Novel molecular hybrids of EGCG and quinoxaline: Potent multi-targeting antidiabetic agents that inhibit α-glucosidase, α-amylase, and oxidative stress.

Diabetes mellitus is a multifactorial disease and its effective therapy often demands several drugs with different modes of action. Herein, we report a rational design and synthesis of multi-targeting novel molecular hybrids comprised of EGCG and quinoxaline derivatives that can effectively inhibit α-glucosidase, α-amylase as well as control oxidative stress by scavenging ROS. The hybrids showed superior inhibition of α-glucosidase along with similar α-amylase inhibition as compared to standard drug, acarbose. Most potent compound, 15c showed an IC50 of 0.50 µM (IC50 of acarbose 190 µM) against α-glucosidase. Kinetics studies with 15c revealed a competitive inhibition against α-glucosidase. Binding affinity of 15c (-9.5 kcal/mol) towards α-glucosidase was significantly higher than acarbose (-7.7 kcal/mol). 15c exhibited remarkably high antioxidant activity (IC50 = 18.84 µM), much better than vitamin C (IC50 = 33.04 µM). Of note, acarbose shows no antioxidant activity. Furthermore, α-amylase activity was effectively inhibited by 15c with an IC50 value of 16.35 µM. No cytotoxicity was observed for 15c (up to 40 µM) in MCF-7 cells. Taken together, we report a series of multi-targeting molecular hybrids capable of inhibiting carbohydrate hydrolysing enzymes as well as reducing oxidative stress, thus representing an advancement towards effective and novel therapeutic approaches for diabetes.

Biochemical exploration of family GH119 reveals a single α-amylase specificity and confirms shared catalytic machinery with GH57 enzymes.

While hundreds of starch- and glycogen-degrading enzymes have been characterized experimentally in historical families such as GH13, GH14, GH15, GH57 and GH126 of the CAZy database (www.cazy.org), the α-amylase from Bacillus circulans is the only enzyme that has been characterized in family GH119. Since glycosidase families have been shown to often group enzymes with different substrates or products, a single characterized enzyme in a family is insufficient to extrapolate enzyme function based solely on sequence similarity. Here we report the rational exploration of family GH119 through the biochemical characterization of five GH119 members. All enzymes shared single α-amylase specificity but display distinct product profile. We also report the first kinetic constants in family GH119 and the first experimental validation of previously predicted catalytic residues in family GH119, confirming that families GH119 and GH57 can be grouped in the novel clan GH-T of the CAZy database.

Inhibition of α-amylase digestion by a Lonicera caerulea berry polyphenol starch complex revealed via multi-spectroscopic and molecular dynamics analyses.

Polyphenol-starch complexes exhibit synergistic and beneficial effects on both polyphenols and resistant starches. This study evaluates the inhibitory effects and mechanisms of α-amylase on a Lonicera caerulea berry polyphenol-wheat starch (LPWS) complex following high hydrostatic pressure treatments of 400 MPa for 30 min and 600 MPa for 30 min. The IC50 values for α-amylase inhibition by the complex were 3.61 ± 0.10 mg/mL and 3.42 ± 0.08 mg/mL at a 10 % (w/w) polyphenol content. This interaction was further supported by Fourier-transform infrared spectroscopy and circular dichroism, which confirmed that the alpha helix component of the secondary structure of α-amylase was reduced due to the complex. Multifluorescence spectroscopy revealed that the complex induces changes in the microenvironment of fluorophores surrounding the α-amylase active site. Molecular dynamics simulations and molecular docking revealed that the active site of amylose within the complex becomes enveloped in polyphenol clusters. This wrapping effect reduced the hydrogen bonds between amylose and α-amylase, decreasing from 16 groups to just one group. In summary, the LPWS complex represents a low-digestible carbohydrate food source, thus laying the groundwork for the research and development of functional foods aimed at postprandial hypoglycemic effects.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Assessment of the Anti-Breast Cancer Effects of Urolithin with Molecular Docking Studies in the In Vitro Condition: Introducing a Novel Chemotherapeutic Drug.

A lot of research has been done on using natural items as diabetes treatment. The molecular docking study was conducted to evaluate the inhibitory activities of urolithin A against α-amylase, α-glucosidase, and aldose reductase. The molecular docking calculations indicated the probable interactions and the characteristics of these contacts at an atomic level. The results of the docking calculations showed the docking score of urolithin A against α-amylase was -5.169 kcal/mol. This value for α-glucosidase and aldose reductase was -3.657 kcal/mol and -7.635 kcal/mol, respectively. In general, the outcomes of the docking calculations revealed that urolithin A can construct several hydrogen bonds and hydrophobic contacts with the assessed enzymes and reduces their activities considerably. The properties of urolithin against common human breast cancer cell lines, i.e., SkBr3, MDA-MB-231, MCF-7, Hs578T, Evsa-T, BT-549, AU565 and 600MPE were evaluated. The IC50 of the urolithin was 400, 443, 392, 418, 397, 530, 566 and 551 against SkBr3, MDA-MB-231, MCF-7, Hs578T, Evsa-T, BT-549, AU565 and 600MPE, respectively. After doing the clinical trial studies, the recent molecule may be used as an anti-breast cancer supplement in humans. IC50 values of urolithin A on α-amylase, α-glucosidase, and aldose reductase enzymes were obtained at 16.14, 1.06 and 98.73 µM, respectively.

Structural engineering and truncation of α-amylase from the hyperthermophilic archaeon Methanocaldococcus jannaschii.

Alpha amylases catalyse the hydrolysis of α-1, 4-glycosidic bonds in starch, yielding glucose, maltose, dextrin, and short oligosaccharides, vital to various industrial processes. Structural and functional insights on α-amylase from Methanocaldococcus jannaschii were computationally explored to evaluate a catalytic domain and its fusion with a small ubiquitin-like modifier (SUMO). The recombinant proteins' production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene (MjAFG), catalytic domain (MjAD) and fusion enzymes (S-MjAD) were thoroughly analysed in this comparative study. The MjAD and S-MjAD showed 2-fold and 2.5-fold higher specific activities (µmol min-1 mg -1) than MjAFG at 95 °C at pH 6.0. Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the MjAFG enzyme. The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. The chimeric fusion of amylases with thermostable α-glucosidases makes it a potential candidate for the starch degrading processes.

Purification and biochemical characterization of α- amylase from Aspergillus tamarii MTCC5152.

The purification and biochemical characterization of the extracellular alpha amylase from A.tamarii MTCC5152 were studied. The combined use of ion exchange and gel filtration chromatographic methods were used for purification studies. The specific activity was significantly increased (33 fold) and 19.41 fold purification of the enzyme α-amylase with 24% yield was achieved. The enzyme had an optimal pH of 6.5 and exhibited its highest activity at 55 °C. It is active over a wide range of pH 5-7 at room temperature. The enzyme is relatively stable in the temperature range of 25-35 °C for a period of 4 h hence, more suitable for industrial applications. Km and Vmax value of the enzyme was to be 5.882 mg/mL and 0.803 mg/mL/min respectively using starch as the substrate. The purified protein showed a single band on native and SDS PAGE and the molecular weight was found to be 31 kDa. Starch zymogram also revealed one clear zone of amylolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The characterization studies showed that the enzyme activity is inhibited by Ca2+, Mn2+, Hg2+, Fe2+.

A starch-binding domain of α-amylase (AmyPG) disrupts the structure of raw starch.

Most raw starch-digesting enzymes possess at least one non-catalytic starch-binding domain (SBD), which enhances enzymatic hydrolysis of insoluble starch granules. Previous studies of SBD-starch interaction mainly focus on binding affinity for substrates, while the mechanism involved disruption of starch granules remains partially understood. Raw starch-digesting α-amylases AmyPG and AmyP were from Photobacterium gaetbulicola and an uncultured marine bacterium, respectively. Here, comparative studies on the two α-amylases and their SBDs (SBDPG and SBDAmyP) with high sequence identity were carried out. The degradation capacity of AmyPG towards raw starch was approximately 2-fold higher than that of AmyP, which was due to the stronger disruptive ability of SBDPG rather than the binding ability. Two non-binding amino acids (K626, T618) of SBDPG that specifically support the disruptive ability were first identified using affinity gel electrophoresis, amylose­iodine absorbance spectra, and differential scanning calorimetry. The mutants SBDPG-K626A and SBDPG-T618A exhibited stronger disruptive ability, while the corresponding mutants of AmyPG enhanced the final hydrolysis degree of raw starch. The results confirmed that the disruptive ability of SBD can independently affect raw starch hydrolysis. This advancement in the functional characterization of SBDs contributes to a better understanding of enzyme-starch granule interactions, pushing forward designs of raw starch-digesting enzymes.

Finding structural requirements of structurally diverse α-glucosidase and α-amylase inhibitors through validated and predictive 2D-QSAR and 3D-QSAR analyses.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemic state. The α-glucosidase and α-amylase are considered two major targets for the management of Type 2 DM due to their ability of metabolizing carbohydrates into simpler sugars. In the current study, cheminformatics analyses were performed to develop validated and predictive models with a dataset of 187 α-glucosidase and α-amylase dual inhibitors. Separate linear, interpretable and statistically robust 2D-QSAR models were constructed with datasets containing the activities of α-glucosidase and α-amylase inhibitors with an aim to explain the crucial structural and physicochemical attributes responsible for higher activity towards these targets. Consequently, some descriptors of the models pointed out the importance of specific structural moieties responsible for the higher activities for these targets and on the other hand, properties such as ionization potential and mass of the compounds as well as number of hydrogen bond donors in molecules were found to be crucial in determining the binding potentials of the dataset compounds. Statistically significant 3D-QSAR models were developed with both α-glucosidase and α-amylase inhibition datapoints to estimate the importance of 3D electrostatic and steric fields for improved potentials towards these two targets. Molecular docking performed with selected compounds with homology model of α-glucosidase and X-ray crystal structure of α-amylase largely supported the interpretations obtained from the cheminformatic analyses. The current investigation should serve as important guidelines for the design of future α-glucosidase and α-amylase inhibitors. Besides, the current investigation is entirely performed by using non-commercial open-access tools to ensure easy accessibility and reproducibility of the investigation which may help researchers throughout the world to work more on drug design and discovery.

Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement.

α-Amylase (EC 3.2.1.1) from Geobacillus stearothermophilus (generally recognized as safe) exhibited thermal inactivation, hampering its further application in starch-based industries. To address this, we performed structural analyses based on molecular dynamics targeting the flexible regions of α-amylase. Subsequently, we rationally designed a thermostable mutant, AmyS1, by introducing disulfide bonds to stabilize the flexible regions. AmyS1 showed excellent thermostability without any stability-activity trade-off, giving a 40-fold longer T1/2 (1359 min) at 90 °C. Thermostability mechanism analysis revealed that the introduction of disulfide bonds in AmyS1 refined weak spots and reconfigured the protein's force network. Moreover, AmyS1 exhibited improved pH compatibility and enhanced corn starch liquefaction at 100 °C with a 5.1-fold increased product concentration. Baking tests confirmed that AmyS1 enhanced bread quality and extended the shelf life. Therefore, mutant AmyS1 is a robust candidate for the starch-based industry.

Challenges and prospects of microbial α-amylases for industrial application: a review.

α-Amylases are essential biocatalysts representing a billion-dollar market with significant long-term global demand. They have varied applications ranging from detergent, textile, and food sectors such as bakery to, more recently, biofuel industries. Microbial α-amylases have distinct advantages over their plant and animal counterparts owing to generally good activities and better stability at temperature and pH extremes. With the scope of applications expanding, the need for new and improved α-amylases is ever-growing. However, scaling up microbial α-amylase technology from the laboratory to industry for practical applications is impeded by several issues, ranging from mass transfer limitations, low enzyme yields, and energy-intensive product recovery that adds to high production costs. This review highlights the major challenges and prospects for the production of microbial α-amylases, considering the various avenues of industrial bioprocessing such as culture-independent approaches, nutrient optimization, bioreactor operations with design improvements, and product down-streaming approaches towards developing efficient α-amylases with high activity and recyclability. Since the sequence and structure of the enzyme play a crucial role in modulating its functional properties, we have also tried to analyze the structural composition of microbial α-amylase as a guide to its thermodynamic properties to identify the areas that can be targeted for enhancing the catalytic activity and thermostability of the enzyme through varied immobilization or selective enzyme engineering approaches. Also, the utilization of inexpensive and renewable substrates for enzyme production to isolate α-amylases with non-conventional applications has been briefly discussed.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Computational Insights into the Selecting Mechanism of α-Amylase Immobilized on Cellulose Nanocrystals: Unveiling the Potential of α-Amylases Immobilized for Efficient Poultry Feed Hydrolysis.

The selection of an appropriate amylase for hydrolysis poultry feed is crucial for achieving improved digestibility and high-quality feed. Cellulose nanocrystals (CNCs), which are known for their high surface area, provide an excellent platform for enzyme immobilization. Immobilization greatly enhances the operational stability of α-amylases and the efficiency of starch bioconversion in poultry feeds. In this study, we immobilized two metagenome-derived α-amylases, PersiAmy2 and PersiAmy3, on CNCs and employed computational methods to characterize and compare the degradation efficiencies of these enzymes for poultry feed hydrolysis. Experimental in vitro bioconversion assessments were performed to validate the computational outcomes. Molecular docking studies revealed the superior hydrolysis performance of PersiAmy3, which displayed stronger electrostatic interactions with CNCs. Experimental characterization demonstrated the improved performance of both α-amylases after immobilization at high temperatures (80 °C). A similar trend was observed under alkaline conditions, with α-amylase activity reaching 88% within a pH range of 8.0 to 9.0. Both immobilized α-amylases exhibited halotolerance at NaCl concentrations up to 3 M and retained over 50% of their initial activity after 13 use cycles. Notably, PersiAmy3 displayed more remarkable improvements than PersiAmy2 following immobilization, including a significant increase in activity from 65 to 80.73% at 80 °C, an increase in activity to 156.48% at a high salinity of 3 M NaCl, and a longer half-life, indicating greater thermal stability within the range of 60 to 80 °C. These findings were substantiated by the in vitro hydrolysis of poultry feed, where PersiAmy3 generated 53.53 g/L reducing sugars. This comprehensive comparison underscores the utility of computational methods as a faster and more efficient approach for selecting optimal enzymes for poultry feed hydrolysis, thereby providing valuable insights into enhancing feed digestibility and quality.

Chemical profile of Roselle extract and its inhibitory activities on three digestive enzymes in vitro and in vivo.

Roselle is rich in an extensive diversity of beneficial substances, including phenolic acids, amino acids, anthocyanins, vitamins, and flavonoids. Herein, the chemical constituents in Roselle extract (RE) were identified by UPLC-DAD-QTOF-MS. Besides, its inhibitory effects on three digestive enzymes, i.e. α-amylase, α-glucosidase, and pancreatic lipase, were investigated in both in vitro and in vivo. Thirty-three constituents including hibiscus acid, 18 phenolic acids, 2 anthocyanins and 12 flavonoids were identified. The anthocyanins content in RE was 21.44 ± 0.68 %, while the contents of chlorogenic acids, rutin and quercetin were 17.76 ± 2.28 %, 0.31 ± 0.01 % and 0.32 ± 0.01 %, respectively. RE inhibited pancreatic lipase in a non-competitive way with an IC50 value of 0.84 mg/mL. Besides, it demonstrated a mixed-type inhibition on both α-glucosidase and α-amylase with IC50 values of 0.59 mg/mL and 1.93 mg/mL, respectively. Fluorescence quenching assays confirmed the binding of RE to the enzyme proteins. Furthermore, rats pre-treated with RE at doses of 50 and 100 mg/kg body weight (bwt) exhibited significant reductions in fat absorption and improvements in fat excretion through feces. Additionally, the in vivo study revealed that RE was effective in suppressing the increase of blood glucose after starch consumption, while its effects on maltose and sucrose consumption were relatively weak.

Red rice starch modification - Combination of the non-thermal method with a pulsed electric field (PEF) and enzymatic method using α-amylase.

The objective of this study was to investigate the dual modification of red rice starch using pulsed electric field (PEF) and α-amylase, focusing on morpho-structural, thermal, and viscoamylographic properties. Native starch (Control) underwent various treatments: PEF at 30 kV cm-1 (PEF30), α-amylase at 9.0 U mg-1 (AA0), and a combination of both (PEF30 + α and α + PEF30). The PEF30 + α treatment exhibited the highest degree of digestion (10.66 %) and resulted in morphological changes in the starch granules, which became elongated and curved, with an increased average diameter of 50.49 µm compared to the control. The starch was classified as type A, with a maximum reduction in crystallinity of up to 21.17 % for PEF30. The deconvolution of FT-IR bands indicated an increase in the double helix degree (DDH) for PEF30 and AA0, while the degree of order (DO) was reduced for PEF30, AA0, and PEF30 + α. DSC analysis revealed significant modifications in gelatinization temperatures, particularly for PEF30, and these changes were supported by a reduction in gelatinization enthalpy (ΔH) of up to 28.05 % for AA0. These findings indicate that both individual and combined treatments promote a decrease in starch gelatinization and facilitate the process, requiring less energy. Differences were observed between the formulations subjected to single and alternating dual treatments, highlighting the influence of the order of PEF application on the structural characteristics of starch, especially when applied before the enzymatic treatment (PEF + α). Regarding the viscoamylographic parameters, it was observed that AA0 presented higher values than the control, indicating that α-amylase enhances the firmness of the paste. The double modification with PEF + α was more effective in reducing syneresis and starch retrogradation, leading to improvements in paste properties. This study provided significant insights into the modification of red rice starch using an efficient and environmentally friendly approach.

A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen.

BACKGROUND AND AIM: The purpose of the current study is to isolate a heavily amylase-producing bacteria of the genus Bacillus from soil samples, optimize the production of the enzyme, purify it, and evaluate its activity against biofilm-producing bacteria. A total of 12 soil samples were collected and screened for promising Bacillus species with good amylolytic activity. Isolation was done by serial dilution and plating technique and amylolytic activity was determined by starch agar plate method. Among the 12 Bacillus isolates recovered from soil samples, 7 showed positive α-amylase production. The best isolate that recorded the greatest amylolytic activity was selected for further studies. This isolate was identified by 16S rRNA sequencing as Bacillus cereus and registered under gene bank accession number OP811897. Furthermore, the α-amylase enzyme was produced by a submerged fermentation technique using best production media and partially purified by ammonium sulfate and chilled ethanol and molecular weight had been determined by SDS-PAGE gel electrophoresis. The production of α-amylase was optimized experimentally by one-factor at a time protocol and statistically by Plackett-Burman design as well as RSM CCD design. Data obtained from OFAT and CCD revealed that α-amylase activities were 1.5- and twofold respectively higher as compared to un-optimized conditions. The most significant factors had been identified and optimized by CCD design. RESULTS: Among the eleven independent variables tested by PBD, glucose, peptone, (NH4)2SO4, and Mg SO4 were the most significant parameters for α-amylase production with an actual yield of 250U/ml. The best physical parameters affecting the enzyme production were incubation time at 35 °C, and pH 5.5 for 48 h. The partially purified enzyme with 60% ammonium sulphate saturation with 1.38- fold purification showed good stability characteristics at a storage temperature of 4 °C and pH up to 8.5 for 21 days. Antibiofilm activity of purified α-amylase was determined against Pseudomonas aeruginosa (ATCC 35659) by spectrophotometric analysis and CLSM microscopic analysis. Results demonstrated biofilm inhibition by 84% of the formed Pseudomonas biofilm using a microtiter plate assay and thickness inhibition activity by 83% with live/Dead cells percentage of 17%/83% using CLSM protocol. CONCLUSIONS: A highly stable purified α-amylase from B. cereus showed promising antibiofilm activity against one of the clinically important biofilm-forming MDR organisms that could be used as a cost-effective tool in pharmaceutical industries.

Regulation of multi-scale structures and retrogradation property of A- and B-type wheat starch granules with maltogenic α-amylase.

Maltogenic α-amylase (MA) is widely used to modify starch for improving properties. In this work, A- and B-type starches were separated from wheat completely and submitted to MA hydrolysis. Firstly, research in morphological features (SEM) suggested that MA treatment did not destroy the granule integrity. Next, crystalline features from XRD and SAXS assay showed that MA treatment did not change the crystal form, but deceased crystalline sheet (dc) and amorphous sheet (da) thickness in both modified starches. And amorphous sheet was more severely destroyed at higher MA dosage than crystalline sheet. Then changes in molecule structure (HPAEC) showed that MA mainly acted on sides chains with degree of polymerization 12-24 in amylopectin for both starches, resulting in a reduce in peak, trough, and final viscosity obtained from RVA assay. At last, RVA and DSC assay showed that the short-term and long-term retrogradation were retarded in both modified starches. This range of techniques covered changes in multi-scale structures and retrogradation property resulted from MA treatment on both starches, which provided references for studying the changes in structures and properties of MA modified starch granules and provided an important method for retarding retrogradation of starchy foods without gelatinization processing.

Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications.

α-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60-80 °C and is active and stable over a wide pH range (4.0-9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry.

Preparation of water-insoluble lignin nanoparticles by deep eutectic solvent and its application as a versatile and biocompatible support for the immobilization of α-amylase.

As one of the most abundant biopolymers, lignin is a widely available resource. However, its potential largely remains untapped, with most of it ending up as waste from industries like paper production, pulp processing, and bio-refining. The research undertaken in this study focused on the extraction of lignin from agroforestry waste using a deep eutectic solvent (DES) as a carrier for α-amylase immobilization, resulting in high stability and reusability. Several techniques, including Nuclear Magnetic Resonance (NMR), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray Spectroscopy (EDS), and the Brunauer-Emmett-Teller (BET) method were employed to examine the structure and morphology of both the extracted lignin and the immobilized enzyme. The temperature used to recover lignin by DES would affect immobilization efficiency and enzyme loading by influencing its specific surface area, pore size, and volume distribution. Investigations using Nuclear Overhauser Effect Spectroscopy (NOESY) uncovered that the hydroxyl groups in G, H, and S units and the ß-O-4 structure of lignin primarily serve as binding sites for enzyme molecules. Immobilized α-amylase demonstrated a higher pH and thermal stability level, with an optimal pH of 7.0 and temperature of 100 °C, compared to the free enzyme, which exhibited optimal activity at a pH of 6.5 and temperature of 90 °C. Importantly, immobilized α-amylase retained >80 % of its initial activity even after 28 days at room temperature, and it maintained 70 % of its activity after being reused 12 times. These findings strongly suggest that lignin derived from agroforestry residues holds promising potential as a future versatile immobilization material, a prospect integral to society's sustainable development.

Exploration of computational approaches to predict the structural features and recent trends in α-amylase production for industrial applications.

Amylases are biologically active enzymes that can hydrolyze starch to produce dextrin, glucose, maltose, and oligosaccharides. The amylases contribute approximately 30% to the global industrial enzyme market. The globally produced amylases are widely used in textile, biofuel, starch processing, food, bioremediation of environmental pollutants, pulp, and paper, clinical, and fermentation industries. The purpose of this review article is to summarize recent trends and aspects of α-amylases, classification, microbial production sources, biosynthesis and production methods, and its broad-spectrum applications for industrial purposes, which will depict the latest trends in α-amylases production. In the present article, we have comprehensively compared the biodiversity of α-amylases in different model organisms ranging from archaea to eukaryotes using in silico structural analysis tools. The detailed comparative analysis: regarding their structure, function, cofactor, signal peptide, and catalytic domain along with their catalytic residues of α-amylases in 16 model organisms were discussed in this paper. The comparative studies on alpha (α) amylases' secondary and tertiary structures, multiple sequence alignment, transmembrane helices, physiochemical properties, and their phylogenetic analysis in model organisms were briefly studied. This review has documented the recent trends and future perspectives of industrially important novel thermophilic α-amylases. In conclusion, this review sheds light on the current understanding and prospects of α-amylase research, highlighting its importance as a versatile enzyme with numerous applications and emphasizing the need for further exploration and innovation in this field.