Search Results (19)

The high amount of starch in fruits is responsible for its post-processing cloudiness. In the current study, α-amylase from porcine pancreases was immobilized onto carboxymethyl cellulose/polyacrylamide (CMC/PAM) hydrogel. This in-house-built CMC/PAM-Amy hydrogel offers a more efficient and sustainable solution for apple juice clarification. To acquire the best immobilization efficiency, the concentration of glutaraldehyde crosslinker was optimized. Biocatalytic characterization studies were brought into consideration for free and immobilized α-amylase. The synthesized native and immobilized CMC/PAM-Amy hydrogels were also characterized using SEM, FTIR and XRD. Under ideal circumstances, the activity of CMC/PAM-Amy was up to 604 μmolmin⁻¹, and its immobilization efficiency was 96.29 ± 1.15%. A kinetic parameters study resulted in a conspicuously lowered Km value for immobilized amylase, signifying its higher affinity for its substrate. CMC/PAM-Amy showed a half-life (t_1/2) 3.5 times higher than free-Amy at 50, 55 and 60 °C. The higher values of the inactivation rate constant (k_d), free energy of inactivation (ΔG*), enthalpy of inactivation (ΔH*) and change in entropy (ΔS*) of CMC/PAM-Amy manifested the enhanced thermal stability of amylase after immobilization. A reusability study revealed that immobilized amylase retained roughly 70% of its initial catalytic activity after six successive repetitions of the process. CMC/PAM-Amy displayed improved recycling ability operational stability and biocatalytic activity, rendering it an auspicious tool in decreasing the starch content of crude apple juice to about 61% of its total starch content before treatment. Moreover, the values of Brix, viscosity, acidity and turbidity were also decreased in CMC/PAM-Amyclarified apple juice. Therefore, immobilized amylases with other industrial enzymes could be an efficient tool for potential industrial application. Full article

The growing demand in industrial and biotechnological settings for more efficient enzymes with enhanced biochemical features, particularly thermostability and thermotolerance, necessitates a timely response. Renowned for their versatility, thermostable enzymes offer significant promise across a range of applications, including agricultural, medicinal, and biotechnological domains. This comprehensive review summarizes the structural attributes, catalytic mechanisms, and connection between structural configuration and functional activity of two major classes of thermostable enzymes: α-amylases and laccases. These enzymes serve as valuable models for understanding the structural foundation behind the thermostability of proteins. By highlighting the commercial importance of thermostable enzymes and the interest these generate among researchers in further optimization and innovation, this article can greatly contribute to ongoing research on thermostable enzymes and aiding industries in optimizing production processes via immobilization, use of stabilizing additives, chemical modification, protein engineering (directed evolution and mutagenesis), and genetic engineering (through cloning and expression of thermostable genes). It also gives insights to the exploration of suitable strategies and factors for enhancing thermostability like increasing substrate affinity; introducing electrostatic, intramolecular, and intermolecular hydrophobic interactions; mitigating steric hindrance; increasing flexibility of an active site; and N- and C-terminal engineering, thus resulting in heightened multipronged stability and notable enhancements in the enzymes’ industrial applicability. Full article

Amylase is an enzyme used to hydrolyze starch in order to obtain different products that are mainly used in the food industry. The results reported in this article refer to the immobilization of α-amylase in gellan hydrogel particles ionically cross-linked with Mg²⁺ ions. The obtained hydrogel particles were characterized physicochemically and morphologically. Their enzymatic activity was tested using starch as a substrate in several hydrolytic cycles. The results showed that the properties of the particles are influenced by the degree of cross-linking and the amount of immobilized α-amylase enzyme. The temperature and pH at which the immobilized enzyme activity is maximum were T = 60 °C and pH = 5.6. The enzymatic activity and affinity of the enzyme to the substrate depend on the particle type, and this decreases for particles with a higher cross-linking degree owing to the slow diffusion of the enzyme molecules inside the polymer’s network. By immobilization, α-amylase is protected from environmental factors, and the obtained particles can be quickly recovered from the hydrolysis medium, thus being able to be reused in repeated hydrolytic cycles (at least 11 cycles) without a substantial decrease in enzymatic activity. Moreover, α-amylase immobilized in gellan particles can be reactivated via treatment with a more acidic medium. Full article

Here, we describe a multianalyte biosensor platform for the fluorescent analysis of different human state biomarkers (α-amylase, phenylalanine, glucose, lactate/pyruvate, alcohol) and some immunosuppressive drugs (cyclosporine A, tacrolimus, methotrexate, rapamycin) using chimeric PQQ- and natural NAD⁺-dependent enzymes. The principle of the approach is based on the analysis of the brightness of photography of a sensor plate taken with a smartphone camera and processed using ImageJ software. The brightness of the image correlates with the fluorescence intensity of the sensor’s spots which is produced by the enzymatic reduction of phenazine methosulfate or its derivative used as a fluorescence probe at UV 356 nm irradiation, where the amount of the reduced dye depends on the concentration of the target analyte (the enzymatic substrate) in the tested sample. The sensor plate is composed of simple and cheap components, and the procedure of its preparation and usage is easy and does not require any specific skills or expensive instrumentation. The proposed sensor platform is characterized by a high selectivity and storage stability depending on the selectivity and stability characteristics of the used enzyme in an immobilized state. The proposed sensor platform could be used for precision quantitative analysis of a single (or several) analytes or used for a simultaneous qualitative multianalyte assay of them using Boolean logic gates. Full article

Maltooligosaccharides (MOS) are homooligosaccharides that consist of 3–10 glucose molecules linked by α-1,4 glycosidic bonds. As they have physiological functions, they are commonly used as ingredients in nutritional products and functional foods. Many researchers have investigated the potential applications of MOS and their derivatives in the pharmaceutical industry. In this review, we summarized the properties and methods of fabricating MOS and their derivatives, including sulfated and non-sulfated alkylMOS. For preparing MOS, different enzymatic strategies have been proposed by various researchers, using α-amylases, maltooligosaccharide-forming amylases, or glycosyltransferases as effective biocatalysts. Many researchers have focused on using immobilized biocatalysts and downstream processes for MOS production. This review also provides an overview of the current challenges and future trends of MOS production. Full article

We set out to isolate alpha-amylase producers from soil samples, optimize the production, and immobilize the enzyme on chitosan-loaded barium ferrite nanoparticles (CLBFNPs). Alpha-amylase producers were isolated on starch agar plates and confirmed by dinitrosalicylic acid assay. The potent isolate was identified by phenotypic methods, 16S-rRNA sequencing, and phylogenetic mapping. Sequential optimization of α-amylase production involved the use of Plackett–Burman (P–BD) and central composite designs (CCD), in addition to exposing the culture to different doses of gamma irradiation. Alpha-amylase was immobilized on CLBFNPs, and the nanocomposite was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy, with energy-dispersive analysis of X-ray analysis. Forty-five α-amylase producers were isolated from 100 soil samples. The highest activity (177.12 ± 6.12 U/mg) was detected in the MS009 isolate, which was identified as Bacillus paramycoides. The activity increased to 222.3 ± 5.07 U/mg when using the optimal culture conditions identified by P–BD and CCD, and to 319.45 ± 4.91 U/mg after exposing the culture to 6 kGy. Immobilization of α-amylase on CLBFNPs resulted in higher activity (246.85 ± 6.76 U/mg) compared to free α-amylase (222.254 ± 4.89 U/mg), in addition to retaining activity for up to five cycles of usage. Gamma irradiation improved α-amylase production, while immobilization on CLBFNPs enhanced activity, facilitated enzyme recovery, and enabled its repetitive use. Full article

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment yield of 19.9%. The identification of the bacteria was carried out using 16s rDNA sequencing. The enzyme was purified through ion exchange chromatography (IEX) in a DEAE-Sephadex column, revealing a protein with a molecular weight of ≈130 kDa. The enzyme was stable at pH 3.0 and heat stable up to 80 °C. However, the optimum conditions were reached at 65 °C and pH 3.0, with a specific activity of 1851.7 U mg⁻¹ ± 1.3. The agar-agarose immobilized α-amylase had a hydrolytic activity nearly 25% higher when compared to the free enzyme. This study provides critical information for the understanding of the enzymatic profile of B. licheniformis strain LB04 and the potential application of the microorganisms at an industrial level, specifically in the food industry. Full article

The design and synthesis of artificial receptors based on molecular imprinting (MI) technology for the development of a new MIP-based biosensor for detection of the stress biomarker α-amylase in human saliva in point-of-care (PoC) applications is described in this work. The portable electrochemical devices for monitoring α-amylase consists of cost-effective and disposable gold screen-printed electrodes (AuSPEs). To build the electrochemical device, the template biomolecule was firstly immobilized directly over the working area of the gold chip previously activated with a self-assembled monolayer (SAM) of cysteamine (CA). Then, pyrrole (Py) monomer was selected as building block of a polymeric network prepared by CV electropolymerization. After the electropolymerization process, the enzyme was removed from the polymer film in order to build the specific recognition sites for the target enzyme. The MIP biosensor showed a very wide linear concentration range (between 3.0 × 10⁻⁴ to 0.60 mg mL⁻¹ in buffer solution and between 3.0 × 10⁻⁴ to 3.0 × 10⁻² mg mL⁻¹ in human saliva) and low detection levels were achieved (LOD < 3.0 × 10⁻⁴ mg mL⁻¹) using square wave voltammetry (SWV) as the electroanalytical technique. Full article

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract. Full article

This work presents an efficient and facile strategy to prepare an α-amylase bioreactor. As enzymes are quite large to be immobilized inside metal-organic frameworks (MOFs), the tertiary and quaternary structures of α-amylase were first disrupted using a combination of urea, dithiothreitol (DTT), and iodoacetamide (IAA). After losing its tertiary structure, the unfolded proteins can now penetrate into the microporous MOFs, affording fragmented α-amylase@MOF bioreactors. Among the different MOFs evaluated, UiO-66 gave the most promising potential due to the size-matching effect of the α-helix of the fragmented α-amylase with the pore size of UiO-66. The prepared bioreactor exhibited high yields of small carbohydrate (maltose) even when reused up to 15 times (>80% conversion). Full article

Silk fibroin is a widely and commercially available natural protein derived from silkworm cocoons. Thanks to its unique amino acid composition and structure, which lead to localized nanoscale pockets with limited but sufficient hydration for protein interaction and stabilization, silk fibroin has been studied in the field of enzyme immobilization. Results of these studies have demonstrated that silk fibroin offers an important platform for covalent and noncovalent immobilization of enzymes through serving as a stabilization matrix/support with high retention of the biological activity of the enzymes of interest. In the hope of providing suggestions for potential future research directions, this review has been written to briefly introduce and summarize key advances in silk fibroin-based materials for immobilization of both enzymes/biocatalysts (including alkaline phosphatase, β-glucosidase, glucose oxidase, lipase, urease, uricase, horseradish peroxidase, catalase, xanthine oxidase, tyrosinase, acetylcholinesterase, neutral protease, α-chymotrypsin, amylase, organophosphorus hydrolase, β-galactosidase, carbonic anhydrase, laccase, zymolyase, phenylalanine ammonia-lyase, thymidine kinase, and several others) and non-enzymatic catalysts (such as Au, Pd, Fe, α-Fe₂O₃, Fe₃O₄, TiO₂, Pt, ZnO, CuO, Cu₂O, Mn₃O₄, and MnO₂). Full article

In this study, Alpha amylase from Bacillus subtilis was immobilized by entrapment in Calcium Alginate beads (CA). To improve the properties of these beads, alginate was blended with Cellulosic Residue (CR) obtained from sorghumstarch extraction. The conditions of entrapment were optimized for a maximum immobilization yield (Y%) by mathematical statistics, where the 2³-full factorial design of experiments was used. The properties of calcium alginate beads were improved by comparing the activity of immobilized enzymes in the hydrolysis of starch. The activity of the immobilized enzyme by Calcium Alginate/Cellulosic Residue (CA/CR) was found to be higher than the Calcium Alginate method. Zn²⁺ and Cu²⁺ have inhibitory effects on both immobilized enzymes. The Bacillus subtilis immobilized in alginate can be reused for 7 cycles with 12.7 μmol of reduced sugars and 6 cycles for the entrapped enzyme in CA/CR with 30 μmol of reduced sugars. Full article

Two commercially available enzymes, Dextrozyme (α-amylase) and Esperase (protease), were covalently immobilized on non-woven electrospun poly(styrene-co-maleic anhydride) nanofiber mats with partial retention of their catalytic activity. Immobilization was achieved for the enzymes on their own as well as in different combinations with an additional enzyme, β-galactosidase, on the same non-woven nanofiber mat. This experiment yielded a universal method for immobilizing different combinations of enzymes with nanofibrous mats containing maleic anhydride (MAnh) residues in the polymer backbone. Full article

The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents (polyethyleneimine or dodecyl aldehyde), co-aggregation with protein co-feeders (bovine serum albumin and/or soy protein), use of silica magnetic nanoparticles functionalized with amino groups (SMNPs) as separation aid, and starch as pore-making agent. The combination of enzyme surface modification with dodecyl aldehyde, co-aggregation with SMNPs and soy protein, in the presence of 0.8% starch (followed by hydrolysis of the starch with α-amylase), yielded CLEAs expressing high activity (immobilization yield around 100% and recovered activity around 80%), high effectiveness factor (approximately 65% of the equivalent free enzyme activity) and high stability at 40 °C and pH 8.0, i.e., PPL CLEAs co-aggregated with SMNPs/bovine serum albumin or SMNPs/soy protein retained 80% and 50% activity after 10 h incubation, respectively, while free PPL was fully inactivated after 2 h. Besides, highly porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches. Full article

Starch is promising candidate material for enhancing the catalytic activity of α-amylase during the crosslinking process. To help meet industrial needs, here we tested the influence of bovine serum albumin (BSA) and starch on the performance of crosslinked α-amylase aggregates (CLEA), α-amylase-prepared as CLEA with starch (CLEA-S), and BSA (CLEA-BSA). Our results showed that the activities of CLEA, CLEA-S, and CLEA-BSA were 1.1-, 1.0-, and 0.74-fold higher than the free α-amylase, respectively. The stability of the immobilized enzyme slightly changed. After immobilization, the enzyme increased its pH and temperature ranges with the optimal pH values of 5.5, 7.5, 5.5, respectively for CLEA, CLEA-S, and CLEA-BSA, and an upper temperature limit of 50 °C for all three immobilized forms. Among the three immobilized forms, the CLEA-S was the most thermostable, losing only 3% of its initial activity during 390 min incubation at 50 °C. Our microscopic observations of CLEA-S showed that porous structures were formed and such structures could help substance diffusion. In addition, there was excellent affinity between CLEA-S and the substrate. The results suggest that CLEA-S have great potential for industrial application, including for use in starch-based alcohol fermentation. Full article