Improved pH Stability Research Articles

Synergistic catalysis of immobilized enzyme and peroxymonosulfate activation: CuFe2O4/kaolin-CTS-Laccase for enhanced performance of reactive blue 19 degradation

This work constructed an immobilized laccase - peroxymonosulfate (PMS) activation synergistic catalytic system for enhancing the efficient removal of reactive blue 19 (RB19). Superparamagnetic CuFe2O4/kaolin (CF/K) composites were prepared by one-step solvothermal and calcination methods and then modified with chitosan as immobilized carriers for laccase. The enzyme activity of CuFe2O4/kaolin-CTS-Laccase (CF/K-CTS-Lac) could reach 316.3 U/g under optimal immobilization conditions of 3 h and 2.6 mg/mL. CF/K-CTS-Lac exhibited improved pH stability and thermal stability in comparison to free laccase. The CF/K-CTS-Lac/PMS synergistic catalytic system achieved 98.4 % decolourization of RB19 within 90 min, which was 1.7 and 1.9 times higher than that of the CF/K-CTS-Lac system and CF/K-CTS/PMS system, respectively. Enhanced RB19 degradation efficiency attributed to synergistic catalysis by immobilized laccase and PMS activation. Free radical quenching experiments showed that hydroxyl radicals (•OH), sulfate radicals (SO4•-), superoxide radicals (•O2-) and singlet oxygen (1O2) were involved in the synergistic catalytic system, with •O2- and 1O2 playing a major role. Our investigations are expected to provide new insights into the construction of synergistic catalytic systems for pollutants.

An Alternative Approach to Plastic Recycling: Fabrication and Characterization of rPET/CA Nanofiber Carriers to Enhance Porcine Pancreatic Lipase Stability Properties.

In response to the increasing demand for sustainable technologies, this study presents a novel approach to plastic recycling. In this study, a method was presented to produce nanofiber carriers by electrospinning using recycled poly(ethylene terephthalate) (rPET) obtained from wastewater bottles and cellulose acetate (CA). These carriers serve as a platform for immobilized porcine pancreatic lipase (PPL), aiming to enhance its stability. The production parameters for the rPET/CA nanofibers were found to be an rPET concentration of 15% (v/v), a CA concentration of 6% (v/v), an electrical voltage of 13 kV, a needle-collector distance of 18 cm, and an injection speed of 0.1 mL/h. The nanofiber structure and morphology were assessed by using attenuated total reflectance-infrared Fourier transform infrared (ATR-FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) analyses. Then, PPL was immobilized onto the nanofibers through adsorption and cross-linking methods. The optimum temperature for free PPL was determined to be 30 °C, and the optimum temperature for PPL immobilized on rPET/CA was determined to be 40 °C. It was observed that, especially under acidic conditions, after the immobilization process, PPL immobilized rPET/CA nanofibers became more resistant to pH changes than free PLL. Furthermore, the immobilized PPL exhibited improved pH stability, reusability, and thermal stability compared to its free counterpart. This innovative approach not only contributes to plastic waste reduction but also opens new avenues for enzyme immobilization with potential applications in biocatalysis and wastewater treatment.

Enhanced stability and bioavailability of mulberry anthocyanins through the development of sodium caseinate-konjac glucomannan nanoparticles

This study was carried out to improve the stability of anthocyanins (ACNs) by developing MA-SC-KGM nanoparticles using a self-assembly method that involved the combination of sodium caseinate (SC) and konjac glucomannan (KGM) with mulberry anthocyanin extract (MA). Atomic force microscopy (AFM) analysis showed SC encapsulated MA successfully. Multispectral techniques demonstrated the presence of hydrogen bonds and hydrophobic interactions in the nanoparticles. MA-SC-KGM ternary mixture improved storage stability, color stability and anthocyanin retention better compared to the MA-SC binary mixture. Notably, MA-SC-KGM nanoparticles significantly inhibited the thermal degradation of ACNs, improved pH stability, and showed stability and a slow-release effect in gastrointestinal digestion experiments. In addition, MA-SC-KGM nanoparticles were effective in scavenging DPPH· and ABTS+ free radicals, with enhanced stability and antioxidant capacity even during the heating process. This study successfully developed a novel MA-SC-KGM protein-polysaccharide composite material that effectively stabilized natural ACNs, expanding the application of ACNs in various industries.

Computation-Aided Phylogeny-Oriented Engineering of β-Xylosidase: Modification of "Blades" to Enhance Stability and Activity for the Bioconversion of Hemicellulose to Produce Xylose.

Hemicellulose is a highly abundant, ubiquitous, and renewable natural polysaccharide, widely present in agricultural and forestry residues. The enzymatic hydrolysis of hemicellulose has generally been accomplished using β-xylosidases, but concomitantly increasing the stability and activity of these enzymes remains challenging. Here, we rationally engineered a β-xylosidase from Bacillus clausii to enhance its stability by computation-aided design combining ancestral sequence reconstruction and structural analysis. The resulting combinatorial mutant rXYLOM25I/S51L/S79E exhibited highly improved robustness, with a 6.9-fold increase of the half-life at 60 °C, while also exhibiting improved pH stability, catalytic efficiency, and hydrolytic activity. Structural analysis demonstrated that additional interactions among the propeller blades in the catalytic module resulted in a much more compact protein structure and induced the rearrangement of the opposing catalytic pocket to mediate the observed improvement of activity. Our work provides a robust biocatalyst for the hydrolysis of agricultural waste to produce various high-value-added chemicals and biofuels.

A straight forward synthetic route for solid-emissive carbon dots from AIE-active precursors

Carbon dots (CDs) with solid-state emission are becoming the eager pursuit for researchers under the continuous and in-depth development of carbon materials. A general strategy for efficiently constructing solid-emissive CDs is of importance. Here, solid-emissive CDs were successfully prepared using aggregation-induced emission (AIE) amino-substituted tetraphenylethylene as precursor. Thus obtained CDs well inherited the AIE properties of precursors with improved pH stability and bio-imaging performance.

Fabrication of a Magnetic Porous Organic Polymer for α-Glucosidase Immobilization and Its Application in Inhibitor Screening

The technology based on immobilized enzymes was employed to screen the constituents inhibiting disease-related enzyme activity from traditional Chinese medicine, which is expected to become an important approach of innovative drug development. Herein, the Fe3O4@POP composite with a core-shell structure was constructed for the first time with Fe3O4 magnetic nanoparticles as the core, 1,3,5-tris (4-aminophenyl) benzene (TAPB) and 2,5-divinylterephthalaldehyde (DVA) as organic monomers, and used as the support for immobilizing α-glucosidase. Fe3O4@POP was characterized by transmission electron microscopy, energy-dispersive spectrometry, Fourier transform infrared, powder X-ray diffraction, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. Fe3O4@POP exhibited a distinct core-shell structure and excellent magnetic response (45.2 emu g-1). α-Glucosidase was covalently immobilized on core-shell Fe3O4@POP magnetic nanoparticles using glutaraldehyde as the cross-linking agent. The immobilized α-glucosidase possessed improved pH stability and thermal stability as well as good storage stability and reusability. More importantly, the immobilized enzyme exhibited a lower Km value and enhanced affinity for the substrate than the free one. The immobilized α-glucosidase was subsequently used for inhibitor screening from 18 traditional Chinese medicines in combination with capillary electrophoresis analysis among which Rhodiola rosea exhibited the highest enzyme inhibitory activity. These positive results demonstrated that such magnetic POP-based core-shell nanoparticles were a promising carrier for enzyme immobilization and the screening strategy based on immobilized enzyme provided an effective way to rapidly explore the targeted active compounds from medicinal plants.

Biocatalytic Profiling of Free and Immobilized Partially Purified Alkaline Protease from an Autochthonous Bacillus aryabhattai Ab15-ES

Partially purified alkaline protease produced by an indigenous bacterial strain, Bacillus aryabhattai Ab15-ES, was insolubilized in alginate beads using an entrapment technique. Maximum entrapped enzyme activities of 68.76% and 71.06% were recorded at optimum conditions of 2% (w/v) sodium alginate and 0.3 M calcium chloride. Biochemical profiling of free and immobilized proteases was investigated by determining their activity and stability as well as kinetic properties. Both enzyme preparations exhibited maximum activity at the optimum pH and temperature of 8.0 and 50 °C, respectively. However, in comparison to the free enzyme, the immobilized protease showed improved pH stability at 8.0–9.0 and thermal stability at 40–50 °C. In addition, the entrapped protease exhibited a higher Vmax and increased affinity to the substrate (1.65-fold) than the soluble enzyme. The immobilized protease was found to be more stable than the free enzyme, retaining 80.88% and 38.37% of its initial activity when stored at 4 °C and 25 °C, respectively, for 30 d. After repeated use seven times, the protease entrapped in alginate beads maintained 32.93% of its original activity. These findings suggest the efficacy and sustainability of the developed immobilized catalytic system for various biotechnological applications.

Extraction of Anthocyanins from Black Grape By-Products and Improving Their Stability Using Cobalt(II) Complexation.

This study was conducted to investigate the effect of cobalt complexation on the spectral properties of anthocyanins (AC) extracted from black grape pomace (Black Magic) and the effect of complexation on the pH stability of AC during storage. Initially, cobalt acetate tetrahydrate aqueous solution was complexed with AC crude extract and diluted separately in buffer solutions with different pH (3.5, 4.5, 5.5, and 6.5). Afterward, spectral changes were determined spectrophotometrically. pH stability was investigated using the same buffer solutions and stored for 7 days in the dark at room temperature, and the absorbance of each solution was measured daily using a spectrophotometer. Results indicated that complexation caused similar hypsochromic and hyperchromic shifts in λmax at all pH values. With regard to pH stability, the degradation of complexed AC followed first-order reaction kinetics causing half-lives to increase up to 80-fold as compared with noncomplexed AC, which was due to the sharp decrease in K (per day), indicating an improved pH stability as compared with noncomplexed AC. Therefore, Co(II) could be used in the stabilization of grape AC for the coloration of a wide range of foods and food products at near-neutral pH environments considering the health benefits of grape AC and the maximum nontoxic dose of Co(II) salt.

Improved pH stability, heat stability, and functionality of phycocyanin after PEGylation

Phycocyanin (PC), a spirulina-derived protein–chromophore complex, suffers from poor techno-functional properties and is highly susceptible to aggregation and color changes upon heating and pH fluctuations. We tackled these issues by modifying PC via PEGylation. Electrophoresis and Fourier transform infrared spectroscopy proved successful conjugation of methoxy PEG (mPEG) chains on PC after PEGylation. Circular dichroism indicated highly ordered folding states adopted by PEGylated PC, which we attributed to the mPEG chains on the protein surface that sterically stabilized the protein structure. Consequently, the mPEG–PC conjugates exhibited high blue color intensity and improved thermodynamic stability. Further, benefit from an electrostatic shielding effect of mPEG chains, surface charges of PEGylated PC were neutralized over pH 2–9 and the blue hue of PC was stabilized against pH variations. Additionally, the flexible and hydrophilic mPEG polymers on the PC surface promoted protein–protein and protein–water interactions. PEGylated PC thus gained increased protein solubility, techno-functionality (emulsifying, foaming, and gelling performance), and antioxidant activities, when compared to unmodified PC. Heat-induced gels formed by mPEG–PC conjugates exhibited increased stiffness, higher water retention, and weak gel-type rheological properties. After PEGylation, the improved functional properties, bioactivity, and color stability against heat and pH fluctuations will facilitate food and pharmaceutical applications of PC.

Sustainable green approach to synthesize Fe3O4/α-Fe2O3 nanocomposite using waste pulp of Syzygium cumini and its application in functional stability of microbial cellulases

Synthesis of nanomaterials following green routes have drawn much attention in recent years due to the low cost, easy and eco-friendly approaches involved therein. Therefore, the current study is focused towards the synthesis of Fe3O4/α-Fe2O3 nanocomposite using waste pulp of Jamun (Syzygium cumini) and iron nitrate as the precursor of iron in an eco-friendly way. The synthesized Fe3O4/α-Fe2O3 nanocomposite has been extensively characterized through numerous techniques to explore the physicochemical properties, including X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, Ultraviolet-Vis spectroscopy, field emission scanning electron microscope, high resolution transmission electron microscope and vibrating sample magnetometer. Further, efficiency of the Fe3O4/α-Fe2O3 nanocomposite has been evaluated to improve the incubation temperature, thermal/pH stability of the crude cellulase enzymes obtained from the lab isolate fungal strain Cladosporium cladosporioides NS2 via solid state fermentation. It is found that the presence of 0.5% Fe3O4/α-Fe2O3 nanocomposite showed optimum incubation temperature and thermal stability in the long temperature range of 50–60 °C for 15 h along with improved pH stability in the range of pH 3.5–6.0. The presented study may have potential application in bioconversion of waste biomass at high temperature and broad pH range.

Modification of DNA regions with metagenomic DNA fragments (MDRMDF): A convenient strategy for efficient protein engineering

In this study, we have established a convenient and efficient approach named Modification of DNA Regions with Metagenomic DNA Fragments (MDRMDF) for protein engineering. Degenerate primers were designed corresponding to conserved regions of the gene of interest which were used for amplification of fragments with template of the metagenomic DNA. The resulting PCR products were used to replace the corresponding regions of the gene of interest to introduce modified gene for function-based screening. Therefore, this method can make full use of the metagenomic DNA sequences with unknown metagenomic gene information for efficient protein engineering. The β-xylosidase BH3683 was used to construct a MDRMDF library which was screened with a newly designed p-NPX-M9 medium-based strategy. As a result, a mutant protein Xyl-M56 showing high activity, improved pH stability and higher tolerance to organic solvents was obtained which may have potential for industrial application. The MDRMDF method may find wide application in enzyme engineering, metabolic engineering and other fields, especially offering a new methodological option for the directed evolution of proteins.

Purification and immobilization of His-tagged organophosphohydrolase on yolk−shell Co/C@SiO2@Ni/C nanoparticles for cascade degradation and detection of organophosphates

The synthesis or decomposition of complex substances through the combination of metals and enzymes is desirable but still a huge challenge. A kind of yolk-shell structured Co/C@SiO2@Ni/C nanocomposites were prepared by the polyhedral ZIF-67@SiO2 composite as a precursor through the extended stöber method and subsequent carbonization treatment. The SiO2 as an intermediate protective layer stabilized the morphology and structure of the carbonized products. The metallic nickel nanoparticles were uniformly distributed on the surface of Co/C@SiO2@Ni/C for highly specific absorption of His-tagged organophosphohydrolase (OpdA) from cell lysates via affinity, and therefore an integrated nanocatalyst (INC) named OpdA@Co/C@SiO2@Ni/C was successfully prepared. The prepared INC was applied to achieve a one-pot chemoenzymatic cascade degradation of organophosphates (OPs) to p-aminophenol (PAP) within 30 min. In addition, the INC proved to be a simple and high-sensitivity nanocatalyst for the detection of methyl-parathion with a low limit of detection (0.30 μM). The INC exhibited improved pH stability, thermal stability, SDS resistance, and storage stability compared to that of free enzymes. When OpdA@Co/C@SiO2@Ni/C was reused for 7 cycles, the cascade degradation efficiency of methyl-parathion could retain more than 60 %. This study provides a strategy for constructing a multifunctional nanocatalyst, and further demonstrated its potential in biocatalysis, accurate detection of OPs and remediation of environmental pollution.

Thermal deactivation of α-amylase immobilized magnetic chitosan and its modified forms: A kinetic and thermodynamic study

The stability and thermodynamic properties of an enzyme are the main factors that governing its applications in industry. With that intention we have immobilized the α-amylase onto synthesized chitosan-magnetite (CSM) composite and its modified forms by gluteraldehyde (CSM-GLA), glyoxal (CSM-GLY) and epichlorohydrin (CSM-ECH). In this study all the immobilized enzymes exhibited improved pH stability about 60–80% of relative activity at pH 9 compared to the free enzyme. The temperature stability at 60 °C is up to 50% of relative activity for covalently immobilized enzymes as enzyme become more rigid by covalent binding and so protected from the conformational changes caused by the environment. The thermal deactivation of the free and immobilized enzymes follows the first order kinetics. The t1/2 and D-values were prolonged considerably in case of covalently immobilized enzymes, indicating better thermal stability than that of free and adsorbed ones. The Ed values 18.71, 32.00, 27.19 and 20.46 KJmol-1 for CSM-E, CSM-GLY-E, CSM-GLA-E and CSM-ECH-E described the high stability and resistance to heat inactivation. The Km values 0.525 ± 0.04, 0.57 ± 0.06, 0.65 ± 0.04 mg/mL and Vmax values 25 ± 0.06, 19.6 ± 0.02, 16.39 ± 0.01 μmol mg−1 min−1for CSM-GLY-E, CSM-GLA-E and CSM-ECH-E showing better substrate affinity. The immobilized enzymes have exhibited about 60% of relative activity after 90 days of storage and very good reuse potential.

Directed mutation of β-glucanases from probiotics to enhance enzymatic activity, thermal and pH stability.

β-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of β-glucanases in probiotics. Here, β-glucanase genes were cloned from Bacillus licheniformis, Lactobacillus fermentum and L. johnsonii. β-glucanase genes, as blg, lfg and ljg isolated from B. licheniformis, L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that β-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of α-helices and β-strands in the three mutants were altered, which might result in β-glucanase activity increasement. Analysis of β-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30-50℃ than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about β-glucanase-encoding genes in L. fermentum and L. johnsonii. These findings provide an efficient way to improve the activity of β-glucanase.

Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei.

Xylanases isolated from microorganisms such as the Trichoderma reesei have attracted considerable research interest because of their potential in various industrial applications. However, naturally isolated xylanases cannot withstand harsh conditions such as high temperature and basic pH. In this study, we performed structural analysis of the major T. reesei xylanase (Xyn2), and novel flexible regions of the enzyme were identified based on B-factor, a molecular dynamics (MD) parameter. To improve thermostability of the Xyn2, disulfide bonds were introduced into the unstable flexible region by using site-directed mutagenesis and two recombinant xylanases, XM1 (Xyn2Cys12-52) and XM2 (Xyn2Cys59-149) were successfully expressed in Pichia pastoris. Secreted recombinant Xyn2 was estimated by SDS-PAGE to be 24 kDa. Interestingly, the half-lives of XM1 and XM2 at 60°C were 2.5- and 1.8- fold higher, respectively than those of native Xyn2. The XM1 also exhibited improved pH stability and maintained more than 60% activity over pH values ranging from 2.0 to 10.0. However, the specific activity and catalytic efficiency of XM1 was decreased as compared to those of XM2 and native Xyn2. Our results will assist not only in elucidating of the interactions between protein structure and function, but also in rational target selection for improving the thermostability of enzymes.

Comparative studies on ZIF-8 and SiO₂ nanoparticles as carrier for immobilized β-glucosidase

The immobilization of enzyme is an important biotechnology in industry. Enzyme immobilization by nanomaterials has attracted interest of researchers due to potentially promising application. Many enzymes had been successfully immobilized by different nanomaterials. In this work, β-glucosidase was immobilized by SiO2 nanoparticles (nano-SiO2) and zeolitic imidazolate framework-8 (ZIF-8), then the enzymatic properties of them were also compared. The nano-SiO2@β-glucosidase and the β-glucosidase@ZIF-8 achieved immobilization yield of 58.5% and 23.0%, respectively. Optimal temperature of the free β-glucosidase, nano-SiO2@β-glucosidase and the β-glucosidase@ZIF-8 were around 50 °C. Both free β-glucosidase and nano-SiO2@β-glucosidase showed maximal catalytic activity at pH 6.0, while the optimal pH of the β-glucosidase@ZIF-8 was 5.4 roughly. The nano-SiO2@β-glucosidase exhibited similar pH stability and thermal stability to those of the free β-glucosidase, and the β-glucosidase@ZIF-8 improved pH stability greatly. Moreover, after the treatment of trypsin for 4 h at 37 °C, the residual activities of the β-glucosidase@ZIF-8 was the highest that retained 94%, which provided huge potential for the application of the β-glucosidase@ZIF-8 in industry. Furthermore, the nano-SiO2@β-glucosidase and the β-glucosidase@ZIF-8 can be reused for 5 cycles with 87.5% and 88.7% residual activity, respectively.

Rational design of Pleurotus eryngii versatile ligninolytic peroxidase for enhanced pH and thermal stability through structure-based protein engineering.

Versatile peroxidase (VP) from Pleurotus eryngii is a high redox potential peroxidase. It has aroused great biotechnological interest due to its ability to oxidize a wide range of substrates, but its application is still limited due to low pH and thermal stability. Since CiP (Coprinopsis cinerea peroxidase) and PNP (peanut peroxidase) exhibited higher pH and thermal stability than VP, several motifs, which might contribute to their pH and thermal stability, were identified through structure and sequence alignment. Six VP variants incorporating the beneficial motifs were designed and constructed. Most variants were nearly completely inactivated except V1 (Variant 1) and V4. V1 showed comparable activity to WT VP against ABTS, while V4 exhibited reduced activity. V1 displayed improved pH stability than WT VP, at pH 3.0 in particular, whereas the pH stability of V4 did not change a lot. The thermal stabilities of V1 and V4 were enhanced with T50 raised by 3°C. The results demonstrated that variants containing the beneficial motifs of CiP and PNP conferred VP with improved pH and thermal stability.

Lipase immobilization on functionalized mesoporous TiO 2 : Specific adsorption, hyperactivation and application in cinnamyl acetate synthesis

Abstract According to the purification characteristics of lipase from Burkholderia ambifaria YCJ01, mesoporous TiO 2 was functionalized by phenylaminopropyl trimethoxysilane (Ph-TiO 2 ) for lipase immobilization. This support permitted one step immobilization and purification of lipase YCJ01 via hydrophobic interactions from crude fermentation supernatant. The activity of immobilized lipase was 6119.2 U/g support with 12.8 mg/g support of enzyme loading. The ratio of the activity of immobilized lipase to decrease activity in the supernatant after immobilization reached 2.2, which indicated that the immobilized lipase molecules exhibited a dramatic hyperactivation. In comparison with free lipase, immobilized lipase showed significantly improved pH stability and thermalstability. The Ph-TiO 2 /YCJ01 biocomposite was analyzed by scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. The result indicated lipase was efficiently immobilized on Ph-TiO 2 surface. Finally, the research about cinnamyl acetate synthesis suggested that Ph-TiO 2 /YCJ01 had enhanced catalytic efficiency compared with free lipase. In solvent free medium, the yield of 96.9% was obtained after one operation, and maintained more than 80% of yield after ten reaction cycles.

Erythromycin Modification That Improves Its Acidic Stability while Optimizing It for Local Drug Delivery.

The antibiotic erythromycin has limited efficacy and bioavailability due to its instability and conversion under acidic conditions via an intramolecular dehydration reaction. To improve the stability of erythromycin, several analogs have been developed—such as azithromycin and clarithromycin—which decrease the rate of intramolecular dehydration. We set out to build upon this prior work by developing a conjugate of erythromycin with improved pH stability, bioavailability, and preferential release from a drug delivery system directly at the low pH of an infection site. To develop this new drug conjugate, adamantane-1-carbohydrazide was covalently attached to erythromycin via a pH-degradable hydrazone bond. Since Staphylococcus aureus infection sites are slightly acidic, the hydrazone bond will undergo hydrolysis liberating erythromycin directly at the infection site. The adamantane group provides interaction with the drug delivery system. This local delivery strategy has the potential of reducing off-target and systemic side-effects. This work demonstrates the synthesis of a pH-cleavable, erythromycin conjugate that retains the inherent antimicrobial activity of erythromycin, has an increased hydrophobicity, and improved stability in acidic conditions; thereby enhancing erythromycin’s bioavailability while simultaneously reducing its toxicity.

Stability of immobilized porcine pancreas lipase on mesoporous chitosan beads: A comparative study

Porcine pancreas lipase was immobilized on mesoporous chitosan beads. Glutaraldehyde as coupling agent was used through several immobilization techniques. With the aid of FESEM, BET and BJH analysis, the effect of glutaraldehyde on porosity of chitosan was evaluated. It was observed that the total surface area and pore volume of the carrier were significantly improved by addition of glutaraldehyde as cross-linking agent. The surface area exposure and pore volume were substantially increased (both by 4.4 folds). In addition, distribution of enzyme on the carrier was illustrated by fluorescence image. The characteristics of the immobilized lipases such as immobilization efficiency, enzyme activity, pH stability, thermal stability, reusability, storage stability and enzyme leakage were evaluated. In kinetic studies of enzyme, Michaelis–Menten kinetic coefficients of the hydrolytic activity for the immobilized lipase were defined using Lineweaver–Burk plot. The low value of ionization constant, Km (∼0.008mM) and high value of specific rate, Vmax(∼200μM/ml.min) indicate strong affinity and high activity of enzyme. The obtained results demonstrate that use of glutaraldehyde has an excellent impact on expansion of the porosity of chitosan and enzyme distribution. The immobilization efficiency increased by1.6 folds and enzyme leakage was minimized (17% reduced to zero); while, glutaraldehyde has improved pH stability (in acidic range), thermal stability, reusability and storage stability of immobilized lipase.