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Microbiology Spectrum Dec 2021α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear....
α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear. α-Glucan was shown to be composed mainly of 1,3-glycosidically linked glucose, with trace amounts of 1,4-glycosidically linked glucose. Besides the α-glucan synthetases, amylase-like proteins were also important for α-glucan synthesis. In our previous work, we showed that Aspergillus nidulans AmyG was an intracellular protein and was crucial for the proper formation of α-glucan. In the present study, we expressed and purified AmyG in an Escherichia coli system. Enzymatic characterization found that AmyG mainly functioned as an α-amylase that degraded starch into maltose. AmyG also showed weak glucanotransferase activity. Most intriguingly, supplementation with maltose in shaken liquid medium could restore the α-glucan content and the phenotypic defect of a Δ strain. These data suggested that AmyG functions mainly as an intracellular α-amylase to provide maltose during α-glucan synthesis in A. nidulans. Short α-1,4-glucan was suggested as the primer structure for α-glucan synthesis. However, the exact structure and its source remain elusive. AmyG was essential to promote α-glucan synthesis and had a major impact on the structure of α-glucan in the cell wall. Data presented here revealed that AmyG belongs to the GH13_5 family and showed strong amylase function, digesting starch into maltose. Supplementation with maltose efficiently rescued the phenotypic defect and α-glucan deficiency in an Δ strain but not in an Δ strain. These results provide the first piece of evidence for the primer structure of α-glucan in fungal cells, although it might be specific to A. nidulans.
Topics: Amino Acid Sequence; Aspergillus nidulans; Cell Wall; Fungal Proteins; Fungi; Glucans; Maltose; Phylogeny; Sequence Alignment; alpha-Amylases
PubMed: 34756063
DOI: 10.1128/Spectrum.00644-21 -
International Journal of Biological... Jan 2021Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the...
Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the active centre of Colorado potato beetle (Leptinotarsa decemlineata) α-amylase (LdAmy). Bond cleavage frequency values for LdAmy were determined by HPLC product analysis on a chromophore labelled maltooligomer substrate series. Binding energies between amino acid moieties of subsites and glucose residues of substrate were calculated. Active site contains six subsites in the binding region of LdAmy; four glycone- (-4, -3, -2, -1) and two aglycone-binding sites (+1, +2). Subsite map calculation resulted in apparent binding energies -11.8 and - 11.0 kJ/mol for subsites (+2) and (-3), respectively, which revealed very favorable interactions at these positions. Structures of binding sites of LdAmy and mammalian α-amylases show similarity, but there are variations in the binding energies at subsite (-2) and (-4). Differences were interpreted by comparison of amino acid sequences of human salivary α-amylase (HSA) and porcine pancreatic α-amylase (PPA) and two insect (Leptinotarsa decemlineata and Tenebrio molitor) enzymes. The observed substitution of positively charged His305 in HSA at subsite (-2) with an acidic Asp in LdAmy in the same position may explain the obtained energy reduction.
Topics: Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; Coleoptera; Humans; Hydrolysis; Protein Binding; Sequence Homology, Amino Acid; Substrate Specificity; Swine; Tenebrio; alpha-Amylases
PubMed: 33310101
DOI: 10.1016/j.ijbiomac.2020.12.071 -
ACS Chemical Biology Feb 2023Acarbose is a well-known microbial specialized metabolite used clinically to treat type 2 diabetes. This natural pseudo-oligosaccharide (PsOS) shows potent inhibitory...
Acarbose is a well-known microbial specialized metabolite used clinically to treat type 2 diabetes. This natural pseudo-oligosaccharide (PsOS) shows potent inhibitory activity toward various glycosyl hydrolases, including α-glucosidases and α-amylases. While acarbose and other PsOSs are produced by many different bacteria, their ecological or biological role in microbial communities is still an open question. Here, we show that several PsOS-producing actinobacteria, i.e., sp. SE50/110 (acarbose producer), GLA.O (acarbose producer), and ATCC 31484 (trestatin producer), can grow in the presence of acarbose, while the growth of the non-PsOS-producing organism M1152 was suppressed when starch is the main source of energy. Further investigations using recombinant α-amylases from M1152 and the PsOS-producing actinobacteria revealed that the α-amylase was inhibited by acarbose, whereas those from the PsOS-producing bacteria were not inhibited by acarbose. Bioinformatic and protein modeling studies suggested that a point mutation in the α-amylases of the PsOS-producing actinobacteria is responsible for the resistance of those enzymes toward acarbose. Converting the acarbose-resistant α-amylase AcbE to its A304H variant diminished its acarbose-resistance property. Taken together, the results suggest that acarbose is used by the producing bacteria as a competitive exclusion agent to suppress the growth of other microorganisms in their natural environment, while the producing organisms equip themselves with α-amylase variants that are resistant to acarbose.
Topics: Humans; Acarbose; Diabetes Mellitus, Type 2; Bacterial Proteins; Actinobacteria; alpha-Amylases
PubMed: 36648321
DOI: 10.1021/acschembio.2c00795 -
Molecules (Basel, Switzerland) Apr 2019The edible and medicinal perennial herb dandelion is known to have antitumor, antioxidant, and anticomplement properties. However, the structural characterization and...
The edible and medicinal perennial herb dandelion is known to have antitumor, antioxidant, and anticomplement properties. However, the structural characterization and biological effects of its polysaccharides are not well understood. Here, we aimed to extract and investigate a novel polysaccharide from dandelion. A water-soluble polysaccharide, PD1-1, was successfully obtained from dandelion through ultrasonic-assisted extraction and purification using diethylaminoethyl (DEAE)-Sepharose fast flow and Sephadex G-75 columns. The results showed that PD1-1 is an inulin-type polysaccharide with a molecular weight of 2.6 kDa and is composed of glucose (52.39%), and mannose (45.41%). Glycosidic linkage analysis demonstrated that PD1-1 contains terminal α-d-Man/Glcp-(1→ and →1)-β-d-Man/Glcf-(2→ glycosidic linkage conformations. A physicochemical analysis indicated that PD1-1 has a triple helix structure and exhibits important properties, including good swelling, water-holding, and oil-holding capacities. Furthermore, PD1-1 showed good antioxidant activities in DPPH and hydroxyl free radical scavenging abilities, with IC values of 0.23 mg/mL and 0.25 mg/mL, respectively, and good hypoglycemic activities in α-amylase and α-glucosidase inhibition, with IC values of 0.53 mg/mL and 0.40 mg/mL, respectively, in a concentration-dependent manner. Results suggest that PD1-1 possesses efficacious antioxidant and hypoglycemic properties and has potential applications as a functional food ingredient.
Topics: Antioxidants; Glycoside Hydrolase Inhibitors; Polysaccharides; Taraxacum; alpha-Amylases; alpha-Glucosidases
PubMed: 30991766
DOI: 10.3390/molecules24081485 -
European Journal of Biochemistry Sep 1994Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to... (Comparative Study)
Comparative Study
Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases.
Topics: Amino Acid Sequence; Animals; Bacteria; Biological Evolution; Fungi; Mammals; Molecular Sequence Data; Plants; Sequence Homology, Amino Acid; alpha-Amylases
PubMed: 7925367
DOI: 10.1111/j.1432-1033.1994.00519.x -
BioMed Research International 2017A continuous research is attempted to fulfil the highest industrial demands of natural amylases presenting special properties. New -amylases extracted from stems and...
A continuous research is attempted to fulfil the highest industrial demands of natural amylases presenting special properties. New -amylases extracted from stems and leaves of , which is widespread and growing spontaneously in Tunisia, were studied by the means of their activities optimization and purification. Some similarities were recorded for the two identified enzymes: (i) the highest amylase activity showed a promoted thermal stability at 50°C; (ii) the starch substrate at 1% enhanced the enzyme activity; (iii) the two -amylases seem to be calcium-independent; (iv) Zn, Cu, and Ag were considered as important inhibitors of the enzyme activity. Following the increased gradient of elution on Mono Q-Sepharose column, an increase in the specific activity of 11.82-fold and 10.92-fold was recorded, respectively, for leaves and stems with the presence of different peaks on the purification profiles. amylases activities were stable and compatible with the tested commercial detergents. The combination of plant amylase and detergent allowed us to enhance the wash performance with an increase of 35.24 and 42.56%, respectively, for stems and leaves amylases. Characterized amylases were reported to have a promoted potential for their implication notably in detergent industry as well as biotechnological sector.
Topics: Apocynaceae; Calcium; Copper; Detergents; Enzyme Inhibitors; Enzyme Stability; Hydrogen-Ion Concentration; Plant Leaves; Plant Stems; Silver; Starch; Temperature; Zinc; alpha-Amylases
PubMed: 29392138
DOI: 10.1155/2017/6712742 -
Microbial Cell Factories Jul 2022Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has...
BACKGROUND
Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients.
RESULTS
In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2-threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar.
CONCLUSIONS
We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.
Topics: Amylases; Bacillus subtilis; Erythromycin; Flagella; alpha-Amylases
PubMed: 35780132
DOI: 10.1186/s12934-022-01861-x -
BMC Complementary and Alternative... Apr 2014The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant....
BACKGROUND
The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste.
METHOD
The purification method included chromatography of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE.
RESULTS
Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.
CONCLUSIONS
From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.
Topics: Chromatography; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glycogen; Humans; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; Plant Extracts; Salvadoraceae; Starch; Temperature; Toothpastes; alpha-Amylases
PubMed: 24690287
DOI: 10.1186/1472-6882-14-119 -
Microbiology Spectrum Aug 2022Halotolerant bacteria capable of starch hydrolysis by their amylases will benefit various industries, specifically since the hydrolytic activity of current industrial...
Halotolerant bacteria capable of starch hydrolysis by their amylases will benefit various industries, specifically since the hydrolytic activity of current industrial amylases is inhibited or even absent in salt-rich or alkaline environments. Seeking novel enzymes, we analyzed the entire genome content of a marine bacterium isolated from the gut of sea urchins to compare it against other bacterial genomes. Conditions underlying α-amylase activity were examined at various salinities (0 to 4%) and temperatures (25°C to 37°C). Genomic analyses revealed the isolated bacterium as a new species of . Comparative analysis of the contents of carbohydrate-active enzymes revealed various α-amylases, each with its respective carbohydrate-binding module for starch hydrolysis. Functional analysis identified the hydrolysis of starch and the maltooligosaccharides maltose and dextrin into d- and UDP-glucose. The fastest growth and α-amylase production occurred at 3% salinity at a temperature of 30°C. The sp. consists of exclusive contents of α-amylases and other enzymes that may be valuable in the hydrolysis of the algal polysaccharides cellulose and laminarin. Toward the discovery of novel carbohydrate-active enzymes that may be useful in the hydrolysis of starch, we examined a halotolerant bacterial isolate of sp. regarding its genomic content and conditions underlying the production of active α-amylases. The production of α-amylases was measured in bacterial cultures at relatively high temperature (37°C) and salinity (4%). The sp. revealed an exclusive content of amylases and other carbohydrate-active enzymes compared to other relevant bacteria. These enzymes may be valuable for the hydrolysis of algal polysaccharides. The enzymatic cascade of the sp. for starch metabolism allows polysaccharide degradation into monosugars while preventing the accumulation of intermediate inhibitors of maltose or dextrin.
Topics: Amylases; Dextrins; Hydrogen-Ion Concentration; Hydrolysis; Maltose; Polysaccharides; Starch; Temperature; alpha-Amylases
PubMed: 35863032
DOI: 10.1128/spectrum.01078-22 -
Planta May 2022α-Amylase synthesis by wheat aleurone during grain development (late maturity α-amylase) appears to be independent of gibberellin unlike α-amylase synthesis by...
α-Amylase synthesis by wheat aleurone during grain development (late maturity α-amylase) appears to be independent of gibberellin unlike α-amylase synthesis by aleurone during germination or following treatment with exogenous GA. Late-maturity α-amylase (LMA) in wheat (Triticum aestivum L.) involves the synthesis of α-amylase by the aleurone tissue during grain development. Previous research identified a putative ent-copalyl diphosphate synthase gene, coding for an enzyme that controls the first step in gibberellin biosynthesis, that underlies the major genetic locus involved in variation in LMA phenotype. The reported results for gene transcript analysis, preliminary gibberellin analysis and the effects of DELLA mutants on LMA phenotype appeared to be consistent with involvement of gibberellin but did not provide definitive proof of a causal link. Conversely, several observations do not appear to be consistent with this hypothesis. In this current study, LMA phenotype, gibberellin profiles and ABA content were recorded for experiments involving susceptible and resistant genotypes, gibberellin biosynthesis inhibitors, genetic lines containing different LMA quantitative trait loci and treatment of distal halves of developing grains with exogenous gibberellin. The results suggested that gibberellin may not be a prerequisite for LMA expression and further that the mechanism involved in triggering α-amylase synthesis did not correspond to the model proposed for germination and gibberellin challenged aleurone of ripe grain. The results provide new insight into LMA and highlight the need to investigate alternate pathways for the induction of α-amylase gene transcription, the function of novel 1-β-OH gibberellins and other functions of DELLA proteins in developing grains.
Topics: Germination; Gibberellins; Seeds; Triticum; alpha-Amylases
PubMed: 35522329
DOI: 10.1007/s00425-022-03899-y