Phytochemical composition, simulated digestive bioaccessibility and cytotoxicity of Ficus auriculata Lour. fruits: In vitro and in silico insights.

Ficus auriculata Lour. (Moraceae) is an underutilized wild edible fruit widely consumed for its nutritional properties. The present study aimed to determine the phytochemical composition and in vitro antioxidant, enzyme inhibitory, anti-inflammatory and anti-cancerous properties of the F. auriculata fruit extracts through in vitro digestion (oral, gastric and intestinal phases). The extracts were obtained by hot extraction and cold maceration methods using aqueous and methanolic solvents. Major phytoconstituents identified through LC-MS was subjected to molecular docking against the target proteins. The elemental analysis shows the presence of major elements; high levels of total phenolics (124.61 ± 0.82 mg gallic acid equivalent/g), flavonoids (76.38 ± 0.82 mg quercetin equivalent/g), vitamin E (32.48 ± 0.09 mg alpha-tocopherol equivalent/g), and carbohydrate (34.59 ± 0.45 mg glucose equivalent/g) in hot extracted methanolic undigested extract (HEM UD) and high level of total protein (124.71 ± 0.34 mg bovine serum albumin equivalent/g) in cold extracted methanolic undigested fruit extract were found. HEM UD showed high antioxidant activity in 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,2-diphenyl-1-picryl-hydrazyl, and superoxide radical scavenging assays with IC50 of 53.30 ± 0.57, 80.69 ± 0.12, and 65.47 ± 1.13 µg/mL, respectively. The HEM UD extract also potentially inhibited the enzyme activity of α-amylase, α-glucosidase, tyrosinase, and protein denaturation (IC50 of 67.76 ± 1.22, 83.18 ± 1.23, 87.24 ± 1.15, and 65.76 ± 0.60 µg/mL). The most potent extract (HEM UD) was studied for its anticancer effects by MTT assay against the MCF-7 and HeLa cell lines and showed the IC50 of 89.80 ± 0.56 and 60.76 ± 0.04 µg/mL, respectively. The LC-MS analysis elucidated ten phytoconstituents. Based on the molecular docking study, querciturone could potentially be an effective constituent in treating diabetes and inflammation-related issues. The findings indicated the ability of F. auriculata fruits as a promising functional food.

Exploring the influence of different enzymes on soy hull polysaccharide emulsion stabilization: A study on interfacial behavior and structural changes.

The interfacial behavior of soy hull polysaccharide (SHP) at the oil-water interface and the stabilization mechanism of high internal phase emulsion (HIPE) with three enzymes (α-amylase, trypsin and papain) were investigated. The diameter of the α-amylase-treated emulsion was the minimum at 40 min, indicating that the carbohydrate portions of SHP form a thick layer on the surface of the droplet to prevent aggregation. Moreover, Raman spectroscopy revealed significantly higher levels of disordered content of SHP emulsion treated with α-amylase at 60 min, potentially affecting the directional movement of SHP molecules in the emulsion. Conversely, the content of ß-sheet and ß-turn was lower than trypsin and papain, possibly due to ion-dipole interaction between the polar group residues within SHP and ions, or protonation with H+.

Insights into Catechin-Copper Complex Structure and Biologic Activity Modulation.

Compounds of natural origin found in varying quantities in plant-based products constitute a highly significant category, possessing structural significance as well as the capacity to regulate oxidative processes. The activity of these compounds may be modulated by the composition of the biological environment in which they operate, the pH of the environment, or the presence of metal cations in plants or plant extracts. A successful complexation reaction was mainly confirmed by FT-IR, observing the shift from the original transmittance of catechin bonds, especially O-H ones. This work shows the synthetic methodology and the optimization process that took place to synthesize a catechin-copper complex, which demonstrated antioxidant activity. It was tested for iron chelation ability, hydroxyl radicals, and the inhibition of lipoxygenase (15-LOX). An antidiabetic assay was performed by determining the inhibition of alpha-amylase and alpha-glucosidase, finding that the synthesized complex had similar inhibitory potential as pure catechin. The antibacterial tests showed results against Staphylococcus aureus and the antifungal properties of the complex against Candida albicans.

Crystals of taka-amylase A, a cornerstone of protein chemistry in Japan.

In 1935, Shiro Akabori began research on the preparation of taka-amylase A with a purity suitable for chemical research, with the intention of elucidating the chemical nature of the enzyme. He succeeded in developing a method to efficiently obtain crystallized taka-amylase A from Aspergillus oryzae. Using crystallized taka-amylase A as the starting material, a series of studies were conducted to determine its amino acid composition and sequence, sugar chain structure, and three-dimensional structure. Based on these results, the molecular structure and catalytic mechanism of taka-amylase A were elucidated. The scientific achievements from research on taka-amylase A significantly enhanced Japan's capabilities in protein research, represented by the fact that taka-amylase A was the first amylase in the world for which both chemical and crystallographic structures were elucidated.

Extraction Method Effects on Structural Properties and Functional Characteristics of Dietary Fiber Extracted from Ginseng Residue.

In this research, the dietary fibers (DFs) from ginseng residue were extracted by employing three different extraction methods (alkaline: AL, acidic: AC, enzymatic: EN). The extracted DFs were characterized in terms of their structural and functional properties. The results clearly showed that, regardless of the extraction methods, all DF samples exhibited representative infrared spectral features. The DF extracted by AC (citric acid) had more porous structures with a looser configuration, in conjunction with high apparent viscosity, whereas the DF extracted by EN (α-amylase and protease) exhibited higher thermal stability. Moreover, the monosaccharide composition of the DF samples was significantly influenced by the extraction method type. The DF from ginseng residue extracted by AC had the highest functional properties, such as water holding capacity (8.16 g/g), oil holding capacity (3.99 g/g), water swelling capacity (8.13 g/g), cholesterol-absorption capacity (12.85 mg/g), bile acid absorption capacity (91.51 mg/g), nitrite ion absorption capacity (124.38 ug/g at pH 2.0), glucose absorption capacity (52.67 mg/g at 150 mmol/L), as compared to those of DF extracted by the EN and AL (sodium hydroxide) methods. Hence, ginseng residue-derived DF extracted by the AC method may be potentially employed in the preparation of functional food ingredients.

Chia gum-gelatin-based encapsulation of chia sprouts phenolic compounds enhanced storage stability, bioavailability, antioxidant, antidiabetic, and antibacterial properties.

Chia seeds are currently gaining popularity as functional and healthy foods. The developed chia 7-day sprout phenolic extract (CSP) is an abundant supply of highly concentrated antioxidant phenolic compounds with health-promoting and antibacterial properties. The easy destruction against different environmental changes and low bioavailability of these phenolic compounds are the main limitations of their applications/utilization. This study aims to microencapsulate the phenolic compounds of developed CSP for use as valuable functional food additives. Three microcapsules were prepared using coating materials, chia gum (CG), gelatin (G), and their mixture (CG/G) via the freeze-drying technique. The prepared CG-, CG/G-, and G-microcapsules demonstrated high encapsulation efficiency percentages of 97.0, 98.1, and 94.5%, respectively. They retained most of the CSP-phenolics (91.4-97.2%) and increased total antioxidant activity (108-127.1%). The prepared microcapsules released more CSP-phenolic compounds into the simulated intestinal stage (70-82%) than the gastric stage (15-24%), demonstrating that the coating materials enhance protection during the gastric stage. The produced microcapsules exhibited higher storage stability at 40 °C for 60 days than the non-capsulated CSP, indicating that the encapsulation provided enhanced stability. The prepared microcapsules microstructures showed uniform, smoother surfaces, and hidden micropores compared to their coating material microstructures. In addition, the connection between the functional groups of coating materials and CSP-phenolic compounds was demonstrated by FTIR analysis. The prepared CG-, CG/G-, and G-microcapsules can perfectly inhibit the α-amylase and α-glucosidase activities by 65, 68, 60 and 74, 78, and 70%, respectively, compared to CSP (54, and 66%). The three prepared microcapsules displayed better antibacterial with low MBC values (0.36-0.68 mg ml-1) compared to CSP (0.53-0.74 mg ml-1). The prepared CSP microcapsules can be incorporated into various food products to enhance their antioxidant, antidiabetic, and antibacterial properties.

Recent developments in enzymatic preparation, physicochemical properties, bioactivity, and application of resistant starch type III from staple food grains.

Resistant starch (RS) was classified into five types and referred to the starch that cannot be digested and absorbed by the small intestine of healthy human beings. Among them, RS3 has received a lot of attention from researchers because of its good functional properties and greater application prospects. Meanwhile, the enzymatic method is widely used in the preparation of RS3 because of its high efficiency and environmental protection. α-Amylase and pullulanase as the main enzymes can effectively improve the yield of RS3. The physical properties of RS3 have an excellent potential for application in improving food crispness, texture and producing low glycemic index (GI) foods. It is more valuable because it has biological activities such as inducing apoptosis in tumor cells, lowering intestinal pH, and regulating blood glucose, etc. This paper summarized the current research progress of RS3 from different staple food grains, including current applications of enzymes commonly used in the preparation of RS3, physical properties and biological activities of RS3, and the application of RS3 in different areas to provide a theoretical basis for future research on RS3 as well as further development and applications based on the market requirement.

Exploring Acyl Thiotriazinoindole Based Pharmacophores: Design, Synthesis, and SAR Studies with Molecular Docking and Biological Activity Profiling against Urease, α-amylase, α-glucosidase, Antimicrobial, and Antioxidant Targets.

A diminutive chemical library of acyl thiotriazinoindole (ATTI) based bioactive scaffolds was synthesized, instigated by taking the economical starting material Isatin, through a series of five steps. Isatin was first nitrated followed by the attachment of pentyl moiety via nucleophilic substitution reaction. The obtained compound was reacted with thiosemicarbazide to obtain thiosemicarbazone derivative, which was eventually cyclized using basic conditions in water as solvent. Finally, the reported series was obtained through reaction of nitrated thiotriazinoindole moiety with differently substituted phenacyl bromides. The synthesized compounds were characterized using NMR spectroscopy and elemental analysis. Finally, the synthesized motifs were scrutinized for their potential to impede urease, α-glucosidase, DPPH, and α-amylase. Compound 5 h with para cyano group manifested the most pivotal biological activity among all, displaying IC50 values of 29.7 ± 0.8, 20.5 ± 0.5 and 36.8 ± 3.9 µM against urease, α-glucosidase, and DPPH assay, respectively. Simultaneously, for α-amylase compound 5 g possessing a p-CH3 at phenyl ring unfolded as most active, with calculated IC50 values 90.3 ± 1.1 µM. The scaffolds were additionally gauged for their antifungal and antibacterial activity. Among the tested strains, 5d having bromo as substituent exhibited the most potent antibacterial activity, while it also demonstrated the highest potency against Aspergillus fumigatus. Other derivatives 5b, 5e, 5i, and 5j also exhibited dual inhibition against both antibacterial and antifungal strains. The interaction pattern of derivatives clearly displayed their SAR, and their docking scores were correlated with their IC50 values. In molecular docking studies, the importance of interactions like hydrogen bonding was further asserted. The electronic factors of various substituents engendered variety of interactions between the ligands and targets implying their importance in the structures of the synthesized heterocyclic scaffolds. To conclude, the synthesized compounds had satisfactory biological activity against various important targets. Further studies are therefore encouraged by attachment of different substitutions in the structure at various positions to enhance the activity of these compounds.

Isolation and Characterization of Novel Pueroside B Isomers and Other Bioactive Compounds from Pueraria lobata Roots: Structure Elucidation, α-Glucosidase, and α-Amylase Inhibition Studies.

Pueraria lobata (Willd.) Ohwi is a traditional medicinal herb that has been extensively used in Chinese medicine for various therapeutic purposes. In this study, twelve chemical constituents were isolated from the roots of P. lobata, comprising three puerosides (compounds 1-3), six alkaloids (compounds 4-9), and three additional compounds (compounds 10-12). Notably, compound 1 (4R-pueroside B) was identified as a novel compound. The structures of all compounds were elucidated using a range of spectroscopic techniques, including CD spectroscopy for the first-time determination of the absolute configurations of pueroside B isomers (compounds 1 and 2). Enzyme inhibition assays revealed that, with the exception of compound 2, all isolated compounds exhibited varying degrees of α-glucosidase and α-amylase inhibitory activity. Remarkably, compound 12 demonstrated IC50 values of 23.25 µM for α-glucosidase inhibition and 27.05 µM for α-amylase inhibition, which are superior to those of the positive control, acarbose (27.05 µM and 36.68 µM, respectively). Additionally, compound 11 exhibited inhibitory activity against α-glucosidase and α-amylase comparable to the positive control, acarbose. Molecular docking studies indicated that compound 12 interacts with the active sites of the enzymes via hydrogen bonds, van der Waals forces, and hydrophobic interactions, which likely contribute to their inhibitory effects. These findings suggest that the chemical constituents of P. lobata could be potential natural sources of α-amylase and α-glucosidase inhibitors, with compound 12 being particularly promising for further investigation.

Enzymatic hydrolysis method for development of low glycemic index rice flour from temperate grown rice (var. Jehlum): Numerical optimization, rheological and spectroscopic characteristics.

This study aimed at optimizing process protocols for development of low glycemic index (GI) rice flour (LGIRF) by employing enzymatic hydrolysis method using central composite rotatable design (CCRD). LGIRF was evaluated for pasting, farinographic, spectroscopic and microbiological attributes. Independent variables for optimization included concentrations of α-amylase (0.02-0.12 %), glucoamylase (0.02-0.24 %), as well as the incubation temperature (55-80°C). Resistant starch (RS), glycemic index (GI) and glycemic load (GL) were investigated as response variables. The optimum conditions for development of LGIRF with better quality were- α-amylase concentration of 0.040 %, glucoamylase concentration of 0.070 % and an incubation temperature of 60 °C. The results of mineral analysis revealed significantly (p < 0.05) lower levels of boron, potassium, zinc, phosphorus, magnesium, and manganese in LGIRF, while iron and copper were significantly higher. The viscosity profile as evident from pasting profile and farinographic characteristics of LGIRF were significantly (p < 0.05) lower than native rice flour. 1H NMR and 13C NMR spectroscopic studies showed an increase in flexible starch segments and a decrease in amorphous portion of starch LGIRF, along with chemical shift alterations in carbons 1 and 4. Free fatty acids and total plate count were significantly (p < 0.05) higher in LGIRF although was within limits.

Evaluation of the Antimicrobial Activity of Triple Enzyme-Embedded Organic-Inorganic Hybrid Nanoflowers (hNFs) in Comparison with Powerful Antimicrobial Agent Chitosan.

Organic-inorganic hybrid nanoflowers (hNFs) have high stability, reusability, low production cost, and overcome substrate/product inhibition. Antimicrobial activity of various hNFs has been reported to overcome environmental microbial contaminations and infections, which are considered major public health problems. α-amylase, protease, and lipase are the most common industrial enzymes exerting antimicrobial activity; therefore, we synthesized triple enzyme (α-amylase, protease, and lipase)-embedded hNFs by using pancreatin to evaluate their antimicrobial activity in comparison with one of the most potent antimicrobial polymer chitosan. The broad spectrum of the antimicrobial properties of chitosan is used in industrial products, including cosmetics, food, agriculture, pharmaceuticals, and textiles. SEM analysis, thermogravimetric analysis (TGA), and the degree of deacetylation (%DD) were performed for chitosan characterization, where SEM, FTIR, EDX, and XRD analyses were performed for the characterization of hNFs. The catalytic activity of pancreatin and hNFs was evaluated by measuring lipase, α-amylase, and protease enzyme activities at 37 °C. Antibacterial activities of hNFs, pancreatin, and chitosan were tested on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria, compared to the pancreatin and chitosan via agar and broth dilution methods. hNFs showed enhanced catalytic activity for protease, lipase, and α-amylase compared to pancreatin at different pH values (pH 8, 9). hNFs showed catalytic activity after being washed and reused up to six times, indicating their reusability and recoverability. hNFs showed significant antimicrobial activity, such as chitosan, Staphylococcus aureus, and Escherichia coli, compared to pancreatin. Our novel hNFs can be used to develop antimicrobial technologies to fight against environmental microbial contaminations and antibiotic resistance-driven environmental pathogens.

Synthesis, molecular docking study, MD simulation, ADMET, and drug likeness of new thiazolo[3,2-a]pyridine-6,8-dicarbonitrile derivatives as potential anti-diabetic agents.

There are numerous uses for the pharmacological effects of thiazolo-pyridine and its derivatives. The main objective of the study was to synthesis 10 novel derivatives of thiazolo[3,2-a] pyridine-6,8-dicarbonitrile with a 22-78% yield, with a focus on their potential anti-diabetic properties. We investigated the interactions between these compounds and the enzyme α-amylase through an in silico study involving molecular docking. According to the docking analysis results, the resulting compounds had advantageous inhibitory properties. With a docking score of -7.43 kcal/mol against the target protein, compound 4e performed best. The stability root-mean-square deviation (RMSD) showed that the complex stabilizes after 25 ns and with minor perturbation at 80. The RMSF values of the ligand-protein complex indicate that the following residues have interacted with compound 4e during the MD simulation: Trp58, Trp59, Tyr62, Gln63, His101, Val107, lle148, Asn152, Leu162, Thr163, Gly164, Leu165, Asp197, Ala198, Asp 236, Leu237, His299, Asp300, and His305. Moreover, the pharmacokinetic and drug-like properties of the synthesized derivatives of 2-arylamino-dihydroindeno[1,2-b] pyrrol-4(1H)-one suggest that they have the potential to be effective inhibitors of α-amylase and should be considered for further research. Nevertheless, it is crucial to ascertain the in vivo and in vitro effectiveness of these compounds through biochemical and structural investigations.

Covalent immobilization of α-amylase on hollow metal organic framework coated magnetic phase-change microcapsules for the improvement of its thermostability.

Exploring efficient immobilized carrier for α-Amylase (α-Amy) to enhance its thermostability has significant influence in starch related industry. Here, hollow ZIF-8 (HZIF-8) with amino groups coated magnetic phase change microcapsules (PCM) was designed for covalent immobilization of α-Amy. Magnetic PCM consisting n-docosane core and SiO2/Fe3O4 hybrid shell were firstly synthesized. Then, HZIF-8 shell with amino groups was coated and α-Amy was subsequently immobilized through covalent cross-linking strategy. The morphology, chemical structure and magnetic property of PCM@HZIF-8@α-Amy (PCMHA) were comprehensively characterized. Moreover, heat control property of PCMHA was studied with encapsulation efficiency and thermal energy-storage efficiency of 50.55 % and 50.59 %, respectively. Catalytic activity of immobilized α-Amy was fully investigated with Km and Vmax of 2.773 mg/mL and 1.853 µmol/mL·min, respectively. From reusability and storage stability study, immobilized α-Amy not only maintained rather high activity after 9 cycles' reuse, but also exhibited good activity under high salt ion condition after 7 days.

Polydopamine-functionalized polyethersulfone membrane: A paradigm advancement in the field of α-amylase stability and immobilization.

Biocatalytic membranes have great potential in various industrial sectors, with the immobilization of enzymes being a crucial stage. Immobilizing enzymes through covalent bonds is a complex and time-consuming process for large-scale applications. Polydopamine (PDA) offers a more sustainable and eco-friendly alternative for enzyme immobilization. Therefore, surface modification with polydopamine as mussel-inspired antifouling coatings has increased resistance to fouling. In this study, α-amylase enzyme was covalently bound to a bioactive PDA-coated polyethersulfone (PES) membrane surface using cyanuric chloride as a linker. The optimal activity of α-amylase enzyme immobilized on PES/PDA membrane was obtained at temperature and pH of 55°C and 6.5, respectively. The immobilized enzyme can be reused up to five reaction cycles with 55 % retention of initial activity. Besides, it maintained 60 % of its activity after being stored for five weeks at 4°C. Additionally, the immobilized enzyme demonstrated increased Michaelis constant and maximum velocity values during starch hydrolysis. The results of the biofouling experiment of various membranes in a dead-end cell demonstrated that the PES membrane's water flux increased from 6722.7 Lmh to 7560.2 Lmh after PDA modification. Although α-amylase immobilization reduced the flux to 7458.5 Lmh due to enhanced hydrophilicity, compared to unmodified membrane. The findings of this study demonstrated that the membrane produced through co-deposition exhibited superior hydrophilicity, enhanced coating stability, and strong antifouling properties, positioning it as a promising candidate for industrial applications.

Ultrasound and enzyme treatments on morphology, structures, and adsorption properties of cassava starch.

Porous starch materials are environmentally friendly and renewable and exhibit high adsorption performances. Ultrasound and compound enzyme (α-amylase and glucoamylase) treatments were applied to prepare modified cassava starch. The granules, crystal morphology, crystal structure, and molecular structure of starch were investigated. The hydrolysis degree, solubility, swelling, and adsorption properties of cassava starch were analyzed. After the cassava starch was modified by ultrasound and enzyme treatments, the granule size of the starch decreased, and the surfaces were eroded to form pits, grooves and cavity structure. The starch spherulites weakened or even disappeared. The functional groups of starch did not change significantly, but the degree of crystal order decreased. The double-helix structure was reduced, and the crystal structure was composed of A + V-type crystals, with a decrease in crystallinity. The gelatinization temperature and thermal degradation temperatures enhanced. The enzymatic hydrolysis degree and solubility of the modified cassava starch increased. The swelling degree decreased, and oil adsorption, water adsorption improved. MB adsorption behavior of modified cassava starch closely followed a pseudo-second-order kinetics model and the Langmuir isotherm equation. These findings could help to understand the relationship between the structure and properties of modified starch, and guide its application in the field of adsorption.

Improved physicochemical and functional properties of dietary fiber from matcha fermented by Trichoderma viride.

The low-grade matcha is rich in insoluble dietary fiber. Trichoderma viride was used to increase the soluble dietary fiber to improve its functional properties. The soluble dietary fiber content increased from 6.74% to 15.24%. Pectin, hemicellulose, maltose, d-xylose, and glucose contents increased by 63.35% and 11.54%, 2.18, 0.11, and 7.04 mg/g, respectively. Trichoderma viride fermentation disrupted the dense structure of insoluble dietary fiber, resulting in a honeycomb structure and improving crystallinity by 22.75%. These structural changes led to an improved cation exchange capacity from 1.69 to 4.22 mmol/g, an increase in the inhibitory effect of α-amylase from 47.38% to 72.04%, and a 2.13-fold in the ferrous ion scavenging ability, and the IC50 values of superoxide anion was reduced from 7.00 to 1.54 mg/mL, respectively. Therefore, Trichoderma viride fermentation is an excellent method for improving the quality of dietary fiber in matcha processing by-products.

Biological activities, Peptidomics and in silico analysis of low-fat Cheddar cheese after in vitro digestion: Impact of blending camel and bovine Milk.

Cheesemaking with camel milk (CM) presents unique challenges and additional health benefits. This study involved preparing low-fat Cheddar cheese (LFCC) by blending bovine milk (BM) with varying levels of CM. Control cheese was made exclusively with BM. After 180 days of ripening, LFCC samples underwent in vitro digestion to determine antioxidant capacities, α-amylase and α-glucosidase inhibition, and angiotensin-converting enzyme inhibition. The peptide profile of LFCC treatments was analyzed using liquid chromatography-quadrupole-time of flight-mass spectrometry. Antioxidant and biological activities were influenced by BM-CM blends and digestion. At days 120 and 180, the number of αs1-casein-derived peptides increased in all samples except for LFCC made with 15% CM. Generally, 88 peptides exhibited ACE inhibition activity after 120 days of ripening, increasing to 114 by day 180. These findings suggest that ripening time positively affects the health-promoting aspects of functional cheese products.

Quercetin slow-release system delays starch digestion via inhibiting transporters and enzymes.

This study investigates the potential of a quercetin-based emulsion system to moderate starch digestion and manage blood glucose levels, addressing the lack of in vivo research. By enhancing quercetin bioaccessibility and targeting release in the small intestine, the emulsion system demonstrates significant inhibition of starch digestion and glucose spikes through both in vitro and in vivo experiments. The system inhibits α-amylase and α-glucosidase via competitive and mixed inhibition mechanisms, primarily involving hydrogen bonds and van der Waals forces, leading to static fluorescence quenching. Additionally, this system downregulates the protein expression and gene transcription of SGLT1 and GLUT2. These findings offer a novel approach to sustaining glucose equilibrium, providing a valuable foundation for further application of quercetin emulsion in food science.

Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae).

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed Km and Vmax to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl2 significantly inhibited the enzyme, while the Na+, Ca++, and Mg++ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.

Probing the function of C-terminal region of recombinant α-amylase BmaN1 from Bacillus megaterium NL3.

The α-amylase BmaN1 from Bacillus megaterium NL3 is a member of GH13_45 subfamily that has a conserved C-terminal region of approximately 30 residues. This region features a motif of five aromatic amino acids predicted to play a role in starch binding. This study aimed to unravel the role of the C-terminal region in starch hydrolysis. The full-length and C-terminally truncated forms of BmaN1 (BmaN1∆C) were expressed in Escherichia coli ArcticExpress (DE3), resulting in proteins with molecular weights of 56 kDa and 49 kDa, respectively. They exhibited comparable enzymatic activity in the hydrolysis of soluble starch, displaying versatility across a wide range of pH values, temperatures, and NaCl concentrations. BmaN1 and BmaN1∆C activities were inhibited by acarbose and were reduced by SDS and EDTA. In terms of binding and degrading the starch granules, BmaN1∆C showed lower affinity and activity in comparison to BmaN1. Our study indicates that the C-terminal region of BmaN1 significantly enhances its binding affinity and degrading the raw starches.IMPORTANCEα-Amylase (EC 3.2.1.1) stands as an endo-acting enzyme, essential for catalyzing the hydrolysis of α-1,4 glycosidic bonds within starch molecules. The relevance of α-amylases in biotechnological applications is substantial, constituting approximately 30% of the global enzyme market. Among these enzymes, BmaN1 was the first α-amylase identified to possess distinct catalytic residues within the GH13 family. BmaN1 from B. megaterium NL3 belongs to the GH13_45 subfamily. This subfamily is characterized by a conserved C-terminal region consisting of approximately 30 residues that contains a motif of five aromatic residues predicted to be involved in starch binding. Our study shows that the C-terminal effectively contributes to binding and degrading the raw starch granules. This pioneering research on BmaN1 expands our understanding of α-amylases and holds promise for innovative biotechnological advancements.