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PloS One 2016We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE...
We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.
Topics: Amino Acid Sequence; Aspergillus oryzae; Molecular Docking Simulation; Proteolysis; Sequence Homology, Amino Acid; Substrate Specificity; alpha-Amylases
PubMed: 27101008
DOI: 10.1371/journal.pone.0153868 -
The FEBS Journal Jul 2009The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment was investigated...
The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment was investigated in the flour moth Ephestia kuehniella, an important stored-product pest. Three digestive alpha-amylases [Ephestia kuehniella alpha-amylase isoenzymes 1-3 (EkAmy1-3)] with an alkaline pH optimum were purified from larvae and biochemically characterized. These isoenzymes differ significantly in their sensitivity to alpha-amylase inhibitors of plant origin that are directed against herbivores as antifeedants. Such functional variability renders the amylolytic system less vulnerable to suppression by plant defensive molecules. Moreover, we found that expression of alpha-amylases is upregulated in larvae feeding on a diet enriched with an alpha-amylase inhibitor. The alpha-amylases are secreted into the larval midgut by an exocytotic mechanism, as revealed by immunogold microscopy. The cDNA sequence of EkAmy3 was determined, and a homology model of EkAmy3 was built in order to analyze the structural features responsible for adaptation to alkaline pH. First, the overall fold was found to be stabilized by remodeling of ion pairs. Second, molecular simulations supported by activity measurements showed that EkAmy3 does not bind a Cl(-), owing to an Arg-to-Gln mutation in a conserved binding site. The Cl(-)-binding residues are in contact with the catalytic residues, and this change might help to fine-tune the catalytic pK(a) values to an alkaline pH optimum. We conclude that lepidopteran alpha-amylases are evolutionarily adapted in terms of structure and expression dynamics for effective functioning in the digestive system.
Topics: Amino Acid Sequence; Animals; Base Sequence; Digestive System; Hydrogen-Ion Concentration; Insect Proteins; Isoenzymes; Models, Molecular; Molecular Sequence Data; Moths; Phylogeny; Plant Proteins; Protein Structure, Tertiary; Sequence Alignment; Sequence Homology, Amino Acid; alpha-Amylases
PubMed: 19476481
DOI: 10.1111/j.1742-4658.2009.07074.x -
European Journal of Biochemistry Apr 2000Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed...
Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.
Topics: Amino Acid Sequence; Animals; Enzyme Inhibitors; Humans; Insecta; Models, Molecular; Molecular Sequence Data; Mutation; Pancreas; Pest Control; Protein Binding; Protein Conformation; Saliva; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine; Triticum; alpha-Amylases
PubMed: 10759839
DOI: 10.1046/j.1432-1327.2000.01199.x -
Journal of Microbiology and... Nov 2009The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes for the degradation of plant...
The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes for the degradation of plant polysaccharide material. Two alpha-amylases designated as ScAmy54 and ScAmy43 were biochemically characterized and predicted to play an important role in starch degradation. Those enzymes produce specific oligosaccharides, essentially maltotriose, that have a considerable commercial interest. The primary structures of the two enzymes were analyzed by N-terminal sequencing, MALDI-TOF mass spectrometry, and cDNA cloning, and implied that the two proteins have the same N-terminal catalytic domain and ScAmy43 was produced from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. The result of genomic analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during the S. sclerotiorum cultivation. The structural gene of ScAmy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 amino acids. ScAmy54 exhibited high amino acid identity to other liquefying fungal alpha-amylases, essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3D structure of 2guy from A. niger as template. ScAmy54 with three domains A, B, and C, including the well-known (beta/alpha)8-barrel motif in domain A, has a typical structure of the alpha-amylase family. ScAmy43 composed only of domains A and B constitutes a smallest fungal alpha-amylase with only a catalytic domain.
Topics: Ascomycota; Base Sequence; Calcium; Catalysis; Catalytic Domain; Cloning, Molecular; DNA, Fungal; Genome, Fungal; Molecular Sequence Data; Protein Binding; Protein Sorting Signals; Sequence Alignment; Sequence Analysis; Sequence Homology, Amino Acid; alpha-Amylases
PubMed: 19996681
DOI: 10.4014/jmb.0903.3013 -
Journal of Visualized Experiments : JoVE Dec 2013Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This...
Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.
Topics: Aged, 80 and over; Caregivers; Circadian Rhythm; Frail Elderly; Humans; Hydrocortisone; Saliva; Specimen Handling; alpha-Amylases
PubMed: 24378361
DOI: 10.3791/50815 -
BMC Complementary Medicine and Therapies Feb 2021Elaeagnus umbellata Thunb. (autumn olive) is a high valued medicinal plant. It belongs to Elaeagnaceae family and is widely distributed in Himalayan regions of Pakistan....
BACKGROUND
Elaeagnus umbellata Thunb. (autumn olive) is a high valued medicinal plant. It belongs to Elaeagnaceae family and is widely distributed in Himalayan regions of Pakistan. In the present study essential oil were extracted from the fruit of this plant and their antioxidant, anticholinesterase and antidiabetic potentials were also evaluated.
METHODS
Essential oils were extracted from the fruit of E. umbellata using hydro-distillation method and were characterized by GC-MS. The extracted oil were tested for its antioxidant, anticholinesterase, and antidiabetic potentials using standard protocols.
RESULTS
About 68 compounds were identified by GC-MS. The extracted oil exhibited a fairly high free radical scavenging activities against DPPH and ABTS radicals with IC values of 70 and 105 μg/mL respectively (for ascorbic acid, used as standard, the IC values were 32 and 29 μg/mL, respectively against the mentioned radicals). The essential oil also exhibited anticholinesterase activities with IC values of 48 and 90 μg/mL respectively against AChE and BChE (for galantamine used as standard, the IC values were 25 and 30 μg/mL respectively). The essential oil also exhibited antidiabetic potential with IC values of 120 and 110 μg/mL respectively against α-glucosidase and α-amylase (IC values for standard acarbose = 28 and 30 μg/mL respectively).
CONCLUSION
Essential oil extracted from the fruits of E. umbellata exhibited reasonable antioxidant, anticholinesterase, and antidiabetic potentials that could be used as alternative medicine in treating diabetes and neurodegenerative disorders. However, further studies are needed to isolate responsible compounds and evaluate the observed potential in animal models.
Topics: Antioxidants; Cholinesterase Inhibitors; Elaeagnaceae; Enzyme Inhibitors; Gas Chromatography-Mass Spectrometry; Humans; Hypoglycemic Agents; Oils, Volatile; Pakistan; Plant Extracts; alpha-Amylases; alpha-Glucosidases
PubMed: 33618705
DOI: 10.1186/s12906-021-03228-y -
FEMS Yeast Research Jul 2022The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding...
The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were reintroduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation), and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. A total of 11 point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication, and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.
Topics: Amylases; CRISPR-Cas Systems; Point Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; alpha-Amylases
PubMed: 35776981
DOI: 10.1093/femsyr/foac033 -
BioMed Research International 2013Diabetes mellitus is a metabolic disorder of glucose metabolism. The management of blood glucose level is the hallmark in the treatment of this disease. This may be...
Diabetes mellitus is a metabolic disorder of glucose metabolism. The management of blood glucose level is the hallmark in the treatment of this disease. This may be achieved through the use of oral hypoglycemic drugs such as biguanides, insulin secretagogues, and α-glucosidase inhibitors. The purpose of the present study was to investigate the inhibitory effect of Morinda lucida leaf extracts on the activities of α-amylase and α-glucosidase. This was performed using α-amylase from Aspergillus oryzae and α-glucosidase from Saccharomyces cerevisiae. Aqueous extract of Morinda lucida gave the highest percentage yield (9.99%) of the plant out of the three extracts (compared to acetone and ethanolic extracts) and possesses the highest inhibitory activity against α-amylase (IC50 value of 2.30 mg/mL) and α-glucosidase (IC50 value of 2.00 mg/mL). Kinetic analysis revealed that the aqueous extract of this plant leaf inhibited the α-amylase competitively but displayed mixed noncompetitive mode of inhibition towards α-glucosidase. It can be concluded that aqueous extract of Morinda lucida exhibited the best inhibitory activity on the two enzymes studied and the presence of phytochemicals like flavonoids, saponins, and tannins may have contributed greatly to the inhibitory activity of the plant extract.
Topics: Aspergillus oryzae; Blood Glucose; Diabetes Mellitus; Glycoside Hydrolase Inhibitors; Humans; Morinda; Plant Extracts; Plant Leaves; Saccharomyces cerevisiae; alpha-Amylases; alpha-Glucosidases
PubMed: 24455701
DOI: 10.1155/2013/527570 -
European Journal of Biochemistry May 1987An alpha-amylase and a glucoamylase were purified to homogeneity from the culture fluid of beta-cyclodextrin-grown Candida antarctica CBS 6678 by protamine sulfate...
An alpha-amylase and a glucoamylase were purified to homogeneity from the culture fluid of beta-cyclodextrin-grown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G-75 sf, Ultrogel AcA 54), DEAE-Sephacel chromatography, hydroxyapatite chromatography and affinity chromatography on acarbose--AH-Sepharose 4B. Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (Szero20,w), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48,500, 4.7 S, 10.1, 1.74 cm2 mg-1, 4.2 and 57 degrees C, respectively, for glucoamylase and as 50,000, 4.9 S, 10.3, 1.53 cm2 mg-1, 4.2 and 62 degrees C, respectively, for alpha-amylase. Kinetic analyses indicated that both enzymes preferentially hydrolyzed high-molecular-mass substrates, including some raw starches. alpha-Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both alpha-amylase (Ki less than 1 microM) and glucoamylase (Ki less than 0.1 microM), being more effective than Bay e 4609 (Ki less than 10 microM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki less than 0.1 microM). Activity of the latter enzyme was also affected by 1-deoxynojirimycin (Ki less than 1 mM), maltitol and amino alcohols (Ki less than 10 mM). Unlike alpha-amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non-identical with the active site.
Topics: Adsorption; Candida; Chemical Phenomena; Chemistry, Physical; Glucan 1,4-alpha-Glucosidase; Glucosidases; Hydrogen-Ion Concentration; Isoelectric Focusing; Starch; Substrate Specificity; Temperature; alpha-Amylases
PubMed: 3106037
DOI: 10.1111/j.1432-1033.1987.tb11175.x -
Journal of Biochemistry Apr 1983A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to...
A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.
Topics: Amylases; Aspergillus; Bacillus subtilis; Chromatography, High Pressure Liquid; Fluorescent Dyes; Oligosaccharides; Rhizopus; Spectrometry, Fluorescence; alpha-Amylases
PubMed: 6190796
DOI: 10.1093/oxfordjournals.jbchem.a134229