Enzymatic Process For Synthesis Research Articles

Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides.

Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni β1-4GalNAcT (CjCgtA) and β1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep one-pot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.

Enzymatic Synthesis Process of EPA- and DHA-Enriched Structured Acylglycerols at the sn-2 Position Starting from Commercial Salmon Oil and Concentrated by Response Surface Methodology under Supercritical Conditions

The bioavailability of n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) has shown to be greatly influenced by their location in the triacylglycerol backbone. Therefore, the synthesis of structured acylglycerols (SAcyl), which include eicosapentaenoic acids (EPAs) or docosahexaenoic acids (DHAs) at the sn-2 position, has attracted a great interest. The objective of this study was to optimize the synthesis process of a SAcyl from commercial refined salmon oil and an EPA/DHA concentrate in order to enhance the positioning of EPA and DHA in the sn-2 location of the glycerol moiety. For this purpose, immobilized lipase B from Candida antarctica (nonspecific) was used for the acidolysis process under the CO2 supercritical condition. As a result of carrying out a Draper-Lin composite design through the response surface methodology of 18 experiments, an optimized extraction including SAcyl compounds was obtained. Mass spectrometry (MALDI-TOF) analysis was employed to identify the EPA/DHA location at the sn-2 position in the resulting glycerol moiety. In the fraction obtained, an increase in the EPA and DHA content at the sn-2 position was detected. Remarkably, the optimized SAcyl obtained after 6 h, 82 bar, and 60 °C led to the highest EPA/DHA yield at the sn-2 position in the resulting molecule.

Development of an enzymatic process for the synthesis of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol, the key intermediate of ticagrelor

Asymmetric bioreductions catalyzed by ketoreductases have shown considerable potential in the synthesis of chiral alcohols. However, the synthesis routes of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL), a key chiral intermediate in the synthesis of ticagrelor, still pose significant challenges in term of unsatisfactory substrate loading and production cycle. In this study, a structure-guided rational design was applied to a ketoreductase from Candida glabrata (CgKR1) for the asymmetric reduction of the ketone precursor 2-chloro-1-(3, 4-difluorophenyl) ethanone (CFPO). A number of mutants were obtained with remarkably improved activity and inversed stereoselectivity compared with CgKR1-WT. In particular, F92V/Y208T mutant and F92T/Y208V mutant exhibited strict S-stereoselectivity and extremely high catalytic activity for CFPO. In addition, CFPO with the concentration of 200 g/L can be completely converted into (S)-CFPL on a preparative scale (5.0 g, 92.8% isolated yield) in 4 h through optimizing some parameters in the reduction process, delivering a high space-time yield (STY) of 1200 g L−1 day−1. This study not only provides insight into the relationship between the structure and function of CgKR1, but also establishes an efficient enzymatic process for the biocatalytic synthesis of crucial chiral intermediate (S)-CFPL of ticagrelor.

Engineering of continuous bienzymatic cascade process using monolithic microreactors – In flow synthesis of trehalose

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 gproduct·L-1·h−1·mgprotein-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN3 solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.

Development of an Enzymatic Process for the Synthesis of the Key Intermediate of Telotristat Ethyl

Abstract(R)‐1‐(4‐chloro‐2‐(3‐methyl‐1H‐pyrazol‐1‐yl)phenyl)‐2,2,2‐trifluoroethan‐1‐ol [(R)‐2] is a key chiral intermediate in the synthesis of telotristat ethyl, an anti‐carcinoid syndrome drug. However, the synthetic methods for (R)‐2 mainly include chemical reductions, rarely involving biocatalysis or bioreduction until now. Here, we report a method for obtaining (R)‐2 by biocatalytic reduction of corresponding prochiral acetophenone (1) with bulky o‐substitution using recombinant Lactobacillus fermentum short‐chain dehydrogenase/reductase 1 (LfSDR1) as a biocatalyst. Further engineering of LfSDR1 variants could access asymmetric reduction of 1 and yield (R)‐2 with a >99% ee and a >99% conversion. In addition, through the test of different co‐solvents and a series of initial substrate concentrations, substrate 1 with the concentration of 60 g/L can be completely converted into (R)‐2 on a preparative scale (1.13 g, 3.93 mmol, 75.6% isolated yield) in 24 hours. This study presents an efficient enzymatic process for the biocatalytic synthesis of key chiral intermediate (R)‐2 in the synthesis of telotristat ethyl.magnified image

A new reactor for enzymatic synthesis of biodiesel from waste cooking oil: A static-mixed reactor pilot study

Nowadays, biodiesel has become a globally accepted renewable liquid fuel. The environmentally friendly enzymatic synthesis process is being widely researched. To solve the problem that production efficiency is low in enzymatic process, we optimized the structure of the static mixer by computational fluid dynamics (CFD) modeling. The optimal static mixer consists of 6 mixer units, and the length-diameter ratio of every mixer unit is 1.5. The enzymatic synthesis of biodiesel was conducted between waste cooking oil (WCO) and methanol by transesterification and esterification. The reaction conditions were also optimized in this designed static-mixed reactor. Under optimal conditions, the reaction time can save half time when compared with the traditional stirred reactor, which indicates that the static mixer has higher production efficiency.

Quantitative analysis of straight-chain/branched-chain Ratio During Enzymatic Synthesis of Dextran Based on Periodate Oxidation

The applications of dextran depend not only on the molecular weight but also on the types and number of branches. In this study, dextran generated from Leuconostoc mesenteroides (L.M.CICC-20724) was characterized by fourier-transform infrared spectrum and nuclear magnetic resonance spectroscopy. Our analyses showed that dextran was a polysaccharide composed of d-glucose units with predominantly α(1 → 6) linkages in the main chain and few α(1 → 3) linkages in the branch. Periodate oxidation, a classic chemical method, was usually combined with Smith degradation and gas chromatography to analyze glycosidic linkages in polysaccharide quantitatively. In this study, we calculated the exact straight-chain/branched-chain ratio in the dextran using periodate oxidation only. The ratios obtained by periodate oxidation only were compared to the ratios obtained by nuclear magnetic resonance. The results showed that the ratios of the two groups were nearly equal, and the average relative error between the two groups was 0.83%. This method was evaluated and found to be accurate and stable. This technique provided a convenient and straightforward chemical method for the quantitative analysis of the straight-chains and branched-chains in polysaccharides which had a similar structure. The ratios during the enzymatic synthesis process of dextran were analyzed by this method and were found to be stable with a high level of approximately 95% on average.

Biodiesel with low temperature properties: Enzymatic synthesis of fusel alcohol fatty acid ester in a solvent free system

Poor low temperature behavior, the critical defect of biodiesel, limits its utilization in cold districts. This research provided a new biodiesel production process by enzymatic synthesis using fusel alcohol and fatty acid esters in a solvent free system with a significant effect on the improvement of biodiesel's low temperature performance. The optimal conditions for the enzymatic reaction of fusel alcohol and waste cooking oil (WCO) esters were established: immobilized lipase 14% (w/w), total water content 5% (w/w), 40 °C, three-times feeding daily, shaking speed 200 rpm. The ester yield reached 90.4% under optimal conditions. The cold filter plugging point (CFPP) of the fusel alcohol esters was −11 °C, 16 °C lower than the methyl esters of the WCO. The green enzymatic synthesis process and the CFPP results were examined in this research.

Enzymatic synthesis of soybean biodiesel using supercritical carbon dioxide as solvent in a continuous expanded-bed reactor

The enzymatic synthesis of soybean biodiesel using supercritical carbon dioxide (SC-CO2) as solvent in a continuous expanded-bed reactor was investigated. For this purpose, the commercial immobilized lipase Novozym 435 was used as catalyst and the variables investigated were the amount of enzyme (10–70g), oil to ethanol molar ratio (1:9–1:21) and substrate to solvent mass ratio (1:1–1:3), pressure ranging from 100 to 200bar at 70°C. Reaction conversions as high as 90% were obtained at 200bar, oil to ethanol molar ratio of 1:9, substrate to solvent mass ratio of 1:3 using 40g of enzyme. The continuous process for enzymatic synthesis of biodiesel using SC-CO2 showed to be a promising technology for large-scale purposes. The proposed system demonstrated to be possible to obtain high conversion using lesser enzyme than in a packed-bed reactor, which might be an important advantage, due to the exhibited enzyme high-costs.

A two-stage enzymatic process for synthesis of extremely pure high oleic glycerol monooleate

This paper presents a research interest concentrating on aims to establish a feasible industrial process for enzymatic production of highly pure glycerol monooleate (GMO). The synthesis of high oleic glycerol monooleate by enzymatic glycerolysis of high oleic sunflower oil, using Novozyme 435 as the biocatalyst, in a binary solvent mixture of tert-butanol and tert-pentanol (80/20, v/v), at a lab scale has been studied. A yield of 75.31% monoacylglycerol has been achieved at the first stage. A yield of 93.3% GMO was finally reached after further purification at the second stage. To evaluate the possibility of the process for industrialization, production of GMO was performed at a pilot-plant scale under the correspondingly adjusted conditions. A yield of 68.17% and 93.4% of GMO was obtained, respectively, at the end of the three stages.

Synthesis of l-theanine using enzyme/mesoporous silica conjugates under high pH conditions

We present a new enzymatic process for synthesis of l-theanine using glutaminase combined with immobilization technique on a mesoporous silica (MPS). The MPS was firstly attempted to modify with zirconia in order to enhance the durability against the reaction under high pH conditions. The glutaminase on the MPS successfully catalyzed the reaction for the synthesis of l-theanine. The glutaminase/MPS conjugate was subsequently recovered and employed for the reaction again. The conjugate showed the corresponding activity to the first synthesis. This indicates that the conjugate functions as a catalyst for synthesis of l-theanine, having the operational stability sufficient for reuse.

Two-step Enzymatic Synthesis of Guanine Arabinoside

The chemical synthesis of Guanine arabinoside (ara-G) is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside (ara-DA) was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L −1 uracil arabinoside as arabinose donor, 10 mmol·L −1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L −1 phosphate buffer, and reacted at 60°C for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.

Highly Efficient Enzymatic Synthesis of 2‐Monoacylglycerides and Structured Lipids and their Production on a Technical Scale

We report here a two-step process for the high-yield enzymatic synthesis of 2-monoacylglycerides (2-MAG) of saturated as well as unsaturated fatty acids with different chain lengths. The process consists of two steps: first the unselective esterification of fatty acids and glycerol leading to a triacylglyceride followed by an sn1,3-selective alcoholysis reaction yielding 2-monoacylglycerides. Remarkably, both steps can be catalyzed by lipase B from Candida antarctica (CalB). The whole process including esterification and alcoholysis was scaled up in a miniplant to a total volume of 10 l. With this volume, a two-step process catalyzed by CalB for the synthesis of 1,3-oleoyl-2-palmitoylglycerol (OPO) using tripalmitate as starting material was established. On a laboratory scale, we obtained gram quantities of the synthesized 2-monoacylglycerides of polyunsaturated fatty acids such as arachidonic-, docosahexaenoic- and eicosapentaenoic acids and up to 96.4% of the theoretically possible yield with 95% purity. On a technical scale (>100 g of product, >5 l of reaction volume), 97% yield was reached in the esterification and 73% in the alcoholysis and a new promising process for the enzymatic synthesis of OPO was established.

A novel strategy for enzymatic synthesis of 4-hydroxyisoleucine: identification of an enzyme possessing HMKP (4-hydroxy-3-methyl-2-keto-pentanoate) aldolase activity

A two-step enzymatic synthesis process of 4-hydroxyisoleucine is suggested. In the first step, the aldol condensation of acetaldehyde and alpha-ketobutyrate catalyzed by specific aldolase results in the formation of 4-hydroxy-3-methyl-2-keto-pentanoate (HMKP). In the second step, amination of HMKP by the branched-chain amino acid aminotransferase leads to synthesis of 4-hydroxyisoleucine. An enzyme possessing HMKP aldolase activity (asHPAL) was purified 2500-fold from a crude extract of Arthrobacter simplex strain AKU 626. Sequencing of the asHPAL structural gene showed that the purified enzyme belongs to the HpcH/HpaI aldolase family. The 4-hydroxyisoleucine was synthesized in vitro from acetaldehyde, alpha-ketobutyrate and l-glutamate using a coupled aldolase/branched-chain amino acid aminotransferase bienzymatic reaction.

Production of Biodiesel by Immobilized <I>Candida</I> sp. Lipase at High Water Content

A new process for enzymatic synthesis of biodiesel at high water content (10-20%) with 96% conversion by lipase from Candida sp. 99-125 was studied. The lipase, a no-position-specific lipase, was immobilized by a cheap cotton membrane and the membrane-immobilized lipase could be used at least six times with high conversion. The immobilized lipase could be used for different oil conversion and preferred unsaturated fatty acids such as oleic acid to saturated fatty acids such as palmitic acid. The changes in concentration of fatty acids, diglycerides, and methyl esters in the reaction were studied and a mechanism of synthesis of biodiesel was suggested: the triglycerides are first enzymatically hydrolyzed into fatty acids, and then these fatty acids are further converted into methyl esters.

A novel process for enzymatic synthesis of N-lauroyl-β-amino propionitrile using packed bed reactor coupled with on-line separation

N-Lauroyl-β-amino propionitrile is an intermediate for synthesis of sodium N-lauroyl-β-alanine, an antimicrobial surfactant. We provide a novel process for enzymatic synthesis of N-lauroyl-β-amino propionitrile, using a cascade connection of an enzyme packed bed reactor (EPBR) with a crystallization separator for on-line separation. The substrate solution was fed to the reactor inlet. High-purity crystal product was obtained from the separator outlet with a yield of 91.7% under the optimum conditions. The immobilized lipase can be utilized repeatedly. The solvent and unreacted substrates were recovered and reused on-line.

Electrochemical regeneration of coenzyme NADH on a histidine modified silver electrode

Abstract l -Histidine was attached to a silver electrode by a covalent bond. Cyclic voltammetric experiments demonstrated that the modified electrode can catalyze the reduction of NAD + to enzymatically active NADH at a substantially lower overpotential (i.e. −0.72 V vs. SCE). The electrochemical reduction mechanism was investigated by in situ spectroelectrochemistry. It is possible to envisage the formation of NADH through direct electron transfer. The modified electrode could be useful in enzymatic synthesis processes and in dehydrogenase-based biosensors.

A facile enzymatic process for the preparation of (s)-Naproxen ester prodrug in organic solvents

A facile enzymatic synthesis process has been developed to directly prepare the 4-morpholinoethyl ester prodrug of (s)-Naproxen from racemic Naproxen using lipases as the biocatalysts in the organic solvent. With the careful selection of lipase (Lipase MY) and reaction medium (cylohexane), a high enantiomeric ratio of 136 for the enzyme was obtained. A Ping-Pong Bi Bi mechanism with competitive inhibition by the alcohol was illustrated from the variation of initial rates with substrate concentrations.

Bioelectrocatalysis of NAD+ reduction

A thermokinetic approach to the catalysis of the electrochemical reduction of NAD+ by the hydrogenase of Alcaligenes eutrophus H16 is presented. The influence of temperature is weak, with an activation energy of 18 kJ/mol, whereas classical NAD+ hydrogenation with gaseous hydrogen shows an activation energy of 34 kJ/mol. The influence of the electric field on the thermal degradation of the enzyme is emphasized: the hydrogenase is clearly activated by the electric field and is shielded against thermal degradation. In contrast, in the absence of an electric field the thermal degradation of the hydrogenase follows rapid first-order kinetics with a high activation energy of the order of 70 kJ/mole. The catalysis of the bioelectrochemical reduction of NAD+ follows a quite different path to that of the hydrogenation reaction. In practice, the temperature is no longer an essential parameter to be considered in the integration of the NAD+ bioelectrochemical reduction within an enzymatic synthesis process. Adsorption phenomena and specific area of the electrode should be preferentially taken into account. The synthesis—regeneration coupling is tested in a thin-layer cell and gives a high NAD turnover number value of 448 cycles/h.

Bioelectrocatalysis of NAD + reduction

A thermokinetic approach to the catalysis of the electrochemical reduction of NAD + by the hydrogenase of Alcaligenes eutruphus H16 is presented. The influence of temperature is weak, with an activation energy of 18 kJ/mol, whereas classical NAD + hydrogenation with gaseous hydrogen shows an activation energy of 34 kJ/mol. The influence of the electric field on the thermal degradation of the enzyme is emphasized: the hydrogenase is clearly activated by the electric field and is shielded against thermal degradation. In contrast, in the absence of an electric field the thermal degradation of the hydrogenase follows rapid first-order kinetics with a high activation energy of the order of 70 kJ/mole. The catalysis of the bioelectrochemical reduction of NAD + follows a quite different path to that of the hydrogenation reaction. In practice, the temperature is no longer an essential parameter to be considered in the integration of the NAD + bioelectrochemical reduction within an enzymatic synthesis process. Adsorption phenomena and specific area of the electrode should be preferentially taken into account. The synthesis-regeneration coupling is tested in a thin-layer cell and gives a high NAD turnover number value of 448 cycles/h.