered after adding 0.020 mg L−1 of Zn(II). These results reveal that Zn(II) limitation seriously affects the start-up of the anammox process while Se(VI) and Ni(II) are not required or need not be continuously added to the anammox process.

Removal of cadmium(II) ions using Saccharomyces cerevisiae and Leuconostoc mesenteroides immobilized in silica materials by two processing methods

Two processing methods for preparation of microbial biomass-silica biosorbent to remove cadmium ions from aqueous solution were developed. The first method involved a slow addition of sulphuric acid into previously prepared dispersion of viable Saccharomyces cerevisiae or Leuconostoc mesenteroides biomass in sodium silicate solution. The obtained material was mesoporous with irregular shape and size between 1 and 10 μm. The second method for the immobilization of microbial biomass by silica gel entrapment was developed in order to restrict desorption of microbial cells and to obtain larger particles with uniform size. The obtained particles were spherical with average particle size of 1.5 mm. Both materials with viable L. mesenteroides cells displayed higher removal efficiency compared the efficiency of materials that containing viable S. cerevisiae cells. Infrared spectra revealed an intensive secretion of dextran by Leuconostoc mesenteoides cells in the presence of cadmium ions. EDS maps clearly showed the dispersion of cadmium in the cross-section of microbial biomass-silica biosorbent. The maximum theoretical binding adsorption capacity for the silica-alginate-S. cerevisiae composite was 54 mg/g. The adsorption capacity for the silica-alginate-L. mesenteroides composite obtained after 24 cycles was about 93 mg/g. The extracellular secretion of dextran by L. mesenteroides cells immobilized in silica-alginate composite enabled efficient removal of cadmium ions. The efficiency of Cd(II) removal by microbial composite was not affected by the presence of co-existing ions at initial cadmium concentration of 1 and 4 mmol/L.

Microbial cellulases immobilized in biopolymer/silica matrices used as enzyme release systems.

Trichoderma viride CMGB 1 cellulases were immobilized by entrapment in silica gels (by sol-gel method), alginate biopolymers and hybrid alginate/silica materials. Tetramethoxysilane (TMOS), tetraethoxysilane (TEOS) and tetrakis (2-hydroxyethyl) orthosilicate (THEOS) were used as organoalkoxysilane precursors and ethanol or ethylene glycol as cosolvents in a two step sol-gel synthesis. Combined alginate/silica matrices resulted by mixing silica sol with sodium alginate or by coating alginate beads with a silica shell. The partial confinement of ethylene glycol in the matrix with consequences on biocatalytic activity was investigated using SEM-EDAX, thermal analysis and FT-IR spectroscopy. The efficiency of the enzyme-matrix biomaterials was tested in controlled enzyme release experiments. The sol-gel method developed using EG as a co-solvent allowed cellulase immobilization yields 1.5-4.5 times higher compared to classical sol-gel methods that use EtOH. The characterization of the gels by microscopic and spectrophotometric analyzes showed that there are similarities between the structure of the gels based on THEOS and those developed by us from TEOS, TMOS and EG as co-solvent. The new developed gels showed good cellulase release properties at acidic pH, comparable to those based on THEOS and alginate. The microbial cellulases immobilized in the matrices obtained and characterized in this work can operate as efficient systems for releasing enzymes, in acidic pH conditions, as feed additives.

Critical Analysis of Biomass Retention Strategies in Mainstream and Sidestream ANAMMOX-Mediated Nitrogen Removal Systems.

ANAMMOX (anaerobic ammonium oxidation) represents an energy-efficient process for biological nitrogen removal, particularly from wastewater streams with low chemical oxygen demand (COD) to nitrogen (C/N) ratios. Its widespread application, however, is still hampered by a lack of access to biomass-enriched with ANAMMOX bacteria (AMX), slow growth rates of AMX, and their sensitivity to inhibition. Although the coupling of ANAMMOX processes with partial nitrification is already widespread, especially for sidestream treatment, maintaining a functional population density of AMX remains a challenge in these systems. Therefore, strategies that maximize retention of AMX-rich biomass are essential to promote process stability. This paper reviews existing methods of biomass retention in ANAMMOX-mediated systems, focusing on (i) granulation; (ii) biofilm formation on carrier materials; (iii) gel entrapment; and (iv) membrane technology in mainstream and sidestream systems. In addition, the microbial ecology of different ANAMMOX-mediated systems is reviewed.

E. coli BL21 Bacterial and Newcastle Pestikal lasota Viral Ghosts Preparation, DNA/RNA Elimination/Purification/Isolation/ Quantification Using Agarose Gel Entrapment Bubble Method

Sponge like protocol is a new protocol able to turn most microbes including viruses to empty cells or empty envelops. The original protocol guarantees full evacuation, however, in some cases it need a validation. In this study, simple tools are described to separate the genetic constituents of both partially prepared E. coli BL21 and Newcastle Pestikal lasota strain ghosts. The used methods describe the preparation of entrapment bubble (in the agarose gel) as a proposed step for separating the genetic materials from the other constituents. That might solve some technical problems particularly during the virus ghost preparation.

Decitabine bioproduction using a biocatalyst with improved stability by adding nanocomposites

A novel IDA-LaNDT derivative was able to reach the highest productivity in the biosynthesis of a well-known antitumoral agent called decitabine. However, the combination of two simple and inexpensive techniques such as ionic absorption and gel entrapment with the incorporation of a bionanocomposite such as bentonite significantly improved the stability of this biocatalyst. These modifications allowed the enhancement of storage stability (for at least 18 months), reusability (400 h of successive batches without significant loss of its initial activity), and thermal and solvent stability with respect to the non-entrapped derivative. Moreover, reaction conditions were optimized by increasing the solubility of 5-aza by dilution with dimethylsulfoxide. Therefore, a scale-up of the bioprocess was assayed using the developed biocatalyst, obtaining 221 mg/L·h of DAC. Finally, green parameters were calculated using the nanostabilized biocatalyst, whose results indicated that it was able to biosynthesize DAC by a smooth, cheap, and environmentally friendly methodology.

Granulation of Drinking Water Treatment Residues: Recent Advances and Prospects

Beneficial reuse of drinking water treatment plant residues (WTRs) has been intensively studied worldwide in the last decades, but few engineering applications can be found. The majority of WTRs were directly reused in cake form (after dewatering), e.g., alum sludge cake as main substrate used in constructed wetlands (CWs), or oven dried and ground powdery form, e.g., sorbent for pollutant removal. However, WTRs reuse in such forms has several drawbacks, i.e., difficulty of recovering and easy clogging (in CWs), which result in limited WTRs engineering applications. Granulation or pelleting could widen and be a wiser WTRs reuse route and also seems to be a promising strategy to overcome the “application bottleneck” issues. In the literature, a number of trials of WTRs granulation have been reported since 2008, including sintering ceramsite, gel entrapment and newly emerged techniques. Hence, there is a need to overlook these studies and promote WTRs granulation for further development. To this end, this review firstly provides a piece of updated comprehensive information and critical analysis regarding WTRs granulation/pelleting technology. It aims to enhance WTRs granulation studies in the developing stage and thus enlarge WTRs engineering applications.

Entrapment of enzyme in the presence of proline: effective approach to enhance activity and stability of horseradish peroxidase

In this report, activity and stability of horseradish peroxidase (HRP) entrapped in polyacrylamide gel in the presence of proline (HEP) are compared with that of enzyme entrapped in absence of proline (HE). Within polyacrylamide (8%) beads, 80% entrapment yield for peroxidase was observed in the presence as well as absence of proline. The HEP (1.5M proline) showed 170% higher activity compared to HE. HEP also showed significant increase in K M, V max and K cat. At 8th cycle of use, HEP retained 40% of its activity, whereas HE retained only 10% of activity. In addition, in comparison with HE, HEP showed increased storage stability and thermo-stability. HEP showed higher activity compared to HE over an extensive range of pH (4-8), temperature (30-80°C) and inhibitors such as NaN3, Cd2+ and Pb2+. Our results suggest that peroxidase entrapment in polyacrylamide gel in the presence of proline can be a useful approach for increasing activity and stability of horseradish peroxidase.

Endoglucanase Produced by Bacillus subtilis Strain CBS31: Biochemical Characterization, Thermodynamic Study, Enzymatic Hydrolysis, and Bio-industrial Applications

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. Here, homogeneous endoglucanase GluCB31 from Bacillus subtilis subsp. inaquosorum CBS31 was studied. GluCB31 was purified to 17.68-fold with an 8.33% yield and a specific activity of 1066.37 U/mg. Biochemical properties of GluCB31 were performed and the results are as follows; molecular mass of 35 kDa with an optimum pH at 7.5 and temperature at 50°C. GluCB31 was immobilized in calcium alginate gel and it exhibited the highest activity at 10°C higher temperature than soluble enzyme, as the entrapment in alginate gel made GluCB31 more stable. Kinetic studies showed the Vmax of 1293.33 ± 2.51 U/mg and Km of 0.0183 mg/mL. Enzymatic activity was activated by Tween-20 (106.7%), Tween-80 (111.6%), Triton X-100 (142.3%), SDS (135.5%), Mg++ (185.7%), Cu++ (167.6%), Zn++ (153.7%), Mn++ (106.3%), Ba++ (181.9%), Ni++ (107.2%) while inhibited by Fe++ (15.8%), β-mercaptoethanol (46.8%), EDTA (54.5%). Enthalpy, free energy, and entropy of activation were calculated to be 38.526 kJmol-1, 44.187 kJmol-1, and -17.518 Jmol-1K-1 respectively. Also, ΔGE-S and ΔGE-T were found to be -10.75 kJmol-1 and -45.92 kJmol-1 respectively. A low ΔS, ΔGE-S, and ΔGE-T values were signified enzyme-catalyzed reaction occurs at a fast rate and the existence of the enzyme in its stable state. Cellobiose was the major end product of hydrolysis. These attributes of GluCB31 demonstrated the diversity of catalytic activities and serve in various biotechnological processes, thus deserve to be developed as a bio-industrial agent.

Enhancement of mainstream nitrogen removal via simultaneous partial nitrification, anammox and denitrification by the gel entrapment technique

A BSgel system was prepared by integrating the gel entrapment technique with a traditional biofilm carrier to realize the SNAD process under mainstream conditions.

Conservation of the Biocatalytic Activity of Whole Yeast Cells by Supported Sol – Gel Entrapment for Efficient Acyloin Condensation

In this study, an efficient and generally applicable 2nd generation sol – gel entrapment method was developed for immobilization of yeastcells. Cells of Lodderomyces elongisporus, Candida norvegica, Debaryomyces fabryi, Pichia carsonii strains in admixture with hollow silica microspheres support were immobilized in sol – gel matrix obtained from polycondensation of tetraethoxysilane. As biocatalysts in theselective acyloin condensation of benzaldehyde catalyzed by pyruvate decarboxylase of the yeast, the novel immobilized whole-cell preparations were compared to other states of the cells such as freshly harvested wet cell paste, lyophilized cells and sol – gel entrapped preparations without hollow silica microspheres support. Reusability and storability studies designated this novel 2nd generation sol – gel method as a promising alternative for solid formulation of whole-cells bypassing expensive and difficult downstream steps while providing easy-to-handle and stable biocatalysts with long-term preservation of the biocatalytic activity.

Extraction and immobilization of Proteolytic Enzyme from local Yemeni Bean Seeds (Dolichos Lablab L.)

The aim of this study is to prepare immobilized protease with high hydrolytic activity forbiotechnological applications. Local Bean Seeds (Dolichos Lablab L.) were used for the extraction of acidprotease using tris-buffer, pH 4.5 as enzyme solvent. Free acidic protease was immobilized on entrapment in calcium alginate gel (in situ activated) by covalent binding method. Their activity and immobilization efficiency for hemoglobin hydrolysis was investigated. Temperature and pH maxima of the immobilized protease showed no changes before and after immobilization. The immobilized protease exhibited good thermal stability and re-usability.

Immobilization and characterization of acid phosphatase from wheat germ (Type I) in agarose gel

Acid phosphatase (specific activity 1.82 U/mg) from wheat germ was immobilized by entrapment in agarose gel. Blocks of 5 × 5 mm dimension were used for characterization. The optimum percent immobilization was 75.25% when agarose–enzyme mixture concentration was 1.35%. The soluble and immobilized acid phosphatase exhibited emission maxima at 335 nm, respectively, when excitation wavelength was 280 nm. The intensity of immobilized enzyme was lower than corresponding soluble enzyme. Fourier Transform Infrared spectrum exhibited bands at wave numbers 3282.2 and 1635.9 cm−1 for immobilized enzyme. The optimum pH and temperature for immobilized acid phosphatase were 5.5 and 60 °C, respectively. The Km values for soluble and immobilized acid phosphatase were 0.068 and 0.232 mM, respectively. The Vmax values for soluble and immobilized acid phosphatase were 1.92 and 2.34 µmol/min/mg protein, respectively. The immobilized enzyme retained 48% activity on 56th day when stored at 4 °C. The same enzyme blocks could be reused up to 8 cycles. The method of immobilization reported here is simple and cost effective and will be suitable for further applications.