Hydrophilic formate dehydrogenase (FDH) from candida boidinii was chemically modified by coating it with didodecyldimethylammonium bromide (DDAB). This coating changed the phase behavior of the enzyme, making it highly soluble in hydrophobic solvents and thereby offering the chance for biphasic enzyme recycling from hydrophilic substrates and products. Different coating procedures of FDH with DDAB were investigated and all proved suitable for efficient coating of the enzyme’s outer surface. A 50 mM Tris- (hydroxymethyl)-amminomethan (tris) buffer at pH 8 was chosen to make DDAB soluble and avoid aggregation. The reaction of NAD+ with uncoated and coated FDH to NADH and CO2 was monitored by UV-vis spectroscopy and kinetic parameters (rmax, Km, KI , EA) for the the FDH were determined. The coated enzyme resulted in a lower relative initial activity between 40-60% compared to the uncoated one. The stability of the coated enzyme (FDH*) was improved significantly and remained stable in long-term experiments, resulting in a deactivation rate kD smaller than 3% per day and a half-life time t1/2largerthan 23 days, while the deactivation rate of the uncoated enzyme was 260% per daywitha t1/2of 0.3 days. Both activation energies were similar, with 42 kJ mol-1 for the coated and 48 kJ mol-1 for the uncoated enzyme.This result suggests that there is not significant transport resistance originating from the DDAB coating layer. The reason for the significantly lower activity of the coated FDH probably stems from accumulation of formed CO2 in the coating layer, thereby preventing high equilibrium conversions
Gelatin-grafted N- proflavine acryl amide was synthesized through two steps; firstly the Gelatin was grafted with
acrylic acid free radically using Ammonium per-sulfate at 60℃, Then it was modified to its corresponding acyl
chloride derivation, second step included the substitution with amino group of proflavine, in this research Gelatin
was used as a natural nontoxic, water soluble polymer as a drug carrier.
The prepared pro drug polymer was characterized by FTIR and 1H-NMR spectroscopies, Controlled drug release
was studied in different pH values at 37℃. Many advantages were obtained comparing with other known
methods.
Gelatin-grafted N- proflavine acryl amide was synthesized through two steps; firstly the Gelatin was grafted with
acrylic acid free radically using Ammonium per-sulfate at 60℃, Then it was modified to its corresponding acyl
chloride derivation, second step included the substitution with amino group of proflavine, in this research Gelatin
was used as a natural nontoxic, water soluble polymer as a drug carrier.
The prepared pro drug polymer was characterized by FTIR and 1H-NMR spectroscopies, Controlled drug release
was studied in different pH values at 37℃. Many advantages were obtained comparing with other known
methods.
In this work a new prodrug polymer was
prepared with two attachment groups (amid-ester), using di
functional spacer such as ethanol amine, which could react with
polyacrylic acid producing amide group, with remain ethanol
terminal group which could react with captopril acyl chloride,
producing ester group with extended the arm substituted drug to
improve the hydrolysis and to prevent the steric effect of polymer
chains. Many advantages enhanced the prodrug of polymer. The
prepared polymers were characterized by FTIR, 1H –NMR
spectroscopies. Controlled drug release was studied in different
pH values at 37℃, using UV. Spectra with comparing with
calibration curve. The modification percentage test was studied,and swelling percentage was calculated and all physical properties were observed.
Ninhydrin Based Visible Spectrophotometric Determination of GemigliptinRatnakaram Venkata Nadh
A simple method is described to determine the amount of gemigliptin in bulk and tablet formulation by visible spectrophotometry. Basis of the proposed method is the reaction of the primary amine present on gemigliptin with ninhydrin in alkaline pH (alkaline borate buffer) medium to produce a purple color (Ruhemann’s purple) which has maximum absorption at 558 nm. The method was validated as per the current ICH guidelines. The obtained regression equation (y = 0.0148x+0.0011) in the range of 5-30 μg mL-1 has a good correlation coefficient (> 0.999). As the method does not require any separation, it is rapid and simple. The recovery levels of the drug were in the range of 99.73 – 99.96. This method is a green method as it no organic solvents were employed
Chapter 13 Determination of riociguat using ninhydrin as a chromogen 2019Ratnakaram Venkata Nadh
A simple method is described to determine the amount of riociguat in bulk and tablet formulation by visible spectrophotometry. Formation of a chromophore with max of 597 nm, due to the reaction between the aromatic amine groups present on riociguat and ninhydrin in citric acid medium forms the basis for the current method. Extension of conjugation due to attachment of two ninhydrin molecules to a riociguat molecule explains the noticed high intensity as well as max of the generated chromophore. Current ICH guidelines were followed to validate the method. The obtained regression equation (y = 0.0316x+0.001) has a good correlation coefficient (> 0.999) in the studied range of 5.0-30.0 μg mL-1. Due to lack of separation steps in the method, it is found to be rapid as well as simple. The recovery levels of riociguat were in the range of 99.87 – 100.06.
Fractionation and characterization of lignins as and efficient tools for thei...Michal Jablonsky
Dissolution and fractionation of lignocellulosic material is a critical step of valorization of lignins. Content of dierent types of lignin precursors and the content of functional groups OH and OCH3 is aecting their utilization. Chemical and physical characterization of isolated lignin fractions is the key tool for further lignins
application. Presented work deals with the isolation of the lignin from the black liquor by the precipitation method, using a variety of acids. Properties of isolated lignin, preparations and different application and the possibilities of using lignins for various industrial sectors are presented.
Applications of Poly (lactic acid) in Tissue Engineering and Delivery SystemsAna Rita Ramos
Applications of Poly (lactic acid) in Tissue Engineering and Delivery Systems
Poly (lactic acid) is a thermoplastic derived from renewable resources and is at present, one of the most promising biodegradable and nontoxic biopolymers. In addition to its versatility and consequent large-scale production, PLA can be processed with a large number of techniques.
Due to its excellent mechanical properties and biocompatibility, this polymer is becoming largely applied in the biomedical field such as in tissue engineering for scaffolds and in delivery systems in the form of micro and nanoparticles. Furthermore, because it’s relatively cheap and an eco-friend, it has been considered as one of the solutions to lessen the dependence on petroleum-based plastics and solid waste problems.
In order to maximize the knowledge and development of this polymer, it is necessary to understand the material synthesis, proprieties, manufacturing processes, main applications, commercialization and its market state, which will be presented in this review.
Sorbead India is one of best supplier of LDPE bags which are USFDA approved and anti static, use to pack pharmaceutical tablets and capsules this plastic low density polyethylene bags.
Conceptualization of a Domain Specific Simulator for Requirements Prioritizationresearchinventy
This paper conceptualizes a domain specific simulator for requirements prioritization; its aims at helping to identify appropriate prioritization strategies for a project in hand. The possible existing scenarios are difficult to analyze; they involve different variables, like the selection of: stakeholders (their availability, expertise, and importance); prioritization criteria; and prioritization methods. To demonstrate the feasibility of the proposed simulator elements, a well established general purpose simulator, called Arena, was used. The results demonstrate that, it is possible to build the suggested scenarios in order to study and make inferences about the prioritization strategies.
Micro-Tensile Behavior of AA7020/Carbon Black Nanoparticle Metal Matrix Compo...researchinventy
The micro-tensile behavior of AA7020/carbon black nanoparticle composites was examined by using finite element analysis and representative volume element models for three different volume fractions. The uniform distributions of the nanoparticles were constructed to produce a representative volume element of the composites. The predicted mechanical properties and overall tensile stress-strain by finite element analysis corresponded well to the experimental results at the elastic region and near the yield surface, but the model slightly overestimated the strength of the composites at higher stress. This deviation possibly occurs from micromechanical factors, which were not considered in the RVE models. In contrast to FE analysis, fracture of the nanoparticle-matrix debonding occurred because of large localized stress. In the case of a high applied load, non-linear behavior of reinforcements and interfacial strength should be considered to obtain accurate results.
Disturbances in Solar Wind Electrons during the Ascending Half of the Solar C...researchinventy
Ulysses achieved a solar orbit with an inclination of 80.200 , perihelion of 1.34 AU, aphelion of 5.4 AU and a period of 6.2 years. By comparing observations taken over nearly all heliolatitudes and two different intervals covering the same radial distances, we are able to separate the radial and latitudinal variations in the solar wind. This paper describes the detailed results of the studies devoted to the solar activity impact on the Earth’s upper atmosphere and ionosphere. The methods used in this study are based on the data driven from the Ulysses spacecraft. The primary objective of the Ulysses solar wind plasma investigation uses SWOOPS (Solar Wind Observations over the Poles of the Sun) instrument to investigate and establish bulk flow parameters and internal state conditions of the solar wind as a function of solar latitude. The electron temperature Te and the electron density Ne are found to exhibit a sharp enhancement during the ascending half of the solar cycle 23 (1996 – 2004)
Comparative study of fluoride with chemical parameters of brine solutions, sa...researchinventy
Variations in the fluoride content of saline water, salt and pan soil from nine different salt pans located at the East Coast belt of Tamil Nadu have been documented. Fluoride which is non-degradable and persists in the environment was estimated by Zirconyl-alizarin method. The fluoride level varied from 0.6 to 1.7 ppm. Fluoride level in the salt of Marakkanam (Bore well) was found to be slightly higher than other salt pans.The chemical parameters which are used to study the level of fluoride in saline waters included chloride, sulphate, sodium, magnesium, potassium and calcium.
The Acute Effects Of Vibroacoustically-Induced Microvibrations On EEG Activit...researchinventy
A 7-13 c/s mechanographic signal (amplitude 1-5μV) is detectable over relaxed limbs, phenomenon termed microvibration (MV). Exposure to mechanographic signals, low frequency noise, on other side, results in vibroacoustic disease. The aim of this study was to investigate the acute effects of vibroacoustically-induced MV in regio nuchae (vibroacoustic device Vitafon, St. Petersburg, Russian Federation) using four different modes of MV of specific amplitude (2.8 μm - 12.3 μm) and frequency (30 Hz – 18 kHz) on native undulating electrical CNS activity (EEG registration) in the adult rats. The experiments were performed on Wistar male rats and were divided into control and experimental groups. Rats subjected to the same experimental protocol, but with vibroacoustic treatment switched off, served as controls. EEG activity was registered during application of vibroacoustic MV and following was observed regardless of working regime: none of the graphoelements indicating hyperexcitability; there were no reduction in vigilance; beta rhythm and moderate transition alpha in beta rhythm were observed. Slightly override the beta waves in relation to alpha EEG band, under the stimulus of vibroacoustically-induced microvibration by Vitafon, suggests that mental alertness increased as well as focused attention, with no signs of hyperexcitability in rats
How to Build a Dynamic Social Media PlanPost Planner
Stop guessing and wasting your time on networks and strategies that don’t work!
Join Rebekah Radice and Katie Lance to learn how to optimize your social networks, the best kept secrets for hot content, top time management tools, and much more!
Watch the replay here: bit.ly/socialmedia-plan
In this work a new prodrug polymer was
prepared with two attachment groups (amid-ester), using di
functional spacer such as ethanol amine, which could react with
polyacrylic acid producing amide group, with remain ethanol
terminal group which could react with captopril acyl chloride,
producing ester group with extended the arm substituted drug to
improve the hydrolysis and to prevent the steric effect of polymer
chains. Many advantages enhanced the prodrug of polymer. The
prepared polymers were characterized by FTIR, 1H –NMR
spectroscopies. Controlled drug release was studied in different
pH values at 37℃, using UV. Spectra with comparing with
calibration curve. The modification percentage test was studied,and swelling percentage was calculated and all physical properties were observed.
Ninhydrin Based Visible Spectrophotometric Determination of GemigliptinRatnakaram Venkata Nadh
A simple method is described to determine the amount of gemigliptin in bulk and tablet formulation by visible spectrophotometry. Basis of the proposed method is the reaction of the primary amine present on gemigliptin with ninhydrin in alkaline pH (alkaline borate buffer) medium to produce a purple color (Ruhemann’s purple) which has maximum absorption at 558 nm. The method was validated as per the current ICH guidelines. The obtained regression equation (y = 0.0148x+0.0011) in the range of 5-30 μg mL-1 has a good correlation coefficient (> 0.999). As the method does not require any separation, it is rapid and simple. The recovery levels of the drug were in the range of 99.73 – 99.96. This method is a green method as it no organic solvents were employed
Chapter 13 Determination of riociguat using ninhydrin as a chromogen 2019Ratnakaram Venkata Nadh
A simple method is described to determine the amount of riociguat in bulk and tablet formulation by visible spectrophotometry. Formation of a chromophore with max of 597 nm, due to the reaction between the aromatic amine groups present on riociguat and ninhydrin in citric acid medium forms the basis for the current method. Extension of conjugation due to attachment of two ninhydrin molecules to a riociguat molecule explains the noticed high intensity as well as max of the generated chromophore. Current ICH guidelines were followed to validate the method. The obtained regression equation (y = 0.0316x+0.001) has a good correlation coefficient (> 0.999) in the studied range of 5.0-30.0 μg mL-1. Due to lack of separation steps in the method, it is found to be rapid as well as simple. The recovery levels of riociguat were in the range of 99.87 – 100.06.
Fractionation and characterization of lignins as and efficient tools for thei...Michal Jablonsky
Dissolution and fractionation of lignocellulosic material is a critical step of valorization of lignins. Content of dierent types of lignin precursors and the content of functional groups OH and OCH3 is aecting their utilization. Chemical and physical characterization of isolated lignin fractions is the key tool for further lignins
application. Presented work deals with the isolation of the lignin from the black liquor by the precipitation method, using a variety of acids. Properties of isolated lignin, preparations and different application and the possibilities of using lignins for various industrial sectors are presented.
Applications of Poly (lactic acid) in Tissue Engineering and Delivery SystemsAna Rita Ramos
Applications of Poly (lactic acid) in Tissue Engineering and Delivery Systems
Poly (lactic acid) is a thermoplastic derived from renewable resources and is at present, one of the most promising biodegradable and nontoxic biopolymers. In addition to its versatility and consequent large-scale production, PLA can be processed with a large number of techniques.
Due to its excellent mechanical properties and biocompatibility, this polymer is becoming largely applied in the biomedical field such as in tissue engineering for scaffolds and in delivery systems in the form of micro and nanoparticles. Furthermore, because it’s relatively cheap and an eco-friend, it has been considered as one of the solutions to lessen the dependence on petroleum-based plastics and solid waste problems.
In order to maximize the knowledge and development of this polymer, it is necessary to understand the material synthesis, proprieties, manufacturing processes, main applications, commercialization and its market state, which will be presented in this review.
Sorbead India is one of best supplier of LDPE bags which are USFDA approved and anti static, use to pack pharmaceutical tablets and capsules this plastic low density polyethylene bags.
Conceptualization of a Domain Specific Simulator for Requirements Prioritizationresearchinventy
This paper conceptualizes a domain specific simulator for requirements prioritization; its aims at helping to identify appropriate prioritization strategies for a project in hand. The possible existing scenarios are difficult to analyze; they involve different variables, like the selection of: stakeholders (their availability, expertise, and importance); prioritization criteria; and prioritization methods. To demonstrate the feasibility of the proposed simulator elements, a well established general purpose simulator, called Arena, was used. The results demonstrate that, it is possible to build the suggested scenarios in order to study and make inferences about the prioritization strategies.
Micro-Tensile Behavior of AA7020/Carbon Black Nanoparticle Metal Matrix Compo...researchinventy
The micro-tensile behavior of AA7020/carbon black nanoparticle composites was examined by using finite element analysis and representative volume element models for three different volume fractions. The uniform distributions of the nanoparticles were constructed to produce a representative volume element of the composites. The predicted mechanical properties and overall tensile stress-strain by finite element analysis corresponded well to the experimental results at the elastic region and near the yield surface, but the model slightly overestimated the strength of the composites at higher stress. This deviation possibly occurs from micromechanical factors, which were not considered in the RVE models. In contrast to FE analysis, fracture of the nanoparticle-matrix debonding occurred because of large localized stress. In the case of a high applied load, non-linear behavior of reinforcements and interfacial strength should be considered to obtain accurate results.
Disturbances in Solar Wind Electrons during the Ascending Half of the Solar C...researchinventy
Ulysses achieved a solar orbit with an inclination of 80.200 , perihelion of 1.34 AU, aphelion of 5.4 AU and a period of 6.2 years. By comparing observations taken over nearly all heliolatitudes and two different intervals covering the same radial distances, we are able to separate the radial and latitudinal variations in the solar wind. This paper describes the detailed results of the studies devoted to the solar activity impact on the Earth’s upper atmosphere and ionosphere. The methods used in this study are based on the data driven from the Ulysses spacecraft. The primary objective of the Ulysses solar wind plasma investigation uses SWOOPS (Solar Wind Observations over the Poles of the Sun) instrument to investigate and establish bulk flow parameters and internal state conditions of the solar wind as a function of solar latitude. The electron temperature Te and the electron density Ne are found to exhibit a sharp enhancement during the ascending half of the solar cycle 23 (1996 – 2004)
Comparative study of fluoride with chemical parameters of brine solutions, sa...researchinventy
Variations in the fluoride content of saline water, salt and pan soil from nine different salt pans located at the East Coast belt of Tamil Nadu have been documented. Fluoride which is non-degradable and persists in the environment was estimated by Zirconyl-alizarin method. The fluoride level varied from 0.6 to 1.7 ppm. Fluoride level in the salt of Marakkanam (Bore well) was found to be slightly higher than other salt pans.The chemical parameters which are used to study the level of fluoride in saline waters included chloride, sulphate, sodium, magnesium, potassium and calcium.
The Acute Effects Of Vibroacoustically-Induced Microvibrations On EEG Activit...researchinventy
A 7-13 c/s mechanographic signal (amplitude 1-5μV) is detectable over relaxed limbs, phenomenon termed microvibration (MV). Exposure to mechanographic signals, low frequency noise, on other side, results in vibroacoustic disease. The aim of this study was to investigate the acute effects of vibroacoustically-induced MV in regio nuchae (vibroacoustic device Vitafon, St. Petersburg, Russian Federation) using four different modes of MV of specific amplitude (2.8 μm - 12.3 μm) and frequency (30 Hz – 18 kHz) on native undulating electrical CNS activity (EEG registration) in the adult rats. The experiments were performed on Wistar male rats and were divided into control and experimental groups. Rats subjected to the same experimental protocol, but with vibroacoustic treatment switched off, served as controls. EEG activity was registered during application of vibroacoustic MV and following was observed regardless of working regime: none of the graphoelements indicating hyperexcitability; there were no reduction in vigilance; beta rhythm and moderate transition alpha in beta rhythm were observed. Slightly override the beta waves in relation to alpha EEG band, under the stimulus of vibroacoustically-induced microvibration by Vitafon, suggests that mental alertness increased as well as focused attention, with no signs of hyperexcitability in rats
How to Build a Dynamic Social Media PlanPost Planner
Stop guessing and wasting your time on networks and strategies that don’t work!
Join Rebekah Radice and Katie Lance to learn how to optimize your social networks, the best kept secrets for hot content, top time management tools, and much more!
Watch the replay here: bit.ly/socialmedia-plan
http://inarocket.com
Learn BEM fundamentals as fast as possible. What is BEM (Block, element, modifier), BEM syntax, how it works with a real example, etc.
Lightning Talk #9: How UX and Data Storytelling Can Shape Policy by Mika Aldabaux singapore
How can we take UX and Data Storytelling out of the tech context and use them to change the way government behaves?
Showcasing the truth is the highest goal of data storytelling. Because the design of a chart can affect the interpretation of data in a major way, one must wield visual tools with care and deliberation. Using quantitative facts to evoke an emotional response is best achieved with the combination of UX and data storytelling.
Content personalisation is becoming more prevalent. A site, it's content and/or it's products, change dynamically according to the specific needs of the user. SEO needs to ensure we do not fall behind of this trend.
Succession “Losers”: What Happens to Executives Passed Over for the CEO Job?
By David F. Larcker, Stephen A. Miles, and Brian Tayan
Stanford Closer Look Series
Overview:
Shareholders pay considerable attention to the choice of executive selected as the new CEO whenever a change in leadership takes place. However, without an inside look at the leading candidates to assume the CEO role, it is difficult for shareholders to tell whether the board has made the correct choice. In this Closer Look, we examine CEO succession events among the largest 100 companies over a ten-year period to determine what happens to the executives who were not selected (i.e., the “succession losers”) and how they perform relative to those who were selected (the “succession winners”).
We ask:
• Are the executives selected for the CEO role really better than those passed over?
• What are the implications for understanding the labor market for executive talent?
• Are differences in performance due to operating conditions or quality of available talent?
• Are boards better at identifying CEO talent than other research generally suggests?
Oxidation of Acetaminophen by Fluidized-bed Fenton Process: Optimization usin...Oswar Mungkasa
prepared by M.C. Lu*, R.M. Briones**, and M.D.G. de Luna**, *** *Department of Environmental Resources Management, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan (E-mail: mmclu@mail.chna.edu.tw) ** Environmental Engineering Graduate Program, University of the Philippines, 1011 Diliman, Quezon City, Philippines (Email: rowenambriones@yahoo.com) *** Department of Chemical Engineering, University for Urban Environments in Asia, 25-28 May 2011, Manila, Philippines. organized by International Water Association (IWA).
International Journal of Engineering Research and Applications (IJERA) is an open access online peer reviewed international journal that publishes research and review articles in the fields of Computer Science, Neural Networks, Electrical Engineering, Software Engineering, Information Technology, Mechanical Engineering, Chemical Engineering, Plastic Engineering, Food Technology, Textile Engineering, Nano Technology & science, Power Electronics, Electronics & Communication Engineering, Computational mathematics, Image processing, Civil Engineering, Structural Engineering, Environmental Engineering, VLSI Testing & Low Power VLSI Design etc.
A short description of thermal technologies for the recovery of ammonia from N-rich wastewaters and expirementing with membrane distillation for getting better results.
The Effect of Formic Acid, Hydrogen Peroxyde and Other Conditions on Epoxidiz...ijtsrd
Epoxidized vegetable oil have drawn much attention in recent yearrs, especially in the polymer industry as they are economical, available, environmentally friendly, non noxious and renewable. Cashew nut shell liquid CNSL , an agricultural by product abundantly available in tropical countries such as Vietnam, India, is one of the major and economical resources of naturally occurring phenols. Cardanol a byproduct of CNSL could be epoxidized by reacting carbon carbon double bonds of long unsaturated chain with peracids via the Prileshajev epoxidation process or the conventional process. This paper deals with the epoxidized reaction of cardanol take place in formic acid and hydrogen peroxyde. The results shown that the conversion efficiency of the epoxidized reaction reacheres 80 at 600C, stirring rates 1800 rpm, 2 p toluenesulfonic acid catalyst and rate of double bonds DB HCOOH AF H2O2 = 1.0 0.5 1.5. The product of epoxidized cardanol is also characterized by FT IR, 1H NMR and13C NMR. Bach Trong Phuc | Nguyen Thanh Liem "The Effect of Formic Acid, Hydrogen Peroxyde and Other Conditions on Epoxidized Reaction of Cardanol Extracted from Cashew Nut Shell Liquid of Vietnam" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-6 | Issue-3 , April 2022, URL: https://www.ijtsrd.com/papers/ijtsrd49624.pdf Paper URL: https://www.ijtsrd.com/chemistry/polymer-chemistry/49624/the-effect-of-formic-acid-hydrogen-peroxyde-and-other-conditions-on-epoxidized-reaction-of-cardanol-extracted-from-cashew-nut-shell-liquid-of-vietnam/bach-trong-phuc
Applicability of Fenton Process for Treatment of Industrial Effluents: A ReviewIJERA Editor
Wastewater and effluent treatment has undergone innovative changes over the years. Traditional wastewater treatment has yielded to modern and path-breaking procedures which are more efficient and effective. The world of difficulties and unknown has opened new avenues and paths to highly feasible effluent treatment procedures, so the vigorous importance of advanced oxidation procedures. This review paper will delineate the increasing importance of various advanced oxidation processes including Fenton treatment for wastewater. Advanced oxidation processes (AOP’s), which involve and includes the in-situ generation of highly potent chemical oxidants such as the hydroxyl radical, have emerged as an important avenue of technologies to accelerate the non-selective oxidation and thus the destruction of a wide range of non-degradable organic contaminants in wastewater which cannot be eliminated biologically. Advanced oxidation processes (AOP) are technologies based on the generation of highly reactive species, the hydroxyl radicals, used in oxidative degradation procedures for organic compounds dissolved or dispersed in aquatic media. These processes are promising alternatives for decontamination of media containing dissolved recalcitrant organic substances, which would not be efficiently removed by conventional methods.
About
Indigenized remote control interface card suitable for MAFI system CCR equipment. Compatible for IDM8000 CCR. Backplane mounted serial and TCP/Ethernet communication module for CCR remote access. IDM 8000 CCR remote control on serial and TCP protocol.
• Remote control: Parallel or serial interface.
• Compatible with MAFI CCR system.
• Compatible with IDM8000 CCR.
• Compatible with Backplane mount serial communication.
• Compatible with commercial and Defence aviation CCR system.
• Remote control system for accessing CCR and allied system over serial or TCP.
• Indigenized local Support/presence in India.
• Easy in configuration using DIP switches.
Technical Specifications
Indigenized remote control interface card suitable for MAFI system CCR equipment. Compatible for IDM8000 CCR. Backplane mounted serial and TCP/Ethernet communication module for CCR remote access. IDM 8000 CCR remote control on serial and TCP protocol.
Key Features
Indigenized remote control interface card suitable for MAFI system CCR equipment. Compatible for IDM8000 CCR. Backplane mounted serial and TCP/Ethernet communication module for CCR remote access. IDM 8000 CCR remote control on serial and TCP protocol.
• Remote control: Parallel or serial interface
• Compatible with MAFI CCR system
• Copatiable with IDM8000 CCR
• Compatible with Backplane mount serial communication.
• Compatible with commercial and Defence aviation CCR system.
• Remote control system for accessing CCR and allied system over serial or TCP.
• Indigenized local Support/presence in India.
Application
• Remote control: Parallel or serial interface.
• Compatible with MAFI CCR system.
• Compatible with IDM8000 CCR.
• Compatible with Backplane mount serial communication.
• Compatible with commercial and Defence aviation CCR system.
• Remote control system for accessing CCR and allied system over serial or TCP.
• Indigenized local Support/presence in India.
• Easy in configuration using DIP switches.
Explore the innovative world of trenchless pipe repair with our comprehensive guide, "The Benefits and Techniques of Trenchless Pipe Repair." This document delves into the modern methods of repairing underground pipes without the need for extensive excavation, highlighting the numerous advantages and the latest techniques used in the industry.
Learn about the cost savings, reduced environmental impact, and minimal disruption associated with trenchless technology. Discover detailed explanations of popular techniques such as pipe bursting, cured-in-place pipe (CIPP) lining, and directional drilling. Understand how these methods can be applied to various types of infrastructure, from residential plumbing to large-scale municipal systems.
Ideal for homeowners, contractors, engineers, and anyone interested in modern plumbing solutions, this guide provides valuable insights into why trenchless pipe repair is becoming the preferred choice for pipe rehabilitation. Stay informed about the latest advancements and best practices in the field.
CFD Simulation of By-pass Flow in a HRSG module by R&R Consult.pptxR&R Consult
CFD analysis is incredibly effective at solving mysteries and improving the performance of complex systems!
Here's a great example: At a large natural gas-fired power plant, where they use waste heat to generate steam and energy, they were puzzled that their boiler wasn't producing as much steam as expected.
R&R and Tetra Engineering Group Inc. were asked to solve the issue with reduced steam production.
An inspection had shown that a significant amount of hot flue gas was bypassing the boiler tubes, where the heat was supposed to be transferred.
R&R Consult conducted a CFD analysis, which revealed that 6.3% of the flue gas was bypassing the boiler tubes without transferring heat. The analysis also showed that the flue gas was instead being directed along the sides of the boiler and between the modules that were supposed to capture the heat. This was the cause of the reduced performance.
Based on our results, Tetra Engineering installed covering plates to reduce the bypass flow. This improved the boiler's performance and increased electricity production.
It is always satisfying when we can help solve complex challenges like this. Do your systems also need a check-up or optimization? Give us a call!
Work done in cooperation with James Malloy and David Moelling from Tetra Engineering.
More examples of our work https://www.r-r-consult.dk/en/cases-en/
Cosmetic shop management system project report.pdfKamal Acharya
Buying new cosmetic products is difficult. It can even be scary for those who have sensitive skin and are prone to skin trouble. The information needed to alleviate this problem is on the back of each product, but it's thought to interpret those ingredient lists unless you have a background in chemistry.
Instead of buying and hoping for the best, we can use data science to help us predict which products may be good fits for us. It includes various function programs to do the above mentioned tasks.
Data file handling has been effectively used in the program.
The automated cosmetic shop management system should deal with the automation of general workflow and administration process of the shop. The main processes of the system focus on customer's request where the system is able to search the most appropriate products and deliver it to the customers. It should help the employees to quickly identify the list of cosmetic product that have reached the minimum quantity and also keep a track of expired date for each cosmetic product. It should help the employees to find the rack number in which the product is placed.It is also Faster and more efficient way.
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Improved Stability of Formate Dehydrogenase by Coating with Didodecyldimethylammonium Bromide
1. Research Inventy: International Journal of Engineering And Science
Vol.6, Issue 8 (September 2016), PP -25-35
Issn (e): 2278-4721, Issn (p):2319-6483, www.researchinventy.com
25
Improved Stability of Formate Dehydrogenase by Coating with
Didodecyldimethylammonium Bromide
Matthias Reicherta
, Marco Haumannb
*, Rainer Buchholza,c
,
Christoph Lindenbergera
*,
a
Friedrich-Alexander Universität Erlangen-Nürnberg, Busan Campus, Bioprocess Engineering, 1276 Jisa-
Dong, Gangseo-Gu, Busan 618-230, Republic of Korea
b
Friedrich-Alexander Universität Erlangen-Nürnberg, Chemical Reaction Engineering, Egerlandstr. 3, 91058
Erlangen, Germany
c
Friedrich-Alexander Universität Erlangen-Nürnberg, Bioprocess Engineering, Paul-Gordan-Straße 3, 91052
Erlangen, Germany
Abstract: Hydrophilic formate dehydrogenase (FDH) from candida boidinii was chemically modified by
coating it with didodecyldimethylammonium bromide (DDAB). This coating changed the phase behavior of the
enzyme, making it highly soluble in hydrophobic solvents and thereby offering the chance for biphasic enzyme
recycling from hydrophilic substrates and products. Different coating procedures of FDH with DDAB were
investigated and all proved suitable for efficient coating of the enzyme’s outer surface. A 50 mM Tris-
(hydroxymethyl)-amminomethan (tris) buffer at pH 8 was chosen to make DDAB soluble and avoid aggregation.
The reaction of NAD+
with uncoated and coated FDH to NADH and CO2 was monitored by UV-vis
spectroscopy and kinetic parameters (rmax, Km, KI, EA) for the the FDH were determined. The coated enzyme
resulted in a lower relative initial activity between 40-60% compared to the uncoated one. The stability of the
coated enzyme (FDH*) was improved significantly and remained stable in long-term experiments, resulting in a
deactivation rate kD smaller than 3% per day and a half-life time t1/2largerthan 23 days, while the deactivation
rate of the uncoated enzyme was 260% per daywitha t1/2of 0.3 days. Both activation energies were similar, with
42 kJ mol-1
for the coated and 48 kJ mol-1
for the uncoated enzyme.This result suggests that there is not
significant transport resistance originating from the DDAB coating layer. The reason for the significantly lower
activity of the coated FDH probably stems from accumulation of formed CO2 in the coating layer, thereby
preventing high equilibrium conversions.
I. Introduction
The highly selective production of chemicals is one of the major challenges for modern chemical
engineering since most feedstock for chemical transformations is based on crude oil or gas (Cornils and
Herrmann, 2002).Catalysis is the key technology to achieve high selectivity and enzymes are superior in this
regard compared to homogeneous and heterogeneous catalysts. Especially for stereoselective reaction many
enzymes produce the target enantiomer in high yields. Introducing enzymatic reactions into chemical production
processes lowered the production complexity and costs significantly in the past, e.g. in the synthesis of L-
Ephedrine or Pregabalin(Mahmoud, El-Sayed and Coughlin, 1990). Due to high enzyme prices and product
purity requirements the separation after reaction and consecutive use of the precious catalyst can help to
improve the enzyme productivity significantly. Biphasic catalysis with enzymes is a promising technique to
achieve this goal. For such recycling to be effective, the enzyme solubility must be low in the product phase and
high in the catalyst solvent. Biphasic catalysis with hydrophilic enzymes is possible if the products are
hydrophobic and the substrates have a good miscibility in the water phase, the most common solvent for
enzymes. If hydrophobic substrates should be converted, the poor miscibility in the enzyme containing water
phase will result in a low conversion, making such processes unfavorable. This is even more critical, if the
products are hydrophilic and need to be separated from the water phase.
One possibility to separate hydrophilic products from the enzyme catalyst would be the modification of
the enzyme’s phase behavior, namely its solubility in water. This could be achieved by coating the enzyme with
a surfactant, so that the polar head group of the surfactant will coordinate to the hydrophilic enzyme surface,
while the nonpolar tail of the surfactant will point away from the enzyme. Such coated enzymes will be soluble
in nonpolar media now and could be easily separated from an aqueous product phase.
2. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
26
Scheme1. Top: reaction of NAD+
and formate to yield NADH and CO2, catalyzed by FDH or
lipid-coated FDH (FDH*). Bottom: Schematic presentation of the FDH* coated with DDAB.
The coating of enzyme surfaces with polymers has been described as an efficient method to prevent
negative interaction of enzyme interfaces (e.g. gas bubbles) that may produce enzyme inactivation (Betancor,
Lopez-Gallego, Hidalgo, Alonso-Morales, Fuentes, Fernandez-Lafuente, and Guisan, 2004). For enzyme
stabilization and solubility in organic media, some chemical modification techniques are promising and known.
The dissociation of multimeric enzymes can be avoided by coating their surface with a large cationic polymer
e.g. polyethylene imine (PEI) (Bolivar, Rocha-Martin, Mateo, Cava, Berenguer, Fernandez-Lafuente, and
Guisan, 2009), polyethylene glycol (PEG) (Turner, Spear, Huddleston, Holbrey, and Rogers, 2003; Laszlo and
Compton, 2001; Maruyama, Nagasawa and Goto, 2001) or with amphiphiles (lipids/surfactants) (Okahata,
Fujimoto and Ijiro, 1995). However, since most of the glycolipids are not available on the market and have to be
newly synthesized, makes them expensive. One of the most extensively investigated double chained cationic
surfactant (Marques, Regev, Khan, and Lindman, 2003), is didodecyldimethylammonium bromide (DDAB). It
has market value between 7 and 15Euro per gram and it is forming micelles in aqueous solution (Lichtenberg,
Opatowski, and Kozlov, 2000).
In this work we present different coating procedures for FDH by the surfactant
didodecyldimethylammonium bromide (DDAB), which is expected to form a two phase emulsion consistent of
bilayer structures (Marques, Regev, Khan and Lindman 2003; Lichtenberg, Opatowski, and Kozlov, 2000). The
coating quality and the performance of the lipid-coated FDH (FDH*) will be investigated for the test reaction of
formate with NAD+
to yield NADH and CO2 as shown in Scheme 1.To the best of our knowledge, this system in
tris-(hydroxymethyl)-amminomethane (tris) buffer has not been described for the particular enzyme in literature
so far.
II. Experimental
Chemicals and materials
The 50 mM Tris-(hydroxymethyl)-amminomethane (tris) buffer (DAE Jung, Korea) was prepared,
adjusted with HCL to a pH of 8, autoclaved and stored at room temperature. For the FDH stock solution the
lyophilized formate dehydrogenase (FDH) from candida boidinii (Sigma-Aldrich, Germany) was weighed and
added to ice cold 50 mM tris buffer of pH 8. The stock solution wasstored at 4 °C for further experiments. For
the NADH stock solution the lyophilizedβ-nicotinamide adenine dinucleotide hydrate (NAD+
) from yeast
(Sigma-Aldrich, Germany) and β-nicotinamide adenine dinucleotide(NADH) reduced dipotassium salt (Sigma-
Aldrich, Germany) solution were weighed, dissolved in 50 mM tris buffer of pH 8, stored at -20 °C and
defrosted for experiment. The DDAB stock solutions containing32 mg of didodecyldimethylammonium
bromide (DDAB) (Steinbeis GmbH, Germany) were dissolved in 1 ml of ethanol and stored at 4 °C.
Additionally, 4 mg of DDAB were dissolved in 1 ml of 50 mM tris buffer of pH 8 and stored at 4 °C.The
formate solution (Dae Jung, Korea) was weighed, dissolved in 50 mM tris buffer of pH 8 and stored at room
temperature.
FDH coating procedures
Three different coating methods were used to examine the influence on the relative activity (a/a0) of
coated-FDH (FHD*). In method 1, the enzyme solution was first added to the vial, followed by the prepared
DDAB-ethanol solution. Method 2 reversed the steps, with the enzyme solution being added to the vial after the
DDAB-ethanol solution. In Method 3, a DDAB tris buffer was used instead of the DDAB-ethanol solution. The
buffer was then added to the enzyme solution, similar to method 1. Both, an unstirred and a stirred enzyme
solution were taken as reference.
3. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
27
The FDH*reaction solutions were obtained by simply mixing prepared FDH* enzyme and DDAB
solutions. 8 mg of lyophilized FDH was mixed in a glass sample vial (Wheaton, USA) with 5 ml of 50 mM tris
buffer pH 8 at 4 °C. Four other vials with magnet stirrers were prepared, one with 2600 μl, twowith 2950 μl and
one with 3000 μl of tris buffer (4 °C, 50 mM and pH 8). 1 ml of the enzyme stock solution was transferred in
different steps, to each of the other fourstirred vials. Under stirring, 50 μl of 32 mgml-1
DDAB ethanol stock
solution was added, in different steps, to two of the vials. 400 μl of 4 mgml-1
DDAB tris buffer stock solution
was added to the last vial. 3 ml of tris buffer (4 °C, 50mM, and pH 8) was given to the unstirred enzyme
reference vial in which the enzyme concentration was determined with Bradford's method (Bradford, 1976).
After the preparation, all vials had a total volume of 4 ml and were stirred at least 20 hours (with 300 min-1
or
500 min-1
at 4 °C) before measurement. The sequence of the different coating steps andthe end concentrations
areitemized in Table 1.
Table 1. Sequence of different coating steps and final concentrations.
FDH FDH*
Step Unstirred FDH Stirred FDH Method 1 Method 2 Method 3
1 5000 μl FDH 3000 μl tris 2950 μl tris 2950 μl tris 2600 μl tris
2 4 x 1000 μl FDH 1000 μl FDH 1000 μl FDH 50 μl DDAB/EtOH 1000 μl FDH
3 3000 μl tris 50 μl DDAB/EtOH 1000 μl FDH 400 μl DDAB/tris
Vtotal 4 ml 4 ml 4 ml 4 ml 4 ml
[FDH] 0.4 mgml-1
0.4 mg ml-1
0.4 mg ml-1
0.4 mg ml-1
0.4 mg ml-1
[DDAB] 0 mg ml-1
0 mg ml-1
0.4 mg ml-1
0.4 mg ml-1
0.4 mg ml-1
Kinetic experiments
FDH concentration
To determine the exact enzyme concentrationthe absorption was measured in a spectrophotometer UV-
1800 (Shimadzu, Japan) at 595 nm. The formate dehydrogenase (FDH) from candida boidinii concentration was
determined and compared with a BSA standard (Sigma, Germany) using Bradford’s method.
NAD+
/ NADH activity
Prior to all measurements exact concentration of the active NAD+
and NADH were determined. The
activities of the FDH preparations were evaluated by their UV absorption, recording the increase of the
absorption at 340 nm (ε=6220 M-1
cm-1
at 340 nm)which resulted from the formation of NADH during the
oxidation of formic acid.The prepared NAD+
stock solution was unfrozen and setwithin the experiment on
flocked ice. 100 μl of 1.67 mM NAD+
stock solution, 400 μl of 1000 mM formate stock solution and 350 μl of
50 mM tris buffer of pH 8 were prepared in a tube. The tube was placed in a thermomixer (compact, Eppendorf,
Germany) and mixed for 10 minutes at 400 min-1
to adjust the 25 °C. As well the precision cells 105.201-QS
(Hella GmbH, Germany)weretempered in an oven (T20, Thermo Scientific, Germany) with the required
temperature for at least 10 minutes. After tempering, the reactive solution was converted into the cell. The cell
was placed in the spectrophotometer. By adding 150 μl of ice cold (4 °C) 0.4 mg ml-1
FDH stock solution, the
reaction was started. The total volume, of 1 ml inside the cell, was gently mixed twice with a pipette (VWR,
Korea). After the reaction was completed (30 minutes) the concentration was calculated from the difference of
the start and end absorption values according to Equation 1.For the determination of the NADH concentration
the stock was unfrozen and diluted by afactor of 10 (100 μl of 1mM NADH stock solution, 900 μl 50 mM tris
buffer of pH 8). A cell filled with tris buffer served as zero value. The NADH concentration was calculated
directly with Lambert Beer's law usingEquation 1. The procedure of allother experiments was handled as
described above.
6220
][
][
][ Absorption
NADH
NAD
(1)
Long term stability of FDH in tris buffer
The stability of FDH in a 50 mM tris buffer of pH 8 was tested under following reaction conditions:
100 μl of 1.67 mM NAD+
stock solution, 400 μl of 1000 mM formate stock solution, 350 μl of 50 mM tris
buffer of pH 8, 50μl of 0.4 mg ml-1
FDHstock solution.
Kinetic parametersof FDH catalyzed reaction
The kinetic parameters of enzymatic reactions were determined in different reaction media set upswith
varying concentrations of substrate (NAD+
, HCOO-
), inhibitor (NADH, EtOH) and DDAB
concentrations.Reactions condition for the determination of the kinetic parameters were as follows:
4. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
28
Km,NAD
+
:10-200 μl of 1.67 mM NAD+
stock solution, 200 μl of 1000 mM formate stock solution, 550-
740 μl of 50 mM tris buffer of pH 8, 50μl of 0.4 mg ml-1
FDHstock solution.
Km,HCOO
-
:600 μl of 1.67 mM NAD+
stock solution, 5-200 μl of 1000 mM formate stock solution, 150-
345μl of 50 mM tris buffer of pH 8, 50μl of 0.4 mg ml-1
FDHstock solution.
KI,NADH: 600 μl of 1.67 mM NAD+
stock solution, 10-200 μl of 1000 mM formate stock solution, 0-150
μl of 1mM NADH stock solution, 0-320μl of 50 mM tris buffer of pH 8, 50μl of 0.4 mg ml-1
FDHstock solution.
KI,EtOH: 400 μl of 1.67 mM NAD+
stock solution, 10-200 μl of 1000 mM formate stock solution, 0-350
μl of EtOH, 0-450 μl of 50 mM tris buffer of pH 8, 50μl of 0.4 mg ml-1
FDHstock solution.
Influence of DDAB: 100 μl of 1.67 mM NAD+
stock solution, 400 μl of 1000 mM formate stock
solution, 0-100μl of 4mg ml-1
DDAB in 50 mM tris buffer of pH 8, 490-540μl of 50 mM tris buffer of pH 8, 50μl
of 0.4 mg ml-1
FDHstock solution.
Activation energy FDH/FDH*
Activation energies were determinedexperimentally measuring at different temperatures T of 10 °C, 15
°C and 25 °C.The experiments at a temperature of 10 °C and15 °Cwere carried out with the complete setup
being placed in a cooling chamber (Coldtop Co., Korea). Reactions conditions for the determination of
activation energies were as follows:
Ea,FDH/Ea,FDH*: 100 μl of 1.67 mM NAD+
stock solution, 25-750 μl of 8000 mM formate stock solution,
0-725 μl of 50 mM tris buffer of pH 8, 150μl of 0.4 mg ml-1
FDH/FDH* stock solution. FDH* was covered with
0.4 mg ml-1
DDAB using coating method 1.
Influence of coating methodson stability of FDH/FDH*
The FDH was coated with 0.4 mg ml-1
DDAB as described at coating procedures. Either the non-coated
enzymes (FDH) or the coated enzymes (FDH*) were stirred at speed of 300 min-1
or 500 min-1
, and the activity
awas determined over 90 hours in order to the initial activity a0of FDH at 0 hours.Reactions conditions for the
determination of the kinetic parameters were as follows: 300 min-1
: 100 μl of 1.67 mM NAD+
stock solution,
400 μl of 1000 mM formate stock solution, 350 μl of 50 mM tris buffer of pH 8, 150 μl of 0.4 mg ml-
1
FDH/FDH*. 500 min-1
: 100 μl of 1.67 mM NAD+
stock solution, 750 μl of 8000 mM formate stock solution,
150 μl of 0.4 mgml-1
FDH/FDH*.
Evaluation of kinetic data
Initial velocity vinitial
The spectrophotometer UV-1800 measured the reaction solution's UV absorption of formed NADH at
340 nm every two seconds over a time period of 200 seconds. The initial velocity was taken by the change in
absorption A over time through linear regression usingEquation (2). In case of FDH the interval was60 seconds
over at least 20 data points and in case of FDH*
within the first 34 seconds over the first ten measured data
points.
0AtvA initial (2)
With A = absorption, vinitial = dA/dt = initial velocity in s-1
, t = time in s, A0 = absorptionat t0.
Kinetic parameters vmax, KM and KI
The activity was calculated with Equation 1 and 2 referred to the FDH concentration (see Equation 3).
The kinetic parameters and inhibition models of enzymatic reactions were determined and evaluated using
SigmaPlot's Enzyme Kinetic Wizard (SigmaPlot 12, Systat Software Inc., USA)plotting activity against
concentration.
][
][
FDH
dt
NADHd
FDH
v
activity initial
(3)
Activation energy
Activation energies were determined by plotting the logarithm of keffagainst T-1
and determining the
slope of the straight line with Equation 4.
TR
E
kk a
eff
1
lnln 0 (4)
With keff = effective rate constant, k0 = frequency factor, Ea = activation energy. The effective rate
constant keffwas calculated from plots of the logarithm of activity against the logarithm of formate concentration
at different temperaturesT.
5. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
29
Deactivation constant kD
In the stirring experiment the deactivation constants were calculated by measuring the activity a of
either FDH or FDH* over the stirring time. This activity was correlated to the initial activity a0 of the non-
coated FDH at time t=0. The decay in activity in % was exponentially fitted and kD determined with Equation 5.
tkD
eAty
a
a
activity
0
0
)( (5)
With t = stirring time in days, a = activity of FDH/FDH* at t in μMmin-1
mg-1
, a0 = activity of FDH at t
= 0 in μM min-1
mg-1
, A0 =initial activity of FDH/FDH* at t = 0 in %, kD = deactivation constant of
FDH/FDH*in day-1
.
FDH/FDH* half-life time
The FDH/FDH* half-life time t1/2was defined as the time when the enzyme (FDH or FDH*)has lost
half of its initial activity A0, and was calculated according to Equation 6.
Dk
t
2ln
2/1 day (6)
FDH/FDH* productivity
FDH and FDH* productivity was calculated by integrating Equation 5 within a determined interval of
time (Equation7).
dteAtyP tk
t
t
FDHFDH
0
0*/ )( (7)
With PFDH/FDH* = productivity, t = stirring time in days, A0 =initial activity of FDH or FDH* at t = 0 in
%, kD = deactivation constant of FDH or FDH* in day-1
.
III. Results
In a preliminary set of experiments the solubility of the DDAB coating agent in different buffer
solutions has been investigated (Figure 1). DDAB was nearly insoluble in 5mM and 50mM potassium-
phosphate buffer at pH 8, yielding white swollen aggregates that could not be resuspended upon prolonged
stirring for 48 h (Figure 1 A and B). However, DDAB readily dissolved in large quantities in ethanol. For that
reason, two ternary systems consisting of either ethanol, DDAB and potassium-phoasphate buffer or ethanol,
DDAB and tris buffer, both at pH 8 and 25 °C were investigated. Only the system using the tris buffer gave an
emulsified and stable system (Figure 1 C and D), while the other systems tended to agglomerate. Hence, all
further experiments were conducted with the tris buffer.
Figure 1. 4 mg ml-1
DDAB in different buffers at pH 8. (A) 50 mM potassium phosphate, (B) 5
mM potassium phosphate, (C) 50 mM tris-(hydroxymethyl)-amminomethan (tris), (D) 50 mM
tris-(hydroxymethyl)-amminomethan (tris) and 1 Vol% EtOH.
6. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
30
Non-coated enzyme
In a first set of experiments the non-coated FDH stability in a 50 mM tris buffer of pH 8 at 25 °C has
been investigated. 400 mg ml-1
FDH was stored in this buffer at 4 °C for 30 days. Kinetic measurements with
0.167 mM NAD+
and 400 mM formate at 25 °Cindicated that the relative enzyme activity remained stable over
three days and that even after 30 days storage time still 94 of the initial activity could be preserved (see Table
2).
Table 2. Stability of 0.4 mgml-1
FDH stored at 4 °C in a tris buffer of pH 8.
Storage time / h Activity (a/a0) / % RSD / %
0 100 ± 2.7
24 102 ± 5.6
48 103 ± 0.4
720 94 ± 2.4
Reaction conditions: 25 °C, 0.167 mM NAD+
, 400 mM formate, 2 μg ml-1
FDH.
Enzymes interact often with substances that affect the reaction rate. These substances (effectors) are
usually low-molecular compounds. Activators increase the reaction rate, whereas inhibitors decrease it. In
general irreversible inhibitors are covalent bound to the enzyme. It is separated between unspecific inhibitions,
where the interaction takes place beyond the active site and specific inhibitions, where the inhibitor has an
affinity to catalytic center. Reversible changes in activity caused by inhibitors are essentially separated in four
types and are derived by the Michaelis-Menten kinetics in Equation (8):
competitive inhibition
non-competitive inhibition
partial-competitive inhibition
uncompetitive inhibition
Competitive inhibition appears if substrate and inhibitor compete against the active site. Mostly they
are of similar structure. There are some possibilities this inhibition can arise:
1. S and I compete against the same binding site
2. Only S or I can bind, cause of their steric interference
3. S and I have an additional common binding site
At our reaction formate serves as substrate and NAD+
is applied as the coenzyme. Since the CO2-
concentration is negligible, the reaction can be seen as irreversible. The byproduct NADH has an inhibitory
character (product inhibition). To simplify the calculations a competitive inhibition model was used.
The kinetic parameter estimation was based on an inhibition model. For this case of competitive
inhibition the classical Michaelis-Menten kinetics in Equation (8) has to be expanded into Equation (9).
SK
S
m
max with
1
21
k
kk
Km (8)
S
K
I
K
S
I
m
1
max with
EI
IE
k
k
K
I
I
I
(9)
Linearization of Equation (9) yields the Lineweaver-Burk Equation (10).
maxmax
11
1
1
vSK
IK
I
m
(10)
The uncoated FDH activity was investigated in a 50 mM tris buffer at a pH value of 8 and a constant
NAD+
concentration of 1 mM with varying formate concentrations from 5 mM to 200 mM. Figure 2 depicts the
corresponding Michaelis-Menten and Lineweaver-Burk plots, from which the kinetic parameters of the uncoated
FDH were determined. The Michaelis-Mentenplot reveals a typical progression of an enzymatic reaction with
Km,HCOO- of 6.18 mM and max of 5.30 µM mg-1
min-1
. Variation of the NAD+
concentration between 16.7 µM
and 334μM at constant FDH of 200 mM yielded a Km,NAD+ of 23.1μM and a max of 5.07 µM mg-1
min-1
.
7. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
31
Figure 2. Michaelis-Menten and Lineweaver-Burk plots for FDH kinetics at varying formate
(left) and NAD+
(right) concentrations. Reaction conditions: (left) 25 °C,1 mM NAD+
, 5-200
mM formate, 2 μg ml-1
FDH and (right) 25 °C, 16.7-334 μM NAD+
, 200 mM formate, 2 μg ml-
1
FDH.
In good agreement with the proposed competitive product inhibition model, the concentration of the
substrate NADH had a negative influence on the enzyme activity indicated by a value for KI,NADH of 14.6 μM,
determined from the Lineweaver-Burk plot shown in Figure 3.
Figure 3. Michaelis-Menten (left) and Lineweaver-Burk (right) plots for FDH kinetics at
varying NADH concentrations. Reaction conditions: 25 °C, 1 mM NAD+
, 10-200 mM formate,
2 μg ml-1
FDH, [NADH] = 0 (●), 20 (○) and 150 (▼) µM.
The influence of the coating agent DDAB and its solvent ethanol on the enzyme activity was also
tested. While ethanol had no significant influence on the activity in the range of 171 μM to 1.71 M, addition of
the coating agent DDAB led to a distinct decrease in activity as shown in Table 3 while Table 4 lists obtained
kinetic data.
Table3. Influence of the DDAB concentration on the activity of FDH
[DAAB] / mg mL-1
Activity (a/a0) / %
0 100
0.04 63.9
0.10 53.6
0.20 50.2
0.40 58.0
Reaction conditions: 25 °C, 167 μM NAD+
, 400 mM formate, 0-0.4 mg/ml DDAB, 2 μg ml-1
FDH.
Table 4. Kinetic parameters determined for various inhibitors.
Substrate max
/ µM mg-1
min-1
Km,NAD+
/ μM
Km,HCOO-
/ mM
KI,NADH
/ μM
KI,EtOH
/ mM
HCOO-
5.30 6.18
NAD+
5.07 23.1
NADH 5.27 5.83 14.6
EtOH 4.69 7.98 11.97
Reaction conditions: [HCOO-
]: 25 °C, 1 mM NAD+, 5-200 mM formate, 2 μg/ml FDH; [NAD+
]; 25
°C, 16.7-334 μM NAD+, 200 mM formate, 2 μgml-1
FDH; [NADH]: 25 °C, 1 mM NAD+, 10-200 mM formate,
0-150 µM NADH, 2 μgml-1
FDH: [EtOH]: 668 μM NAD+, 10-200 mM formate, 0-6 M EtOH, 2 μgml-1
FDH.
8. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
32
Lipid coated enzyme
Three coating methods were tested that differed according to whether the enzyme was coated directly
with DDAB (method 1 and method 3) or method 2. Furthermore, no ethanol was present in the coating solution
when using method 3. All three coating methods resulted in similar initial activity of the FDH*, yielding 38.8 %
(method 1), 40.8 % (method 2) and 39.0 % (method 3) of the non-coated FDH activity, respectively.
Interestingly, the non-coated FDH gradually lost activity over time.After 20 hours a relative FDH activity of
60.3 % was obtained which decreased to 12.3 %, after 90 hours. In contrast, all coated FDH* resulted in stable,
yet lower activity as shown in Figure 4.
Figure 4. Stability studies of non-coated (●) FDH and coated FDH* using different coating
methods.Coating conditions: 4 °C, stirring rate 300 min-1
,0.4 mg ml-1
FDH coated with 0.4 mg
mL-1
DDAB according to methods 1 (○), 2 (▼) and 3 (∆).Reaction conditions: 25°C, 167 μM
NAD+, 400 mM formate,6μgml-1
FDH/FDH*.
To further elucidate the influence of coating on overall kinetics, the reaction temperature was varied in
the concentration regime between 200 and 800 mM of formate. For the non-coated FDH an effective activation
energy of 41.7 kJ mol-1
was determined. Applying the coated FDH* under identical reaction conditions resulted
in a similar activation energy of 47.3 kJ mol-1
. With both activation energies being similar, limitation of the
overall kinetics by transport of substrate through the coating layer to the active site of the enzyme can be
excluded.
The half-life timet1/2of the enzyme (FDH) was strongly decreased from 1.3 days to 0.3 days when the
stirring rate was increased from 300 min-1
to 500 min-1
as shown in Figure 5. In contrast, coated FDH* was
stable even under conditions of more sheer stress and showed a t1/2 of more than 23 days when stirring at 300
min-1
and a t1/2 of more than 43 days at a stirring rate of 500 min-1
. The results indicate that the coating layer
somehow protectedthe FDH* against mechanical stress from stirring and cavitationand didnot hamper the
transport of substrate from the organic phase to the active site of the FDH*.
Figure 5. Half-life time of non-coated FDH and coated FDH*. Reaction conditions: 4 °C,
stirring rate 300-500 min-1
, 0.4 mg ml-1
FDH coated with 0.4 mg mL-1
DDAB using method 1.
Reaction conditions: 300 min-1
:25°C, 167 μM NAD+
, 400 mM formate,6μgml-1
FDH/FDH*;
500 min-1
:25°C, 167 μM NAD+
, 6 M formate,6μgml-1
FDH/FDH*.
9. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
33
According to Equation 5 an exponential fit was used to describe the decay in activity of un-coated FDH
and coated FDH* with stirring time. The deactivation rate kDof non-coated FDH increased from 53.5% per day
to 261% per day when increasing the stirring speed from 300 to 500 min-1
. In contrast, the coated FDH*
remained stable under these conditions with a kD of 3% per day (300 min-1
) and a kD of 1.6% per day (500 min-
1
). However, the initial activity of FDH* dropped from 100% to values around 50 %. The productivity PFDH* of
DDAB coated enzyme (method 1) was enhanced by a factor of 3.8 (at 300 min-1
) and even 31.7 (at 500 min-
1
)with respect to un-coated FDH.
Figure 6. Stability and productivityPFDH/FDH*of non-coated FDH and coated FDH* and
exponential fits with R2
.Coating conditions: 4 °C, stirring rate 300-500 min-1
, 0.4 mg ml-1
FDH coated with 0.4 mg mL-1
DDAB using method 1. Reaction conditions: 300 min-1
:25°C,
167 μM NAD+
, 400 mM formate,6μgml-1
FDH/FDH*; 500 min-1
:25°C, 167 μM NAD+
, 6 M
formate,6μgml-1
FDH/FDH*.
IV. Discussion
Kinetic data
The first step to be able developing a new coating method of a hydrophilic enzyme was using a tris
buffer. FDH remained stable over three days and that even after 30 days storage. The obtained kinetic data are in
good agreement with literature data using the same enzyme system (see Table 5).
Table 5. Comparison ofobtained kinetic datawith literature.
max
/ µM mg-1
min-1
Km,NAD+
/ μM
Km,HCOO-
/ mM
KI,NADH
/ μM
pH T
/ °C
Ref.
5.30 23.1 6.18 14.6 8 25 This work
3.42 39.0 8.70 30.0 8 25 [1]
n.a. 40.0 2.42 n.a. 7.6 25 [2]
6.00 45.0 5.60 n.a. 7.5 30 [3]
[1] Kragl, Vasic-Racki, and Wandrey, 1996; [2] Labrou and Ridgen, 2001; [3] Slusaarczyk, Felber,
Kula, and Pohl, 2000.
Coating procedure and stability
To simplify the coating method used in this work DDAB was dissolved in EtOH. Converting the
concentration, used in the developed coating method, to wt% shows that the experiment produces a two-phase
emulsion of isotropic liquid L1 in the water corner (xbuffer=98.98%;xDDAB=0.04%; xEtOH=0.98%).However results
indicate that adding a DDAB/ethanol solution to a tris buffer produces a trinary phase system DDAB/EtOH/tris
buffer. This system is meta stable and strongly dependent on phase composition. Moreover, adding ethanol in a
certain amount seems to stabilize this system, shown by the fact that a gel is formed in the DDAB/tris buffer
stock solution without adding ethanol after two weeks. These observations are also established by the work of
Friberg, Hasinovic, Yin, Zhang and Patel. The authors have shown that the addition of the ethanol to the
surfactant-water lamellar liquid crystal caused a disordering of it to an isotropic liquid, a micro emulsion, which
can be separated easily in a process.
A comparison of the method developed by Okahata, Fujimoto and Ijiro with the method developed
furtherin this thesis, shows some remarkable improvements. Okahata et al. mixed anaqueous solution (25 ml,
0.01 M acetate buffer, pH 5.6) containing lipase (50 mg)with an aqueous solution dispersion (25 ml) of
dioctadecyldimethylammoniumbromide (50 mg). The preparation was stirred for 20 hours at 4°C. Precipitates
were gathered by centrifugation at 4°C (5000 rpm, 5min) and lyophilized. The lyophilized powder was taken for
kinetic measurements. However precipitates indicate that no emulsion has been formed which is similar to the
10. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
34
observation when using a potassium phosphate buffer. This shows that, if at all, only a very low enzyme amount
was coated and that an adsorption of the lipase on the surfactant surface is more likely. Results have been shown
that the yield was 34.7mg containing a protein content of 10 wt% (obtained from UV absorption). In other
words 93.06 wt% of the used lipase could not be used. In contrast we have coated all FDH present in the stock
solution. This assumption seems justified by a lower conversion of FDH* since free uncoated-FDH present in
the reaction media always lead to total conversion. However, this is more time consuming and total conversion
could be achieved adding FDH.
Application
1000 units (U) of FDH are necessary to form 1 mmole of NADH. If we want to produce NADH in a
stirred tank reactor (STR) with a stirring rate between 300-500 min-1
and assume the FDH hold a deactivation
rate k of 50% a day, with an initial activity of 100%,then, a one year repeated fed batch process would consume
181.000 U of FDH. The current market price of 1000 U of FDH is EUR 2000. In the described process the
annual cost of enzyme refilling is EUR 362.000 Euro. Coating the enzyme with DDAB using our method,
would reduce the deactivation rate k from 50% to 2% a day with respect to un-coated FDH, however with a
reduced initial activity of 50%. To produce 1 mmole of NADH would now require 2000 units (U) of FDH* in
the STR to achieve 100% initial activity but only 32.000 U of FDH* would have been consumed per year. This
leads to an around 11 fold annual cost reduction from EUR 362.000 Euro to EUR 32.800. Figure 7 shows
described example process of NADH production in a STR.
However it is known that the deactivation rate of uncoated enzyme can be reduced to 3% a day by
pressurizing the reactor system to avoid cavitation and maintain the protective and catalytic required hydrate
shell. Assuming the latter system would reduce the deactivation rate of FDH* from 1.6% to 1% a day, enzyme
consumption and costs still could be reduced by a factor 1.3 per year. Neglecting the fact that hydrophobic
coated FDH* easily could be recycled with a settler.
Figure 7. Example process of NADH production in a stirred tank reactor (STR) in comparison
with FDH and coated FDH* shown as annual cumulative enzyme consumption per mmole
NADH and cumulative cost in Euro per consumed enzyme.
V. Conclusion
A coating method was developed which enables hydrophilic enzymes being solvable in hydrophobic solvents
and improved long-term stability due to protection against mechanical stress. There is not a significant transport
resistance originating from the coating layer, however FDH* showed a lower initial activity between 40-60%.
The deactivation rate of hydrophobic coated enzyme was reduced to less 1.6% a day which makes developed
method economic attractive when running a stirred process in which the enzyme-emulsion could be easily
recycled. It would also be interesting to test this coating technology with an alcohol dehydrogenase (ADH) and
a glutamate dehydrogenase (GDH). Their products, aldehyde andα-Ketoglutarate, are interesting as precursor
for chemical industry.
Acknowledgements
We thank Prof. Christian Wandrey for fruitful discussions.
11. Improved Stability Of Formate Dehydrogenase By Coating With Didodecyldimethylammonium…
35
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