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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 -
European Journal of Biochemistry Dec 1987The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the...
The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Risø mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.
Topics: Amino Acid Sequence; Amylases; Base Sequence; DNA; Gene Expression Regulation; Genes; Hordeum; Molecular Sequence Data; Mutation; Peptide Mapping; Plants; beta-Amylase
PubMed: 2446870
DOI: 10.1111/j.1432-1033.1987.tb13640.x -
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 -
International Journal of Environmental... Aug 2022Stress plays a significant role in competitions and in the training of sports participants, and coaches are no exception. To better cope with stressful situations, close...
Stress plays a significant role in competitions and in the training of sports participants, and coaches are no exception. To better cope with stressful situations, close monitoring of coaches' stress levels before, during, and after training and competitions is recommended. According to studies, the use of cortisol (C) and alpha-amylase (AA) as biomarkers for monitoring acute stress is recommended. Therefore, the aim of our study was to compare HR, salivary C and AA, and STAI scores before, during, and after handball matches and training sessions. The study examined one professional handball coach, aged 37, in stress markers (salivary cortisol (C) and alpha-amylase (AA) concentrations), heart rate (HR), and the State-Trait Anxiety Inventory (STAI) scores in five matches/training sessions in the First Qatar Handball League. Statistical analysis included the calculation of descriptive statistic parameters, Mann-Whitney U test for differences between match-training time points, and the effect size analysis (Cohen's d) to calculate the magnitude of differences between match-training time points. Presented markers (C and AA) had statistically stronger reactions before, during, and after the matches than the corresponding time points of the training sessions, similar to HR data and STAI scores. Results indicate that, before and during the matches, the analyzed markers of stress increased, which might lead to the conclusion that coaches are more anxious than frightened before and during matches. Thus, stress-coping strategies for handball coaches should be more focused on stress anticipation and anxiety control.
Topics: Amylases; Biomarkers; Humans; Hydrocortisone; Mentoring; Saliva; Sports; alpha-Amylases
PubMed: 36011885
DOI: 10.3390/ijerph191610251 -
World Journal of Microbiology &... Feb 2016Amylase is one of the earliest characterized enzymes and has many applications in clinical and industrial settings. In biotechnological industries, the amylase activity...
Amylase is one of the earliest characterized enzymes and has many applications in clinical and industrial settings. In biotechnological industries, the amylase activity is enhanced through modifying amylase structure and through cloning and expressing targeted amylases in different species. It is important to understand how engineered amylases can survive from generation to generation. This study used phylogenetic and statistical approaches to explore general patterns of amylases evolution, including 3118 α-amylases and 280 β-amylases from archaea, eukaryota and bacteria with fully documented taxonomic lineage. First, the phylogenetic tree was created to analyze the evolution of amylases with focus on individual amylases used in biofuel industry. Second, the average pairwise p-distance was computed for each kingdom, phylum, class, order, family and genus, and its diversity implies multi-time and multi-clan evolution. Finally, the variance was further partitioned into inter-clan variance and intra-clan variance for each taxonomic group, and they represent horizontal and vertical gene transfer. Theoretically, the results show a full picture on the evolution of amylases in manners of vertical and horizontal gene transfer, and multi-time and multi-clan evolution as well. Practically, this study provides the information on the surviving chance of desired amylase in a given taxonomic group, which may potentially enhance the successful rate of cloning and expression of amylase gene in different species.
Topics: Amino Acid Sequence; Amylases; Archaea; Bacteria; Data Interpretation, Statistical; Eukaryota; Evolution, Molecular; Gene Transfer, Horizontal; Genetic Engineering; Genetic Variation; Molecular Sequence Data; Phylogeny; Sequence Alignment
PubMed: 26745984
DOI: 10.1007/s11274-015-1979-y -
Journal of Biochemistry Jul 1983Cyclohexadextrin and maltose bound to soybean beta-amylase and affected the environments of tryptophan and tyrosine residues, producing characteristic difference spectra...
Cyclohexadextrin and maltose bound to soybean beta-amylase and affected the environments of tryptophan and tyrosine residues, producing characteristic difference spectra in the ultraviolet region. The difference spectrum produced by cyclohexadextrin, a competitive inhibitor, had peaks at 285, 292, and 299 nm, while that by maltose, a reaction product, had peaks at 285 and 292 nm and a small trough at around 300 nm. By using the peaks at 292 and 299 nm, the dissociation constants of enzyme-cyclohexadextrin and enzyme-maltose complexes were calculated to be 0.35 mM and 8.1 mM, respectively. The effects of modification of SH groups of beta-amylase on the interaction of the enzyme with these sugars were examined by using beta-amylase carboxymethylated at the SH1 site and the enzyme modified at SH1 and SH2 sites with iodoacetamide or with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The dissociation constants of the enzyme-cyclohexadextrin and enzyme-maltose complexes were not changed by the modification of these SH groups, but the modification of SH2, the so-called essential SH group of soybean beta-amylase, strongly affected the difference spectra produced by maltose. The spectrophotometric titration of beta-amylase by cyclohexadextrin in the presence of maltose showed that cyclohexadextrin and maltose bind to the enzyme competitively, regardless of the modification of SH2. These results indicated that SH2 is located near the binding site of cyclohexadextrin and maltose, but is not involved in the binding of these sugars.
Topics: Amylases; Binding, Competitive; Circular Dichroism; Cyclodextrins; Dextrins; Kinetics; Maltose; Protein Binding; Glycine max; Spectrophotometry, Ultraviolet; Starch; Sulfhydryl Compounds; beta-Amylase
PubMed: 6194150
DOI: 10.1093/oxfordjournals.jbchem.a134317 -
Carbohydrate Polymers Nov 2022The objective of this study was to evaluate the modification of red rice starch by a combination of hydrothermal pretreatments and α-amylase hydrolysis. In vitro...
The objective of this study was to evaluate the modification of red rice starch by a combination of hydrothermal pretreatments and α-amylase hydrolysis. In vitro digestibility and the morphological, structural, functional, thermal, textural and rheological properties of red rice starch were evaluated. The starch submitted to autoclave (A3) obtained the highest hydrolysis yield (37.66 %) after 300 min. The morphological analysis showed that for the native starch, the granules presented a polyhedral shape and increased in diameter (2.36-394.12 μm) due to hydrothermal pre-treatments. α-Amylase (9 U mg) from Aspergillus oryzae modified the structure of red rice starch, presenting technological properties different from native starch. X-ray diffraction (XDR) were altered after the starch granules were cooked, showing a rupture in the amylose and amylopectin molecules, which justifies the greater absorption capacity of oil and milk. Cohesiveness, adhesiveness and apparent viscosity decreased according to HPT temperature and pressure, as well as α-amylase action.
Topics: Amylases; Hydrolysis; Oryza; Starch; alpha-Amylases
PubMed: 36088003
DOI: 10.1016/j.carbpol.2022.119963 -
International Journal of Molecular... Nov 2023The acini-islet-acinar (AIA) axis concept justifies the anatomical placement of the Langerhans islets within the exocrine pancreatic parenchyma and explains the...
The acini-islet-acinar (AIA) axis concept justifies the anatomical placement of the Langerhans islets within the exocrine pancreatic parenchyma and explains the existence of the pancreas as a single organ. Amylase has been suggested to play a key role as an anti-incretin factor. Oral glucose tolerance tests (OGTT) were performed on 18 piglets in both a healthy (prior to pancreatic duct ligation (PDL) surgery, study Day 10) and an exocrine pancreatic insufficient (EPI) state (30 days after PDL, study Day 48)). Amylase (4000 units/feeding) or Creon (100,000 units/feeding) was administered to pigs with the morning and evening meals, according to study design randomization, for 37 days following the first OGTT. Blood glucose levels, as well as plasma levels of insulin, GLP-1, and GIP, were measured, and the HOMA-IR index was calculated. EPI status did not affect the area under the curve (AUC) of insulin release, fasting insulin levels, or the HOMA-IR index, while amylase supplementation led to a significant ( < 0.05) decrease in the above-mentioned parameters. At the same time, EPI led to a significant ( < 0.05) increase in GLP-1 levels, and neither amylase nor Creon supplementation had any effects on this EPI-related increase. Fasting plasma levels of GIP were not affected by EPI; however, the GIP response in EPI and Amylase-treated EPI animals was significantly lower ( < 0.05) when compared to that of the intact, healthy pigs. Orally administered amylase induces gut anti-incretin action, normalizing glucose homeostasis and reducing HOMA-IR as a long-term outcome, thus lowering the risk of diabetes type II development. Amylase has long-lasting anti-incretin effects, and one could consider the existence of a long-lasting gut memory for amylase, which decreases hyperinsulinemia and hyperglycemia for up to 16 h after the last exposure of the gut to amylase.
Topics: Animals; Swine; Incretins; Blood Glucose; alpha-Amylases; Pancrelipase; Insulin; Glucagon-Like Peptide 1; Amylases; Dietary Supplements; Gastric Inhibitory Polypeptide
PubMed: 38003366
DOI: 10.3390/ijms242216177 -
Journal of Animal Science Oct 2020Developed initially for use in fuel ethanol production, Enogen Feed Corn (EFC; Syngenta Crop Protection) is genetically modified to express high concentrations of...
Developed initially for use in fuel ethanol production, Enogen Feed Corn (EFC; Syngenta Crop Protection) is genetically modified to express high concentrations of α-amylase in the corn kernel. Experiments were conducted to evaluate processing characteristics of EFC, in vitro digestion, and effects on feedlot performance, carcass characteristics, and liver abscess incidence. Experiment 1 used a randomized complete block design (3 × 3 × 5 factorial) to evaluate starch availability, in situ dry matter disappearance (ISDMD), in vitro gas production (IVGP), and volatile fatty acid (VFA) profiles of in vitro cultures. Grains (EFC or mill-run control [CON]) were flaked to a density of 360 g/L, and mixtures with 0%, 25%, 50%, 75%, or 100% EFC were prepared. Grains were tempered with added moisture (0%, 3%, or 6%) prior to steam conditioning for 15, 30, or 45 min. No two- or three-way interactions were observed. Adding moisture improved starch availability (linear; P < 0.01), and tended to improve ISDMD (linear, P = 0.06). Steam conditioning for 30 min improved starch availability, IVGP, and production of acetate, propionate, butyrate, valerate, and total VFA (P < 0.01) compared with conditioning for 15 or 45 min. Starch availability, ISDMD, IVGP, acetate, propionate, valerate, and total VFA production increased with an increasing proportion of EFC (linear, P < 0.01). Experiment 2 used 700 beef heifers (394 ± 8.5 kg initial body weight [BW]) fed finishing diets with steam-flaked corn as CON or EFC for 136 d. Targeting similar starch availabilities, grains were processed to 360 g/L (CON) and 390 g/L for CON and EFC, respectively. Heifers were blocked by BW, stratified, and then randomly assigned to 28 dirt-surfaced pens (25 animals per pen). Dry matter intakes were similar between treatments (P = 0.78), but cattle fed EFC had greater average daily gain (P < 0.01), improving feed efficiency by 5% (P < 0.01). Hot carcass weight was 6 kg greater for EFC cattle (P <0.01) than CON. No differences were observed for longissimus muscle area (P = 0.89), 12th-rib fat thickness (P = 0.21), or USDA yield grade (P = 0.13). Cattle fed CON had greater marbling scores than EFC (P = 0.04), but this did not affect the USDA quality grade (P > 0.33). Cattle fed EFC had 23% fewer abscessed livers than CON (P = 0.03). High-amylase corn may be used to improve microbial digestion, mill-throughput, and cattle performance, and it may mitigate liver abscesses.
Topics: Amylases; Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Body Composition; Cattle; Diet; Digestion; Fatty Acids, Volatile; Female; Starch; Steam; Zea mays
PubMed: 32910163
DOI: 10.1093/jas/skaa302 -
Journal of Clinical Laboratory Analysis Jun 2019The underlying interactions between ABO blood group antigens and pancreatic exocrine tissue have been demonstrated, and serum amylase was synthesized by pancreatic...
OBJECTIVE
The underlying interactions between ABO blood group antigens and pancreatic exocrine tissue have been demonstrated, and serum amylase was synthesized by pancreatic ductal cells. Thus, we investigated the link between ABO blood type and serum amylase activity in Chinese subjects.
METHODS
Our study included 343 relatively healthy Chinese individuals, and the data were retrieved from electronic medical record database.
RESULTS
A increased trend was observed for serum amylase activity in ABO blood type distribution, and we found that serum amylase activity was remarkable increased in subjects with O blood type compared to those with non-O blood type (P = 0.013). Logistic regression analysis indicated that serum amylase was independently associated with individuals with O blood group (adjusted odds ratio 1.574; 95% CI, 1.022-2.425, P = 0.039).
CONCLUSIONS
The present evidence suggests a significant link between serum amylase activity and ABO blood type in the study population, indicating ABO blood type may be associated with the susceptibility of pancreatic disease.
Topics: ABO Blood-Group System; Adolescent; Adult; Amylases; Asian People; Female; Humans; Logistic Models; Male; Middle Aged; Pancreatic alpha-Amylases; Retrospective Studies
PubMed: 30938472
DOI: 10.1002/jcla.22883