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BMC Microbiology Aug 2022Bacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental...
BACKGROUND
Bacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental adaptability. Bacillus cereus 0-9, a biocontrol bacterial strain isolated from wheat roots, has three genes annotated as encoding amylases in the genome, but their functions are unknown, and whether they are involved in the colonization process of the bacterium remains to be further studied.
METHODS
Mutant gene strains and fluorescently tagged strains were constructed by homologous recombination, and amylase protein was expressed in the prokaryotic Escherichia coli BL21(DE3) expression system. The iodine staining method was used to measure the activity of amylase proteins. We further observed the colonization abilities of the test strains in wheat roots through frozen section technology.
RESULTS
The results showed that there were three amylase-encoding genes, amyC, amyP and amyS, in the B. cereus 0-9 genome. Among the three amylase encoding genes, only amyS produced extracellular amylase whose secretion was related to signal peptide at position 1-27. The AmyS protein encoded by the amyS gene is an α-amylase. The growth of Rhizoctonia cerealis was inhibited 84.7% by B. cereus 0-9, but the biocontrol ability of the ΔamyS strain decreased to 43.8% and that of ΔamyS/amyS was restored when the amyS gene was complemented. Furthermore, the biocontrol ability of the ΔamySec strain was decreased to 46.8%, almost the same as that of the ΔamyS mutant. Due to the deletion of the amyS gene, the colonization capacities of ΔamyS (RFP) and ΔamySec (RFP) in wheat roots decreased, while that of ΔamyS/amyS (RFP) was restored after the amyS gene was complemented, indicating that the amyS gene influences the colonization of B. cereus 0-9 in wheat roots. In addition, the colonization and biocontrol abilities of the mutant were restored after the addition of sugars, such as glucose and maltose.
CONCLUSIONS
B. cereus 0-9 encodes three genes annotated as amylases, amyC, amyP and amyS. Only the deletion of the amyS gene with a signal peptide did not produce extracellular amylase. The AmyS protein encoded by the amyS gene is an α-amylase. Our results indicated that the amyS gene is closely related to the colonization abilities of B. cereus 0-9 in wheat roots and the biocontrol abilities of B. cereus 0-9 to fight against R. cerealis. The extracellular amylase produced by B. cereus 0-9 can hydrolyze starch and use glucose, maltose and other nutrients to meet the needs of bacterial growth. Therefore, it is very possible that the secretion and hydrolytic activities of extracellular amylase can promote the colonization of B. cereus 0-9 in wheat roots and play important roles in the prevention and control of plant diseases. Our results contribute to exploring the mechanisms of microbial colonization in plant roots.
Topics: Amylases; Bacillus cereus; Bacterial Proteins; Escherichia coli; Glucose; Maltose; Plant Roots; Protein Sorting Signals; Triticum; alpha-Amylases
PubMed: 35996113
DOI: 10.1186/s12866-022-02618-7 -
Gut May 1997A six year old boy underwent extensive investigation for recurrent abdominal pain and was found to have a persistently raised serum amylase. Endoscopic retrograde...
A six year old boy underwent extensive investigation for recurrent abdominal pain and was found to have a persistently raised serum amylase. Endoscopic retrograde cholangiopancreatography was normal and macroamylasaemia was excluded. Serum amylase concentrations were found to be raised in other family members spanning three generations, all of whom were asymptomatic. Clearance studies suggested no evidence of a renal tubular defect and serum lipase concentrations were normal. This is the first report of apparently familial hyperamylasaemia and the mode of inheritance is consistent with an autosomal dominant pattern.
Topics: Abdominal Pain; Amylases; Child; Genes, Dominant; Humans; Male; Pedigree
PubMed: 9203953
DOI: 10.1136/gut.40.5.689 -
PloS One 2018The purpose of this research is to explore the link between plasma amylase and insulin levels in growing pigs. Blood was obtained from piglets ranging in age from...
The purpose of this research is to explore the link between plasma amylase and insulin levels in growing pigs. Blood was obtained from piglets ranging in age from preterm (8 days to full gestation period), up to postnatal day 90 (2 months post-weaning) that underwent either duodenal-jejunal bariatric interposition surgery or a sham-operation. Plasma amylase activities in preterm and full-term neonates ranged between 500-600 U/L and were decreased by 50% two months post-weaning. Preprandial insulin and C-peptide levels in neonate piglets were not detectable, however they rose gradually after weaning. An increase in plasma amylase activity was observed in the young pigs that underwent duodenal-jejunum bypass (metabolic) surgery. The increase in blood pancreatic amylase activity after an intravenous amylase infusion lowered the subsequent glucose-stimulated insulin/C-peptide release. We suggest a role for blood amylase in the regulation of glucose homeostasis after observing high blood amylase levels in neonate pigs, in pigs that underwent metabolic surgery, and as a result of the reduced glucose-stimulated insulin response following intravenous amylase administration. Blood amylase level is a dynamic physiological parameter, which is not merely a consequence of exocrine pancreatic digestive enzyme production, but rather a regulated factor involved in glucose assimilation and prandial insulin regulation.
Topics: Amylases; Animals; Animals, Newborn; Bariatric Surgery; Infusions, Intravenous; Insulin; Models, Animal; Sus scrofa
PubMed: 29874296
DOI: 10.1371/journal.pone.0198672 -
Molecules (Basel, Switzerland) Oct 2022Amylase and glucosidase enzymes are the primary harmful source in the development of the chronic condition known as diabetes mellitus. The main function of these enzymes...
New Biologically Hybrid Pharmacophore Thiazolidinone-Based Indole Derivatives: Synthesis, In Vitro Αlpha-Amylase and Αlpha-Glucosidase Along with Molecular Docking Investigations.
Amylase and glucosidase enzymes are the primary harmful source in the development of the chronic condition known as diabetes mellitus. The main function of these enzymes is to break the macromolecules into simple sugar units which are directly involved in the solubility of blood, hence increasing blood glucose levels. To overcome this effect, there is a need for a potent and effective inhibitor that inhibits the conversion of macromolecules of sugar into its smaller units. In this regard, we synthesized thiazolidinone-based indole derivatives (1−20). The synthesized derivatives were evaluated for α-amylase and α-glucosidase inhibitory activity. Different substituted derivatives were found with moderate to good potentials having IC50 values ranging, for α-amylase, from 1.50 ± 0.05 to 29.60 ± 0.40 μM and, for α-glucosidase, from IC50 = 2.40 ± 0.10 to 31.50 ± 0.50 μM. Among the varied substituted compounds, the most active analogs four (1.80 ± 0.70 and 2.70 ± 0.70), five (1.50 ± 0.05 and 2.40 ± 0.10, respectively) of the series showed few folds better inhibitory activity than standard drug acarbose (IC50 = 10.20 ± 0.10 and 11.70 ± 0.10 μM, respectively). Moreover, structure−activity relationship (SAR) was established and binding interactions were analyzed for ligands and proteins (α-amylase and α-glucosidase) through a molecular docking study.
Topics: Acarbose; Amylases; Blood Glucose; Glucosidases; Glycoside Hydrolase Inhibitors; Indoles; Ligands; Molecular Docking Simulation; Molecular Structure; Receptors, Drug; Structure-Activity Relationship; alpha-Amylases; alpha-Glucosidases
PubMed: 36235098
DOI: 10.3390/molecules27196564 -
PloS One 2022The kinetics of growth and α-amylase production of a novel Candida wangnamkhiaoensis yeast strain were studied in single-stage steady-state continuous cultures. This...
The kinetics of growth and α-amylase production of a novel Candida wangnamkhiaoensis yeast strain were studied in single-stage steady-state continuous cultures. This was performed in a split-cylinder internal-loop airlift bioreactor, using a variety of carbon sources as fermentation substrates. Results showed that the steady-state yields of cell mass from carbohydrates were practically constant for the range of dilution rates assayed, equaling 0.535 ± 0.030, 0.456 ± 0.033, and 0.491 ± 0.035 g biomass/g carbohydrate, when glucose, maltose, and starch, respectively were used as carbon sources. No α-amylase activity was detected when glucose was used as the carbon source in the influent medium, indicating that α-amylase synthesis of C. wangnamkhiaoensis is catabolically repressed by glucose. Contrastingly, maltose and starch induce synthesis of α-amylase in C. wangnamkhiaoensis, with starch being the best α-amylase inducer. The highest α-amylase volumetric and specific activities (58400 ± 800 U/L and 16900 ± 200 U/g biomass, respectively), and productivities (14000 ± 200 U/L·h and 4050 ± 60 U/g biomass·h, respectively) were achieved at a dilution rate of 0.24 h-1 using starch as the carbon source. In conclusion, single-stage steady-state continuous culture in an airlift bioreactor represents a powerful tool, both for studying the regulatory mechanisms of α-amylase synthesis by C. wangnamkhiaoensis and for α-amylase production. Furthermore, results showed that C. wangnamkhiaoensis represents a potential yeast species for the biotechnological production of α-amylase, which can be used for the saccharification of starch. This offers an attractive renewable resource for the production of biofuels (particularly bioethanol), representing an alternative to fossil fuels with reduced cost of substrates.
Topics: Amylases; Bioreactors; Carbohydrates; Carbon; Glucose; Maltose; Saccharomycetales; Starch; alpha-Amylases
PubMed: 35231077
DOI: 10.1371/journal.pone.0264734 -
Journal of Cellular Biochemistry Feb 2020α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other...
α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.
Topics: Caco-2 Cells; Cell Differentiation; Cell Proliferation; Epithelial Cells; Gene Expression; Humans; Intestine, Small; Pancreas; Pancreatic alpha-Amylases; RNA Interference; RNA, Messenger; Salivary Glands; Salivary alpha-Amylases; Transfection
PubMed: 31478242
DOI: 10.1002/jcb.29357 -
American Journal of Human Genetics Jul 1976Prior genetic studies of the human pancreatic amylase (Amy2) locus have been directed principally to the electrophoretic analysis of serum and urine, on the assumption...
Prior genetic studies of the human pancreatic amylase (Amy2) locus have been directed principally to the electrophoretic analysis of serum and urine, on the assumption that these fluids receive negligible contributions from the salivary (Amy 1) locus. In support of that assumption was the observation that the isozyme bands were lacking in patients with cystic fibrosis and in a postpancreatectomy patient. We have examined the sera of 97 patients having cystic fibrosis and find normal levels of serum amylase. On electrophoresis, three-quarters of the cystic fibrosis patients have a pattern (F-pattern) not observed in normal sera. The pattern is characterized by the absence of Pa 1. Comparative electrophoresis and mixing experiments indicate that the F-pattern is of salivary origin and is unmasked in cystic fibrosis by the absence of a pancreatic contribution. The normal serum pattern is considered to be an admixture of salivary and pancreatic amylase. On the assumption that duodenal fluids might more closely reflect the pancreatic (Amy 2) locus, electrophoretic studies were performed on 148 normal individuals and 37 individuals with cystic fibrosis. Electrophoretic phenotypes in duodenal aspirates are more complex than previously reported in studies of urine and serum; presumably because of the higher concentrations of amylase in the aspirates. Comparative electrophoresis and mixing experiments indicate that the phenotypes observed in duodenal aspirates also reflect admixture of pancreatic and salivary amylase. This recognition of pancreatic and salivary admixture in sera fortunately does not alter our prior understanding of the genetics of the Amy 2 polymorphism. The extensive studies which led to the delineation of the Amy 2 polymorphism were essentially based on the presence or absence of a variant band which proves now to be outside the zone of admixture.
Topics: Amylases; Cystic Fibrosis; Duodenum; Electrophoresis, Polyacrylamide Gel; Humans; Pancreas; Polymorphism, Genetic; Saliva
PubMed: 941905
DOI: No ID Found -
BioMed Research International 2015An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium...
Purification and Characterization of a Polyextremophilic α -Amylase from an Obligate Halophilic Aspergillus penicillioides Isolate and Its Potential for Souse with Detergents.
An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U · mg(-1) and Vmax and Km values of 1.05 µmol · min(-1) · mg(-1) and 5.41 mg · mL(-1), respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80 °C, and 300 g · L(-1) NaCl. The addition of CaCl2 at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe(2+) or Zn(2+)) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g · L(-1). Accordingly, it has a good potential for use as an α-amylase in a low water activity (high salt concentration) and at high pH and temperatures.
Topics: Aspergillus; Enzyme Stability; Fungal Proteins; Hot Temperature; Hydrogen-Ion Concentration; alpha-Amylases
PubMed: 26180787
DOI: 10.1155/2015/245649 -
Human Molecular Genetics Jun 2015The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in...
The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations.
Topics: Amylases; DNA Copy Number Variations; Gene Order; Genetic Loci; Haplotypes; Humans; Obesity; Pancreatic alpha-Amylases; Salivary alpha-Amylases; Starch
PubMed: 25788522
DOI: 10.1093/hmg/ddv098 -
Maternal & Child Nutrition Apr 2019Adding amylase to Super Cereal Plus (SC+A) improves energy and nutrient intake as porridge energy density reaches 1.0 kcal/g, meeting the recommended ≥0.8 kcal/g for...
Adding amylase to Super Cereal Plus (SC+A) improves energy and nutrient intake as porridge energy density reaches 1.0 kcal/g, meeting the recommended ≥0.8 kcal/g for prepared foods for young children. Caregiver response to SC+A in terms of adjusting porridge preparation using printed pictogram instructions was not yet investigated. The study assessed (a) porridge preparation by caregivers; (b) porridge energy density; (c) sensory porridge acceptability; and (d) understanding of preparation instructions. An 8-day follow-up intervention study was conducted amongst caregivers of children aged 6-23 months (n = 238) in Rwanda. Caregivers prepared porridge using SC+A whilst referring to printed pictogram instructions at the study site on Days 1 and 8 and received flour for preparation at home on Days 2-7. At the site, data were collected on porridge preparation procedures, energy density, consistency, acceptability, and interviews (n = 12), and focus group discussions (n = 6) were conducted. Mean porridge dry matter (DM) increased from 21.3 ± 4.4% (Day 1) to 25.1 ± 4.8% (Day 8; p < 0.0005). Flour and water were mixed before cooking by 95% of the participants, as per printed instructions. Sensory porridge acceptability was high, and the printed instructions enabled caregivers to prepare an accepted and energy dense porridge. The preferred water/flour volume ratio was 2.5 instead of 3. In conclusion, Rwandan caregivers prepared well-accepted SC+A porridges with a preferred consistency and mean DM content of 25.1% (1.0 kcal/g), after 1 week practicing at home. This supports introducing SC+A with the tested instructions at scale.
Topics: Amylases; Caregivers; Cooking; Edible Grain; Energy Intake; Female; Flour; Humans; Infant; Infant Food; Male; Nutritive Value; Rwanda
PubMed: 30375162
DOI: 10.1111/mcn.12742