substrate concentrations
Recently Published Documents


TOTAL DOCUMENTS

525
(FIVE YEARS 80)

H-INDEX

45
(FIVE YEARS 6)

2021 ◽  
Author(s):  
Timm Bayer ◽  
Elizabeth Tomaszewski ◽  
Casey Bryce ◽  
Andreas Kappler ◽  
James Byrne

Laboratory-based studies on microbial Fe(II) oxidation are commonly performed over just a few weeks in small volumes with high substrate concentrations, resulting in geochemical gradients and volumetric effects caused by sampling. We used a chemostat to enable uninterrupted supply of medium, and investigated autotrophic growth of the nitrate-reducing Fe(II)-oxidizing culture KS for 24 days. We analysed Fe- and N-speciation, cell-mineral associations, and the identity of minerals. Results were compared to different batch systems (50 and 700 ml – static/shaken). The Fe(II) oxidation rate was highest in the chemostat with 7.57 mM Fe(II) d-1, while the extent was similar (averaged 92% of all Fe(II)). Short-range ordered Fe(III) phases, presumably ferrihydrite, precipitated and later goethite was detected in the chemostat. 1 mM solid phase Fe(II) remained in the chemostat, up to 15 µM of reactive nitrite was measured, and 42% of visualized cells were partially or completely mineral-encrusted, likely caused by abiotic oxidation of Fe(II) by nitrite. Despite (partial) encrustation, cells were still viable. Our results show that even with similar oxidation rates as in batch cultures, cultivating Fe(II)-oxidizing microorganisms under continuous conditions reveals mechanistic insights on the role of reactive intermediates for Fe(II) oxidation, mineral formation and cell-mineral interactions.


2021 ◽  
Vol 11 (23) ◽  
pp. 11493
Author(s):  
Marlene Vuillemin ◽  
Jesper Holck ◽  
Martin Matwiejuk ◽  
Eduardo S. Moreno Prieto ◽  
Jan Muschiol ◽  
...  

The lacto-N-biosidase LnbB from Bifidobacterium bifidum JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-N-tetraose (LNT; Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide in itself and constitutes the structural core of a range of more complex human milk oligosaccharides as well. Thirteen different LnbB variants were expressed and screened for transglycosylation activity by monitoring transglycosylation product formation using lacto-N-biose 1,2-oxazoline as donor substrate and lactose as acceptor substrate. LNT was the major reaction product, yet careful reaction analysis revealed the formation of three additional LNT isomers, which we identified to have a β1,2-linkage, a β1,6-linkage, and a 1,1-linkage, respectively, between lacto-N-biose (Gal-β1,3-GlcNAc) and lactose. Considering both maximal transglycosylation yield and regioselectivity as well as minimal product hydrolysis, the best variant was LnbB W394H, closely followed by W465H and Y419N. A high transglycosylation yield was also obtained with W394F, yet the substitution of W394 and W465 of the subsite −1 hydrophobic platform in the enzyme with His dramatically impaired the undesirable product hydrolysis as compared to substitution with Phe; the effect was most pronounced for W465. Using p-nitrophenyl-β-lacto-N-bioside as donor substrate manifested W394 as an important target position. The optimization of the substrate concentrations confirmed that high initial substrate concentration and high acceptor-to-donor ratio both favor transglycosylation.


2021 ◽  
Vol 1 ◽  
Author(s):  
Laurine Ducrot ◽  
Megan Bennett ◽  
Adam A. Caparco ◽  
Julie A. Champion ◽  
Andreas S. Bommarius ◽  
...  

Small optically active molecules, and more particularly short-chain chiral amines, are key compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-biocatalytic production on a commercial scale is already established, mainly through lipase-catalyzed resolutions leading to ChiPros™ products among others. Nevertheless, their biocatalytic synthesis remains challenging for very short-chain C4 to C5 amines due to low enantiomeric excess. To complement the possibilities recently offered by transaminases, this work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, MsmeAmDH, MicroAmDH, and MATOUAmDH2) were shown to be efficient for the synthesis of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol (99.4%), and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale-up experiments were successfully performed at 150 mM substrate concentrations for the synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-MOIPA”. Modeling studies provided some preliminary results explaining the basis for the challenging discrimination between similarly sized substituents in the active sites of these enzymes.


Energies ◽  
2021 ◽  
Vol 14 (22) ◽  
pp. 7752
Author(s):  
Eunjin Jwa ◽  
Mijin Kim ◽  
Ji-Hyung Han ◽  
Namjo Jeong ◽  
Hyun-Chul Kim ◽  
...  

Decreasing the Pt loading and surface area of the cathode was found to accelerate the hydrogen evolution reaction in microbial electrolysis cells (MEC) at low substrate concentrations. The experimental wire cathode used in this study had a reduced Pt loading of 20 µg Pt/cm2 and only 14% of the surface area of the control disk-type cathode. With the wire cathodes, peak current densities of 33.1 ± 2.3 A/m2 to 30.4 ± 0.5 A/m2 were obtained at substrate concentrations of 0.4 g/L and 1.0 g/L, respectively, which were 5.4 to 6.2 times higher than those obtained with the disk electrode (5.1–5.7 A/m2). The higher cathode overpotentials and higher current densities obtained with the wire electrode compared to those observed with the disk electrode were advantageous for hydrogen recovery, energy recovery efficiencies, and the hydrogen volume produced (8.5 ± 1.2 mL at 0.4 g/L to 23.0 ± 2.2 mL at 1.0 g/L with the wire electrode; 6.8 ± 0.4 mL at 0.4 g/L to 21.8 ± 2.2 mL at 1.0 g/L with the disk electrode). Therefore, the wire electrode, which used only 0.6% of the Pt catalyst amount in typical disk-type electrodes (0.5 mg Pt/cm2), was effective at various substrate concentrations. The results of this study are very promising because the capital cost of the MEC reactors can be greatly reduced if the wire-type electrodes with ultralow Pt loading are utilized in field applications.


Processes ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 2050
Author(s):  
Mohammed Hanaki ◽  
Jérôme Harmand ◽  
Zoubida Mghazli ◽  
Alain Rapaport ◽  
Tewfik Sari ◽  
...  

A two-step model of the anaerobic digestion process is mathematically and numerically studied. The focus of the paper is put on the hydrolysis and methanogenesis phases when applied to the digestion of waste with a high content of solid matter: existence and stability properties of the equilibrium points are investigated. The hydrolysis step is considered a limiting step in this process using the Contois growth function for the bacteria responsible for the first degradation step. The methanogenesis step being inhibited by the product of the first reaction (which is also the substrate for the second one), and the Haldane growth rate is used for the second reaction step. The operating diagrams with respect to the dilution rate and the input substrate concentrations are established and discussed.


2021 ◽  
Author(s):  
Andrea Söllinger ◽  
Joana Séneca ◽  
Mathilde Borg Dahl ◽  
Liabo L. Motleleng ◽  
Judith Prommer ◽  
...  

Abstract How soil microorganisms respond to global warming is key to infer future soil-climate feedbacks, yet poorly understood. Here we applied metatranscriptomics to investigate microbial physiological responses to medium- (8 years) and long-term (>50 years) subarctic grassland soil warming of +6 °C. Besides indications for a community-wide upregulation of central metabolisms and cell replication we observed a downregulation of the protein biosynthesis machinery in the warmed soils, coinciding with a lower microbial biomass, RNA, and soil substrate content. We conclude that permanently accelerated reaction rates at higher temperatures and reduced substrate concentrations results in a cellular reduction of ribosomes, the macromolecular complexes carrying out protein biosynthesis. Later efforts to test this, including a short-term warming experiment (6 weeks, +6 °C), further supported our conclusion. Downsizing the protein biosynthesis machinery facilitates liberation of energy and matter, allowing microorganisms to maintain high metabolic activities and cell division rates even after decades of warming.


Author(s):  
Taresh P. Khobragade ◽  
Sharad Sarak ◽  
Amol D. Pagar ◽  
Hyunwoo Jeon ◽  
Pritam Giri ◽  
...  

Herein, we report the development of a multi-enzyme cascade using transaminase (TA), esterase, aldehyde reductase (AHR), and formate dehydrogenase (FDH), using benzylamine as an amino donor to synthesize the industrially important compound sitagliptin intermediate. A panel of 16 TAs was screened using ethyl 3-oxo-4-(2,4,5-trifluorophenyl) butanoate as a substrate (1). Amongst these enzymes, TA from Roseomonas deserti (TARO) was found to be the most suitable, showing the highest activity towards benzylamine (∼70%). The inhibitory effect of benzaldehyde was resolved by using AHR from Synechocystis sp. and FDH from Pseudomonas sp., which catalyzed the conversion of benzaldehyde to benzyl alcohol at the expense of NAD(P)H. Reaction parameters, such as pH, buffer system, and concentration of amino donor, were optimized. A single whole-cell system was developed for co-expressing TARO and esterase, and the promoter engineering strategy was adopted to control the expression level of each biocatalyst. The whole-cell reactions were performed with varying substrate concentrations (10–100 mM), resulting in excellent conversions (ranging from 72 to 91%) into the desired product. Finally, the applicability of this cascade was highlighted on Gram scale, indicating production of 70% of the sitagliptin intermediate with 61% isolated yield. The protocol reported herein may be considered an alternative to existing methods with respect to the use of cheaper amine donors as well as improved synthesis of (R) and (S) enantiomers with the use of non-chiral amino donors.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Alberto Rodriguez ◽  
Jamie A. Meadows ◽  
Ning Sun ◽  
Blake A. Simmons ◽  
John M. Gladden

AbstractHydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.


Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 322
Author(s):  
Raouia Attaallah ◽  
Aziz Amine

An amperometric biosensor based on tyrosinase, immobilized onto a carbon black paste electrode using glutaraldehyde and BSA was constructed to detect competitive inhibitors. Three inhibitors were used in this study: benzoic acid, sodium azide, and kojic acid, and the obtained values for fifty percent of inhibition (IC50) were 119 µM, 1480 µM, and 30 µM, respectively. The type of inhibition can also be determined from the curve of the degree of inhibition by considering the shift of the inhibition curves. Amperometric experiments were performed with a biosensor polarized at the potential −0.15 V vs. Ag/AgCl and using 0.1 M phosphate buffer (pH 6.8) as an electrolyte. Under optimized conditions, the proposed biosensor showed a linear amperometric response toward catechol detection from 0.5 µM to 38 µM with a detection limit of 0.35 µM (S/N = 3), and its sensitivity was 66.5 mA M−1 cm−2. Moreover, the biosensor exhibited a good storage stability. Conversely, a novel graphical plot for the determination of reversible competitive inhibition was represented for free tyrosinase. The graph consisted of plotting the half-time reaction (t1/2) as a function of the inhibitor concentration at various substrate concentrations. This innovative method relevance was demonstrated in the case of kojic acid using a colorimetric bioassay relying on tyrosinase inhibition. The results showed that the t1/2 provides an extended linear range of tyrosinase inhibitors.


2021 ◽  
Vol 118 (35) ◽  
pp. e2111257118
Author(s):  
Yuki Toyama ◽  
Robert W. Harkness ◽  
Lewis E. Kay

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy–based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding–incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson’s disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.


Sign in / Sign up

Export Citation Format

Share Document