scholarly journals A Novel D-Allulose 3-Epimerase Gene from the Metagenome of a Thermal Aquatic Habitat and D-Allulose Production by Bacillus subtilis Whole-Cell Catalysis

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Satya Narayan Patel ◽  
Girija Kaushal ◽  
Sudhir P. Singh
Keyword(s):  
Thermal Stability ◽  
Bacillus Subtilis ◽  
Water Reservoir ◽  
Heat Stability ◽  
Thermal Spring ◽  
Hot Water ◽  
Whole Cell ◽  
Content Type ◽  
Extraction And Purification ◽  
Whole Cell Catalysis

ABSTRACT A novel d-allulose 3-epimerase gene (daeM) has been identified from the metagenomic resource of a hot-water reservoir. The enzyme epimerizes d-fructose into d-allulose, a functional sugar of rare abundance in nature. The metagenomic DNA fragment was cloned and expressed in Escherichia coli. The purified recombinant protein (DaeM) was found to be metal dependent (Co2+ or Mn2+). It displayed the maximal levels of catalytic activity in a pH range of 6 to 11 and a temperature range of 75°C to 80°C. The enzyme exhibited remarkably high thermal stability at 60°C and 70°C, with half-life values of 9,900 and 3,240 min, respectively. To the best of our knowledge, this is the highest thermal stability demonstrated by a d-allulose 3-epimerase that has been characterized to date. The enzymatic treatment of 700 mg·ml−1 d-fructose yielded about 217 mg·ml−1 d-allulose, under optimal condition. The catalytic product was purified, and its nuclear magnetic resonance (NMR) spectra were found to be indistinguishable from those of standard d-allulose. For biomolecule production, the whole-cell catalysis procedure avoids the tedious process of extraction and purification of enzyme and also offers better biocatalyst stability. Further, it is desirable to employ safe-grade microorganisms for the biosynthesis of a product. The daeM gene was expressed intracellularly in Bacillus subtilis. A whole-cell catalysis reaction performed with a reaction volume of 1 liter at 60°C yielded approximately 196 g·liter−1 d-allulose from 700 g·liter−1 d-fructose. Further, the whole recombinant cells were able to biosynthesize d-allulose in apple juice, mixed fruit juice, and honey. IMPORTANCE d-Allulose is a noncaloric sugar substitute with antidiabetes and antiobesity potential. With several characteristics of physiological significance, d-allulose has wide-ranging applications in the food and pharmacology industries. The development of a thermostable biocatalyst is an objective of mainstream research aimed at achieving industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered a potential metagenomic resource of heat-tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of the Tattapani geothermal region, Chhattisgarh, India, discovering a novel d-allulose 3-epimerase gene, daeM, encoding an enzyme of high-level heat stability. The daeM gene was expressed in the microbial cells of a nonpathogenic and safe-grade species, B. subtilis, which was found to be capable of performing d-fructose to d-allulose interconversion via a whole-cell catalysis reaction. The results indicate that DaeM is a potential biocatalyst for commercial production of the rare sugar d-allulose. The study established that extreme environmental niches represent a genomic resource of functional sugar-related biocatalysts.

Effect of preparation process on elevated temperature tribological properties of composite polyurea grease

2016 ◽  
Vol 68 (5) ◽  
pp. 611-616 ◽  
Author(s):  
Zujian Shen ◽  
Fei Geng ◽  
Xinxin Fan ◽  
Zhichen Shen ◽  
Haiyan Wang
Keyword(s):  
Thermal Stability ◽  
Elevated Temperature ◽  
Tribological Properties ◽  
Water Resistance ◽  
Physical Method ◽  
Heat Stability ◽  
Wear Properties ◽  
Excellent Thermal Stability ◽  
Content Type ◽  
Polyurea Grease

Purpose This paper aims to investigate and prepare the composite polyurea greases with excellent thermal stability and tribological properties. Design/methodology/approach In this paper, composite Ba-based (Ba, barium) tetra-polyurea lubricating greases were prepared with two different methods: mixing Ba-based gelatinizer and tetra-polyurea gelatinizer by a physical method; and introducing barium carboxylate into tetra-polyurea molecules by a chemical method. The properties of the products, such as heat stability, water resistance and friction performance, were analyzed with thermogravimetry, water-resistance test and four-ball friction test. Findings The results indicated that the products obtained by chemically introducing barium carboxylate into tetra-urea molecules showed better elevated temperature tribological properties, and the disadvantages of the polyurea greases with high temperature hardening were also obviously improved. The cone penetration rate at 180°C for 24 h is only 3 per cent. The friction coefficient can be decreased to 0.44 and the last non-seizure load value was increased from 560 N to 1,120 N without any other additives. Originality/value The research is significant because the prepared composite grease showed excellent performances, such as the outstanding thermal stability, water resistance and excellent extreme pressure and anti-wear properties, which may be widely applied in steel, metallurgy, bearings and other industrial fields.

scholarly journals Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry

2011 ◽  
Vol 77 (17) ◽  
pp. 6225-6232 ◽  
Author(s):  
N. Parthuisot ◽  
M. Binet ◽  
A. Touron-Bodilis ◽  
C. Pougnard ◽  
P. Lebaron ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Natural Waters ◽  
Solid Phase ◽  
Environmental Water ◽  
Thermal Spring ◽  
Hot Water ◽  
Content Type ◽  
Thermal Spring Water ◽  
Plate Counts

ABSTRACTA new method was developed for the rapid and sensitive detection of viableLegionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viableL. pneumophilacells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter−1, and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viableL. pneumophilacells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 103viable but nonculturableL. pneumophilacells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viableL. pneumophilain both man-made water systems and environmental water samples.

scholarly journals An Alkylpyrazine Synthesis Mechanism Involving L-Threonine-3-Dehydrogenase Describes the Production of 2,5-Dimethylpyrazine and 2,3,5-Trimethylpyrazine by Bacillus subtilis

2019 ◽  
Vol 85 (24) ◽  
Author(s):  
Lijie Zhang ◽  
Yanli Cao ◽  
Jianan Tong ◽  
Yan Xu
Keyword(s):  
Bacillus Subtilis ◽  
Fermented Foods ◽  
Synthesis Mechanism ◽  
Content Type ◽  
Coa Ligase ◽  
Starting Point ◽  
Whole Cell Catalysis ◽  
Ph Dependent ◽  
Traditional Fermented Foods ◽  
Substrate Addition

ABSTRACT Alkylpyrazines are important contributors to the flavor of traditional fermented foods. Here, we studied the synthesis mechanisms of 2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP). Substrate addition, whole-cell catalysis, stable isotope tracing experiments, and gene manipulation revealed that l-threonine is the starting point involving l-threonine-3-dehydrogenase (TDH) and three uncatalyzed reactions to form 2,5-DMP. TDH catalyzes the oxidation of l-threonine. The product of this reaction is l-2-amino-acetoacetate, which is known to be unstable and can decarboxylate to form aminoacetone. It is proposed that aminoacetone spontaneously converts to 2,5-DMP in a pH-dependent reaction, via 3,6-dihydro-2,5-DMP. 2-Amino-3-ketobutyrate coenzyme A (CoA) ligase (KBL) catalyzes the cleavage of l-2-amino-acetoacetate, the product of TDH, into glycine and acetyl-CoA in the presence of CoA. Inactivation of KBL could improve the production of 2,5-DMP. Besides 2,5-DMP, TMP can also be generated by Bacillus subtilis 168 by using l-threonine and d-glucose as the substrates and TDH as the catalytic enzyme. IMPORTANCE Despite alkylpyrazines' contribution to flavor and their commercial value, the synthesis mechanisms of alkylpyrazines by microorganisms remain poorly understood. This study revealed the substrate, intermediates, and related enzymes for the synthesis of 2,5-dimethylpyrazine (2,5-DMP), which differ from the previous reports about the synthesis of 2,3,5,6-tetramethylpyrazine (TTMP). The synthesis mechanism described here can also explain the production of 2,3,5-trimethylpyrazine (TMP). The results provide insights into an alkylpyrazine’s synthesis pathway involving l-threonine-3-dehydrogenase as the catalytic enzyme and l-threonine as the substrate.

scholarly journals Two different isoschizomers of the type-II restriction endonuclease Taq I (T/CGA) within the same Thermus isolate: Tsp32 I, an enzyme with similar heat stability properties to the prototype enzyme Taq I, and Tsp32 II, a hyperthermostable isoschizomer of Taq I

1995 ◽  
Vol 312 (2) ◽  
pp. 505-510 ◽  
Author(s):  
S G Welch ◽  
R A D Williams
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Cleavage Site ◽  
Heat Stability ◽  
Hot Water ◽  
Heat Inactivation ◽  
Type Ii ◽  
Magnesium Ions ◽  
Water Springs

We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I. Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation. Subsequently it became clear that the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I. One of the enzymes, Tsp32 I, has similar thermal stability characteristics to Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C. Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals Streptomycin-Induced Expression in Bacillus subtilis of YtnP, a Lactonase-Homologous Protein That Inhibits Development and Streptomycin Production in Streptomyces griseus

2011 ◽  
Vol 78 (2) ◽  
pp. 599-603 ◽  
Author(s):  
Johannes Schneider ◽  
Ana Yepes ◽  
Juan C. Garcia-Betancur ◽  
Isa Westedt ◽  
Benjamin Mielich ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signaling Pathway ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Induced Expression ◽  
Content Type ◽  
Homologous Protein

ABSTRACTBacillus subtilisinduces expression of the geneytnPin the presence of the antimicrobial streptomycin, produced by the Gram-positive bacteriumStreptomyces griseus.ytnPencodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium inS. griseus.

scholarly journals Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

2016 ◽  
Vol 198 (21) ◽  
pp. 2925-2935 ◽  
Author(s):  
Heng Zhao ◽  
Yingjie Sun ◽  
Jason M. Peters ◽  
Carol A. Gross ◽  
Ethan C. Garner ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Repression ◽  
Cell Envelope ◽  
Turgor Pressure ◽  
Teichoic Acid ◽  
Lethal Gene ◽  
Genetic Redundancy ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Lipid Ii

ABSTRACTThe integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. InBacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimizedclusteredregularlyinterspacedshortpalindromicrepeat (CRISPR) system with catalytically inactive (“dead”)CRISPR-associated protein9(dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate thatB. subtilisrequires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the σM-dependent cell envelope stress response, includingbcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of theB. subtilisUPP-Pase enzymes, and provide further evidence linking the σMregulon to cell envelope homeostasis pathways.IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

scholarly journals In VitroandIn VivoCharacterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase

2016 ◽  
Vol 60 (8) ◽  
pp. 4830-4839 ◽  
Author(s):  
Christopher M. Tan ◽  
Charles J. Gill ◽  
Jin Wu ◽  
Nathalie Toussaint ◽  
Jingjun Yin ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Antibacterial Agents ◽  
Cell Activity ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Bacterial Dna ◽  
Whole Cell ◽  
Content Type

ABSTRACTOxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, andin vivocharacterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV fromStaphylococcus aureusandEscherichia coliand displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergencein vitroat concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activityin vitroandin vivo.

Experimental investigation of a pyramid type solar still with porous material: productivity assessment

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Naresh Yarramsetty ◽  
Naveen Sharma ◽  
Modumudi Lakshmi Narayana
Keyword(s):  
Porous Material ◽  
Saline Water ◽  
Base Plate ◽  
Hot Water ◽  
Feed Water ◽  
Solar Still ◽  
Absorber Plate ◽  
Content Type ◽  
Water Distillation ◽  
Clay Pots

Purpose This study aims to investigate the effect of porous material (clay pots) and it is facing on the productivity performance of a pyramid type solar still. The clay pots are placed in the basin facing up and facing down. The numbers of clay pots considered were 9 and 25, and its performance was compared with normal (0 clay pots) solar still. Design/methodology/approach The pyramid solar water distillation system has been designed, fabricated and tested under the actual environmental conditions of Kanchikacherla (16.6834 0N, 80.3904 0E), Andhra Pradesh, India. The solar still is used to produce the fresh water and hot water simultaneously from the brackish (i.e. containing dissolved salts) feed water for domestic applications. From open literature, it was established that the rate of evaporation is higher when the flowing water is held for a longer duration on the black color absorber plate, thereby leading to an increase in productivity of freshwater. Therefore, the pyramid solar still has been tested for smooth absorber plate and the absorber plate with porous heat storage material. Findings The porous material increases the production rate of freshwater compared to a base plate. However, the pyramid still with clay pots has higher productivity at a lower temperature because of the porosity effect. Originality/value The total dissolved solids, electrical conductivity and pH of the distilled water and the saline water have also been measured and compared.

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