scholarly journals Highly Efficient Synthesis of Glutathione via a Genetic Engineering Enzymatic Method Coupled with Yeast ATP Generation

Catalysts ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 33
Author(s):  
Chen Huang ◽  
Zhimin Yin
Keyword(s):  
Yeast Cell ◽  
Feedback Inhibition ◽  
Enzymatic Reaction ◽  
Reaction System ◽  
Enzyme Reaction ◽  
Catalytic Efficiency ◽  
Yeast Cells ◽  
Glutathione Synthetase ◽  
Oxidized Glutathione ◽  
Separation And Purification

Glutathione is a tripeptide compound with many important physiological functions. A new, two-step reaction system has been developed to efficiently synthesize glutathione. In the first step, glutamate and cysteine are condensed to glutamyl-cysteine by endogenous yeast enzymes inside the yeast cell, while consuming ATP. In the second step, the yeast cell membrane is lysed by the permeabilizing agent CTAB (cetyltrimethylammonium bromide) to release the glutamyl-cysteine, upon which added glutathione synthetase converts the glutamyl-cysteine and added glycine into glutathione. The ATP needed for this conversion is supplied by the permeabilized yeast cells of glycolytic pathway. This method provided sufficient ATP, and reduced the feedback inhibition of glutathione for the first-step enzymatic reaction, thereby improving the catalytic efficiency of the enzyme reaction. In addition, the formation of suitable oxidative stress environment in the reaction system can further promote glutathione synthesis. By HPLC analysis of the glutathione, it was found that 2.1 g/L reduced glutathione is produced and 17.5 g/L oxidized glutathione. Therefore, the new reaction system not only increases the total glutathione, but also facilitates the subsequent separation and purification due to the larger proportion of oxidized glutathione in the reaction system.

scholarly journals Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

2019 ◽  
Author(s):  
Josef R Bober ◽  
Nikhil Nair
Keyword(s):  
Enzymatic Reaction ◽  
Reaction System ◽  
Catalytic Efficiency ◽  
Batch Process ◽  
Product Formation ◽  
Cell System ◽  
Fast Kinetics ◽  
Equilibrium Conversion ◽  
Stability Issues ◽  
Arabinose Isomerase

AbstractThere are many enzymes that are relevant for making rare and valuable chemicals that while active, are severely limited by thermodynamic, kinetic, or stability issues (e.g. isomerases, lyases, transglycosidase etc.). In this work, we study an enzymatic reaction system −Lactobacillus sakeiL-arabinose isomerase (LsLAI) for D-galactose to D-tagatose isomerization – that is limited by all three reaction parameters. The enzyme has a low catalytic efficiency for non-natural substrate galactose, has low thermal stability at temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies to overcome these limitations, we finally show that encapsulating the enzyme in a gram-positive bacterium (Lactobacillus plantarum) that is chemically permeabilized can enable reactions at high rates, high conversion, and at high temperatures. The modified whole cell system stabilizes the enzyme, differentially partitions substrate and product across the membrane to shift the equilibrium toward product formation enables rapid transport of substrate and product for fast kinetics. In a batch process, this system enables approximately 50 % conversion in 4 h starting with 300 mM galactose (an average productivity of 37 mM/h), and 85 % conversion in 48 h, which are the highest reported for food-safe mesophilic tagatose synthesis. We suggest that such an approach may be invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-, and/or stability-limited.

scholarly journals Production, Purification, and Identification of Cholest-4-en-3-one Produced by Cholesterol Oxidase from Rhodococcus sp. in Aqueous/Organic Biphasic System

2015 ◽  
Vol 8s1 ◽  
pp. BCI.S21580 ◽  
Author(s):  
Ke Wu ◽  
Wei Li ◽  
Jianrui Song ◽  
Tao Li
Keyword(s):  
Cholesterol Oxidase ◽  
Enzymatic Reaction ◽  
Reaction System ◽  
Phase System ◽  
Separation And Purification ◽  
Two Phase ◽  
Whole Cells ◽  
Steroid Drugs ◽  
Set Up ◽  
The Cost

Cholest-4-en-3-one has positive uses against obesity, liver disease, and keratinization. It can be applied in the synthesis of steroid drugs as well. Most related studies are focused on preparation of cholest-4-en-3-one by using whole cells as catalysts, but production of high-quality cholest-4-en-3-one directly from cholesterol oxidase (COD) using an aqueous/organic two-phase system has been rarely explored. This study set up an enzymatic reaction system to produce cholest-4-en-3-one. We developed and optimized the enzymatic reaction system using COD from COX5-6 (a strain of Rhodococcus) instead of whole-cell biocatalyst. This not only simplifies and accelerates the production but also benefits the subsequent separation and purification process. Through extraction, washing, evaporation, column chromatography, and recrystallization, we got cholest-4-en-3-one with purity of 99.78%, which was identified by nuclear magnetic resonance, mass spectroscopy, and infrared spectroscopy. In addition, this optimized process of cholest-4-en-3-one production and purification can be easily scaled up for industrial production, which can largely decrease the cost and guarantee the purity of the product.

scholarly journals Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

2021 ◽  
Vol 7 (7) ◽  
pp. 520
Author(s):  
Jianmin Fu ◽  
Nohelli E. Brockman ◽  
Brian L. Wickes
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Yeast Cell ◽  
Transformation Efficiency ◽  
Transformation Frequency ◽  
Yeast Cells ◽  
Agar Concentration ◽  
Genetic Tool ◽  
Number Of Factors ◽  
Cryptococcus Spp ◽  
Transformation Systems

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

Phospholipid-bound Tissue Factor Modulates both Thrombin Generation and APC-mediated Factor Va Inactivation

1999 ◽  
Vol 82 (12) ◽  
pp. 1673-1679 ◽  
Author(s):  
Katalin Váradi ◽  
Jürgen Siekmann ◽  
Peter Turecek ◽  
H. Peter Schwarz ◽  
Victor Marder
Keyword(s):  
Tissue Factor ◽  
Surface Density ◽  
Protein C ◽  
Thrombin Generation ◽  
Feedback Inhibition ◽  
Activated Protein C ◽  
Reaction System ◽  
Inverse Correlation ◽  
High Concentration ◽  
Factor Va

SummaryHemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

scholarly journals Construction of a novel UDP-rhamnose regeneration system by a two-enzyme reaction system and application in glycosylation of flavonoid

2018 ◽  
Vol 139 ◽  
pp. 33-42 ◽  
Author(s):  
Jianjun Pei ◽  
Anna Chen ◽  
Qing Sun ◽  
Linguo Zhao ◽  
Fuliang Cao ◽  
...  
Keyword(s):  
Reaction System ◽  
Enzyme Reaction ◽  
Regeneration System

scholarly journals Bioaccessibility of curcumin encapsulated in yeast cells and yeast cell wall particles

2020 ◽  
Vol 309 ◽  
pp. 125700 ◽  
Author(s):  
Stephen Young ◽  
Rewa Rai ◽  
Nitin Nitin
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Yeast Cells ◽  
Yeast Cell Wall

scholarly journals Abundances of transcripts, proteins, and metabolites in the cell cycle of budding yeast reveal coordinate control of lipid metabolism

2020 ◽  
Vol 31 (10) ◽  
pp. 1069-1084 ◽  
Author(s):  
Heidi M. Blank ◽  
Ophelia Papoulas ◽  
Nairita Maitra ◽  
Riddhiman Garge ◽  
Brian K. Kennedy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Lipid Metabolism ◽  
Cell Division ◽  
Yeast Cell ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Dynamic Changes ◽  
Coordinate Control ◽  
Cycle Dependent ◽  
Budding Yeast Cell Cycle

In several systems, including budding yeast, cell cycle-dependent changes in the transcriptome are well studied. In contrast, few studies queried the proteome during cell division. There is also little information about dynamic changes in metabolites and lipids in the cell cycle. Here, the authors present such information for dividing yeast cells.

scholarly journals Ionic Liquids as Bifunctional Cosolvents Enhanced CO2 Conversion Catalysed by NADH-Dependent Formate Dehydrogenase

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 304 ◽  
Author(s):  
Zhibo Zhang ◽  
Bao-hua Xu ◽  
Jianquan Luo ◽  
Nicolas Solms ◽  
Hongyan He ◽  
...  
Keyword(s):  
Ionic Liquid ◽  
Ionic Liquids ◽  
Dft Calculations ◽  
Formic Acid ◽  
Formate Dehydrogenase ◽  
Enzymatic Reaction ◽  
Reaction System ◽  
Co2 Concentration ◽  
Co2 Conversion ◽  
Low Co2

Efficient CO2 conversion by formate dehydrogenase is limited by the low CO2 concentrations that can be reached in traditional buffers. The use of ionic liquids was proposed as a manner to increase CO2 concentration in the reaction system. It has been found, however, that the required cofactor (NADH) heavily degraded during the enzymatic reaction and that acidity was the main reason. Acidity, indeed, resulted in reduction of the conversion of CO2 into formic acid and contributed to overestimate the amount of formic acid produced when the progression of the reaction was followed by a decrease in NADH absorbance (method N). Stability of NADH and the mechanism of NADH degradation was investigated by UV, NMR and by DFT calculations. It was found that by selecting neutral–basic ionic liquids and by adjusting the concentration of the ionic liquid in the buffer, the concentration of NADH can be maintained in the reaction system with little loss. Conversion of CO2 to methanol in BmimBF4 (67.1%) was more than twice as compared with the conversion attained by the enzymatic reaction in phosphate buffer (24.3%).

An enhanced enzymatic reaction using a triphase system based on superhydrophobic mesoporous nanowire arrays

2019 ◽  
Vol 4 (1) ◽  
pp. 231-235 ◽  
Author(s):  
Fengying Guan ◽  
Jun Zhang ◽  
Heming Tang ◽  
Liping Chen ◽  
Xinjian Feng
Keyword(s):  
Mass Transport ◽  
Aqueous Solutions ◽  
Reaction Kinetics ◽  
Enzymatic Reaction ◽  
Reaction System ◽  
Nanowire Arrays ◽  
System P ◽  
Wide Range

Gaseous reactants play a key role in a wide range of biocatalytic reactions, however reaction kinetics are generally limited by the slow mass transport of gases (typically oxygen) in or through aqueous solutions. Herein we address this limitation by developing a triphase reaction system.

Sign in / Sign up

Export Citation Format

Share Document