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 Galactose to tagatose isomerization at moderate temperatures with high conversion and productivity

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Josef R. Bober ◽  
Nikhil U. Nair
Keyword(s):  
Lactobacillus Plantarum ◽  
Elevated Temperatures ◽  
Catalytic Efficiency ◽  
Batch Process ◽  
High Conversion ◽  
Lactobacillus Sakei ◽  
Reaction Parameters ◽  
Equilibrium Conversion ◽  
Stability Issues ◽  
Arabinose Isomerase

Abstract There are many industrially-relevant enzymes that while active, are severely limited by thermodynamic, kinetic, or stability issues (isomerases, lyases, transglycosidases). In this work, we study Lactobacillus sakeil-arabinose isomerase (LsLAI) for d-galactose to d-tagatose isomerization—that is limited by all three reaction parameters. The enzyme demonstrates low catalytic efficiency, low thermostability at temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies to overcome these limitations, we show that encapsulating LsLAI in gram-positive Lactobacillus plantarum that is chemically permeabilized enables reactions at high rates, high conversions, and elevated temperatures. In a batch process, this system enables ~ 50% conversion in 4 h starting with 300 mM galactose (an average productivity of 37 mM h−1), and 85% conversion in 48 h. We suggest that such an approach may be invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-, and/or stability-limited.

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.

PENGGUNAAN AIR SUPER KRITIS PADA DEPOLIMERISASI NYLON-12 (BATCH PROCESS)

2018 ◽  
Vol 5 (3) ◽  
Author(s):  
Mohamad Yusman
Keyword(s):  
Supercritical Water ◽  
Scale Up ◽  
Experimental Studies ◽  
Reaction System ◽  
Batch Process ◽  
Chemical Recycling ◽  
Water Decomposition ◽  
Product Distributions ◽  
New Process ◽  
Nylon 12

Water at the supercritical state is a new process for the chemical recycling. At this thermodynamic state i.e. Pc = 218 atmospheres and Tc = 374oC , water behaves very differently from its everyday temperament and it is a very good solvent for organic components. Experimental studies show that supercritical water can decompose hydrocarbons/polymers and produce useful products like 2-Azacyclotridecanone /lactam-1 from Nylon-12 (batch process). The decomposition process itself was carried out in batch reaction system in order to get more information about product distributions, time dependence, and scale-up possibilities.Keywords: supercritical water, decomposition, batch, polymer, hydrocarbon

scholarly journals Exploiting RNA thermometer-driven molecular bioprocess control as a concept for heterologous rhamnolipid production

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Philipp Noll ◽  
Chantal Treinen ◽  
Sven Müller ◽  
Lars Lilge ◽  
Rudolf Hausmann ◽  
...  
Keyword(s):  
High Titer ◽  
Production Capacity ◽  
Batch Process ◽  
Product Formation ◽  
Effect Of Temperature ◽  
Industrial Biotechnology ◽  
Temperature Sensitive ◽  
Translation Control ◽  
Rhamnolipid Production ◽  
Rna Thermometer

AbstractA key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.

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.

scholarly journals Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

2010 ◽  
Vol 76 (5) ◽  
pp. 1653-1660 ◽  
Author(s):  
Ponnandy Prabhu ◽  
Marimuthu Jeya ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Catalytic Efficiency ◽  
Homology Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
High Turnover ◽  
Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

Bistability and the ordered bimolecular mechanism

1991 ◽  
Vol 69 (9) ◽  
pp. 661-664 ◽  
Author(s):  
K. W. Raymond ◽  
Y. Pocker
Keyword(s):  
Steady State ◽  
Kinetic Constant ◽  
Substrate Inhibition ◽  
Enzymatic Reaction ◽  
Reaction System ◽  
Computational Approach ◽  
Instantaneous Velocity ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
Bimolecular Mechanism

An equation describing the instantaneous velocity of an ordered bimolecular enzymatic reaction that exhibits inhibition by substrate and product was derived. Using kinetic constant values for horse liver alcohol dehydrogenase, the velocity expression was applied to an open-reaction system. The calculated steady-state surfaces displayed regions of bistability, which further substantiates the link between substrate inhibition and multiple steady states. This general computational approach may be applied to any system that can be described by an instantaneous velocity equation.Key words: bistability, steady state, enzyme kinetics.

scholarly journals A Marine Fibrinolytic Compound FGFC1 Stimulating Enzymatic Kinetic Parameters of a Reciprocal Activation System Based on a Single Chain Urokinase-Type Plasminogen Activator and Plasminogen

2017 ◽  
Author(s):  
Guo Ruihua ◽  
Duan Dong ◽  
Shaotong Hong ◽  
Yu Zhou ◽  
Fang Wang ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Plasminogen Activator ◽  
Enzymatic Reaction ◽  
Catalytic Efficiency ◽  
Single Chain ◽  
Enzyme Substrate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Activation System ◽  
Bisindole Alkaloid

A marine fibrinolytic compound FGFC1 enhancing fibrinolysis was obtained involving in enzymatic kinetic parameters of reciprocal activation system with single chain urokinase type plasminogen activator and plasminogen. FGFC1, a kind of bisindole alkaloid from a metabolite of rare marine fungi Starchbotrys longispora FG216, modulated enzymatic kinetic parameters including fibrinolytic reaction rate and fibrin degradation characteristics. The enzymatic kinetics of fibrinolysis was described based on enzymatic reaction of chromogenic-substrate associated with p-nitroaniline (p-NA). While single chain urokinase-type plasminogen activator (pro-uPA) actived plasminogen, Kcat and kcat/km increased significantly with increase of FGFC1 concentration. Moreover,&nbsp;Kcat and&nbsp;kcat/km exhibited 26.5-fold and 22.8-fold enhanced activity at the concentration of 40 &mu;g&bull;mL&minus;1 of FGFC1, respectively. The results suggested that FGFC1 improved significantly the maximum catalytic efficiency and the total catalytic activity of fibrinolysis base on the reciprocal activation of pro-uPA and plasminogen. Km increased with increasing FGFC1 concentration, which indicated that FGFC1 decreased slightly the affinity activity of pro-uPA and plasminogen versus enzyme substrate. The marine bisindole alkaloid FGFC1 enhanced fibrinolysis which was taken on enzymatic kinetic characteristics.

scholarly journals Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tao Wu ◽  
Xiaoqing Mu ◽  
Yuyan Xue ◽  
Yan Xu ◽  
Yao Nie
Keyword(s):  
Steric Hindrance ◽  
Degrees Of Freedom ◽  
Glucose Dehydrogenase ◽  
Reaction System ◽  
Docking Simulation ◽  
Catalytic Efficiency ◽  
Space Time ◽  
Enzyme Engineering ◽  
Phenylalanine Dehydrogenase ◽  
Phenylbutyric Acid

Abstract Background Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block l-homophenylalanine (l-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. Results In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space–time conversion was 30.9 mmol g−1 L−1 h−1. The substrate loading and specific space–time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. Conclusions Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

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