scholarly journals β-1,3-Glucanase fromDelftia tsuruhatensisStrain MV01 and Its Potential Application in Vinification

2010 ◽  
Vol 77 (3) ◽  
pp. 983-990 ◽  
Author(s):  
V. Blättel ◽  
M. Larisika ◽  
P. Pfeiffer ◽  
C. Nowak ◽  
A. Eich ◽  
...  
Keyword(s):  
Cell Walls ◽  
Hydrolytic Enzyme ◽  
Glycolytic Enzyme ◽  
Wild Type ◽  
Microbial Activities ◽  
Yeast Growth ◽  
Wine Quality ◽  
Ph Values ◽  
Delftia Tsuruhatensis ◽  
Metabolic Activities

ABSTRACTDuring vinification microbial activities can spoil wine quality. As the wine-related lactic acid bacteriumPediococcus parvulusis able to produce slimes consisting of a β-1,3-glucan, must and wine filtration can be difficult or impossible. In addition, the metabolic activities of several wild-type yeasts can also negatively affect wine quality. Therefore, there is a need for measures to degrade the exopolysaccharide fromPediococcus parvulusand to inhibit the growth of certain yeasts. We examined an extracellular β-1,3-glucanase fromDelftia tsuruhatensisstrain MV01 with regard to its ability to hydrolyze both polymers, the β-1,3-glucan fromPediococcusand that from yeast cell walls. The 29-kDa glycolytic enzyme was purified to homogeneity. It exhibited an optimal activity at 50°C and pH 4.0. The sequencing of the N terminus revealed significant similarities to β-1,3-glucanases from different bacteria. In addition, the investigations indicated that this hydrolytic enzyme is still active under wine-relevant parameters such as elevated ethanol, sulfite, and phenol concentrations as well as at low pH values. Therefore, the characterized enzyme seems to be a useful tool to prevent slime production and undesirable yeast growth during vinification.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis

Science ◽  
2013 ◽  
Vol 341 (6150) ◽  
pp. 1103-1106 ◽  
Author(s):  
Ruben Vanholme ◽  
Igor Cesarino ◽  
Katarzyna Rataj ◽  
Yuguo Xiao ◽  
Lisa Sundin ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Walls ◽  
Metabolite Profiling ◽  
Biosynthetic Pathway ◽  
Wild Type ◽  
Secondary Cell Walls ◽  
Phenolic Metabolite ◽  
In The Wild ◽  
Lignin Biosynthetic Pathway

Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.

scholarly journals Hexokinase is necessary for glucose-mediated photosynthesis repression and lipid accumulation in a green alga

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Melissa S. Roth ◽  
Daniel J. Westcott ◽  
Masakazu Iwai ◽  
Krishna K. Niyogi
Keyword(s):  
Green Alga ◽  
Lipid Droplets ◽  
Fatty Acid Biosynthesis ◽  
Glycolytic Enzyme ◽  
Model System ◽  
Forward Genetics ◽  
Oxygenic Photosynthesis ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
Metabolic Switch

Abstract Global primary production is driven largely by oxygenic photosynthesis, with algae as major contributors. The green alga Chromochloris zofingiensis reversibly switches off photosynthesis in the presence of glucose in the light and augments production of biofuel precursors (triacylglycerols) and the high-value antioxidant astaxanthin. Here we used forward genetics to reveal that this photosynthetic and metabolic switch is mediated by the glycolytic enzyme hexokinase (CzHXK1). In contrast to wild-type, glucose-treated hxk1 mutants do not shut off photosynthesis or accumulate astaxanthin, triacylglycerols, or cytoplasmic lipid droplets. We show that CzHXK1 is critical for the regulation of genes related to photosynthesis, ketocarotenoid synthesis and fatty acid biosynthesis. Sugars play fundamental regulatory roles in gene expression, physiology, metabolism, and growth in plants and animals, and we introduce a relatively simple, emerging model system to investigate conserved eukaryotic sugar sensing and signaling at the base of the green lineage.

scholarly journals H2-Independent Growth of the Hydrogenotrophic Methanogen Methanococcus maripaludis

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Kyle C. Costa ◽  
Thomas J. Lie ◽  
Michael A. Jacobs ◽  
John A. Leigh
Keyword(s):  
Carbon Monoxide ◽  
Slow Growth ◽  
Glycolytic Enzyme ◽  
Wild Type ◽  
Methanococcus Maripaludis ◽  
Content Type ◽  
Hydrogenotrophic Methanogen ◽  
Alternative Pathways ◽  
Lyase Activity ◽  
First Time

ABSTRACTHydrogenotrophic methanogenicArchaearequire reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H2oxidation by the hydrogenase Eha provides these electrons, consistent with an H2requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction inMethanococcus maripaludisthat operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H2-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases ofM. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F420, should be abundant. Indeed, when F420-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H2production via an F420-dependent formate:H2lyase activity shifted markedly toward H2compared to the wild type.IMPORTANCEHydrogenotrophic methanogens are thought to require H2as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H2requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H2. The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

scholarly journals A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

2012 ◽  
Vol 25 (4) ◽  
pp. 443-452 ◽  
Author(s):  
Akhil Srivastava ◽  
Robin A. Ohm ◽  
Lindsay Oxiles ◽  
Fred Brooks ◽  
Christopher B. Lawrence ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Late Stage ◽  
Hydrolytic Enzyme ◽  
Alternaria Brassicicola ◽  
Predicted Amino Acid Sequence ◽  
Broad Host Range ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Reporter Protein

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall–degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19-reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

scholarly journals Variation in pH Optima of Hydrolytic Enzyme Activities in Tropical Rain Forest Soils

2010 ◽  
Vol 76 (19) ◽  
pp. 6485-6493 ◽  
Author(s):  
Benjamin L. Turner
Keyword(s):  
Soil Ph ◽  
Forest Soils ◽  
Extracellular Enzymes ◽  
Hydrolytic Enzyme ◽  
Hydrolytic Enzymes ◽  
Acidic Ph ◽  
Acidic Soils ◽  
Fluorogenic Substrates ◽  
Ph Values ◽  
Optimum Ph

ABSTRACT Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.

Studies of the activity of chymotrypsin

1970 ◽  
Vol 257 (813) ◽  
pp. 89-104 ◽  
Keyword(s):  
Equilibrium Constants ◽  
Hydrolytic Enzyme ◽  
Enzyme Activation ◽  
Ethyl Esters ◽  
Activation Mechanism ◽  
Relaxation Techniques ◽  
Kinetic Measurements ◽  
Ph Values ◽  
Solution Studies ◽  
Hydrolysis Of

Physical and chemical studies of chymotrypsin in solution and kinetic measurements of individual steps in reactions catalysed by this hydrolytic enzyme are reported. The solution studies also provide a basis for correlation of individual reaction steps with structural information obtained by Drs Blow and Hartley and discussed at this Discussion. The rate and equilibrium constants pertaining to the three-step mechanism shown below have been determined for the chymotrypsin catalysed hydrolysis of three specific ester substrates—the ethyl esters of V-acetyl-L-tyrosine, N -acetyl-L-tryptophan, and N -acetyl-L-phenylalanine—at selected pH values with use of both flow and relaxation techniques. iV-acetyl-L-phenylalanine—at selected pH values with use of both flow and relaxation techniques. / * E + S ^ 4 E S -y » EP 2 ,---► E + P 2 , * 2 3 £ 3 4 where E is enzyme, S is substrate, ES is a complex, EP 2 is an intermediate compound, and the products P 1 and P 2 are, respectively, an alcohol or amine and an acid. The hydrolysis of chymotrypsin specific substrates is shown to be considerably more complex than is indicated by this previously proposed mechanism. In addition to the steps shown, there exist a number of ionization and conformational equilibria which account for the bell-shaped pH-rate profiles observed for chymotrypsin catalysed reactions. A catalytically active enzyme conformation, which is in pH dependent equilibrium with a catalytically inactive conformation, is shown to have its origin in the enzyme activation mechanism. In the chymotrypsin catalysed hydrolysis of an amide substrate, furylacryloyltryptophanamide, there has been detected an additional intermediate which differs from the intermediate EP 2 observed in ester hydrolysis.

Role of different Escherichia coli hydrogenases in H+ efflux and F1Fo-ATPase activity during glycerol fermentation at different pH values

2011 ◽  
Vol 31 (3) ◽  
pp. 179-184 ◽  
Author(s):  
Syuzanna Blbulyan ◽  
Arev Avagyan ◽  
Anna Poladyan ◽  
Armen Trchounian
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Membrane Vesicle ◽  
Wild Type ◽  
Glucose Fermentation ◽  
Glycerol Fermentation ◽  
Whole Cells ◽  
Ph Values ◽  
F1fo Atpase

Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild–type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

scholarly journals The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar

2021 ◽  
Vol 6 (12) ◽  
pp. 1583-1592
Author(s):  
Tanner G. DeHart ◽  
Mara R. Kushelman ◽  
Sherry B. Hildreth ◽  
Richard F. Helm ◽  
Brandon L. Jutras
Keyword(s):  
Cell Wall ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Cell Walls ◽  
Structural Strength ◽  
Cell Envelope ◽  
Wild Type ◽  
Tick Vector ◽  
Cell Wall Alteration ◽  
Main Component

AbstractPeptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) β-(1–4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc–GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility.

scholarly journals The Synthesis of Modified Trehalose Glycolipids: Towards Understanding Mincle and MCL Binding

2021 ◽  
Author(s):  
◽  
Jessica Helen Bird
Keyword(s):  
Cell Walls ◽  
Biological Activities ◽  
Chain Fatty Acid ◽  
Wild Type ◽  
Mycobacterium Species ◽  
Immunostimulatory Activity ◽  
Long Chain Fatty Acid ◽  
Mycolic Acids ◽  
Adjuvant Therapies ◽  
Long Chain

<p>Trehalose glycolipids are a diverse family of long-chain fatty acid diesters isolated from the cell walls of bacteria, in particular Mycobacterium species including M. tuberculosis. These molecules possess an array of biological activities which contribute to the survival and virulence of the organism,however, it is their activity as potent stimulators of innate and early adaptive immunity for which they are of interest. In particular, trehalose glycolipids have an application as adjuvants in vaccines and immunotherapies, for diseases such as tuberculosis (TB) and cancer. Recently, the macrophage-inducible C-type lectin, Mincle, and the macrophage C-type lectin, MCL, were identified as receptors for trehalose glycolipids, however, the exact mechanisms by which these receptors recognise and bind glycolipids is, as yet, unknown.This thesis presents the synthesis of a variety of structurally diverse trehalose glycolipid analogues. As such, three mycolic acids bearing a C22 α-chain and diversified meromycolate branches were prepared from an epoxide intermediate, itself prepared in eight steps from commercially available starting materials. The mycolic acids were then coupled to TMS-trehalose and subsequently deprotected to give the mono-and diester derivatives, 1a-cand 2c, which will be assessed for their immunostimulatory activity through the activation of wild type and Mincle-/-murine macrophages. This work provides a first step towards determining how the structures of trehalose glycolipids influence Mincle and MCL binding and activity, and allow for the development of improved trehalose glycolipids for use in adjuvant therapies.</p>

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