Production Of L-carnitine Research Articles

Ascophyllum nodosum polysaccharide regulates gut microbiota metabolites to protect against colonic inflammation in mice.

Ascophyllum nodosum polysaccharide (ANP) can protect against colonic inflammation but the underlying mechanism is still unclear. This study has determined the metabolites of gut microbiota regulated by ANP to reveal the mechanism of the anti-inflammation effect of ANP. Using an in vitro colonic fermentation model, the results indicate that gut microbiota could utilize a proportion of ANP to increase the concentrations of short-chain fatty acids (SCFAs) and decrease ammonia content. Metabolomics revealed that 46 differential metabolites, such as betaine, L-carnitine, and aminoimidazole carboxamide ribonucleotide (AICAR), could be altered by ANP. Metabolic pathway analysis showed that ANP mainly up-regulated the phenylalanine, tyrosine, and tryptophan biosynthesis and aminoacyl-tRNA biosynthesis, which were negatively correlated with inflammation progression. Interestingly, these metabolites associated with inflammation were also up-regulated by ANP in colitis mice, including betaine, L-carnitine, AICAR, N-acetyl-glutamine, tryptophan, and valine, which were mainly associated with amino acid metabolism and aminoacyl-tRNA biosynthesis. Furthermore, the metabolites modulated by ANP were associated with the relative abundances of Akkermansia, Bacteroides, Blautia, Coprobacillus, Enterobacter, and Klebsiella. Additionally, based on VIP values, betaine is a key metabolite after the ANP supplement in vitro and in vivo. As indicated by these findings, ANP can up-regulate the production of SCFAs, betaine, L-carnitine, and AICAR and aminoacyl-tRNA biosynthesis to protect against colonic inflammation and maintain intestinal health.

LITERATURE STUDY OF L-CARNITINE PREPARATION METHODS FOR 1000 TONS OF ANNUAL PRODUCTION

L-carnitine is a gamma-hydroxy amino acid and also known as vitamin Bγ. L-carnitine has many benefits as well as uses. Supplements with L-carnitine content are much needed in the field of health. L-carnitine is obtained using a variety of methods. They are the chemical optic resolution method, the biological method, and those that use chiral materials from natural sources. The purpose of this literature study is to determine the best method to be used in the design of the L-carnitine production plant with a capacity of 1000 tons per year by comparing various existing methods from various aspects. Based on the results of the comparison, it was found that the production of L-carnitine from (S)-3-activated hydroxybutyrolactone is the best method to be used in the production of L-carnitine capacity of 1000 tons per year for 300 days. This method produces a lot-more L carnitine products at a lower cost than other methods that have a higher yield.

Protective Effects and Metabolic Regulatory Mechanisms of Shenyan Fangshuai Recipe on Chronic Kidney Disease in Rats.

Background Chronic kidney disease (CKD) is one of the major causes of renal damage. Shenyan Fangshuai Recipe (SFR), a modified prescription of traditional medicine in China, showed potent effects in alleviating edema, proteinuria, and hematuria of CKD in clinical practices. In this study, we aimed to investigate scientific evidence-based efficacy as well as metabolic regulations of SFR in CKD treatment. Materials and Methods The effect of SFR on CKD was observed in a rat model which is established with oral administration of adenine-ethambutol mixture for 21 days. Further, metabolites in serum were detected and identified with ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Metabolomics study was performed using Ingenuity Pathway Analysis (IPA) software. Results With H&E staining and Masson's trichrome, the results showed that chronic kidney damage is significantly rescued with SFR treatment and recovered to an approximately normal condition. Along with 44 differential metabolites discovered, the regulation of SFR on CKD was enriched in glycine biosynthesis I, mitochondrial L-carnitine shuttle pathway, phosphatidylethanolamine biosynthesis III, sphingosine-1-phosphate signaling, L-serine degradation, folate transformations I, noradrenaline and adrenaline degradation, salvage pathways of pyrimidine ribonucleotides, cysteine biosynthesis III (Mammalia), glycine betaine degradation, and cysteine biosynthesis/homocysteine degradation. Further, TGFβ-1 and MMP-9 were observed playing roles in this regulatory process by performing immunohistochemical staining. Conclusion SFR exerts potent effects of alleviating glomerular sclerosis and interstitial fibrosis in the kidney, mainly via integrated regulations on metabolism and production of homocysteine, L-carnitine, and epinephrine, as well as the expression of TGFβ-1. This study provides evidence for SFR's protective effects on CKD and reveals the metabolic mechanism behind these benefits for the first time.

Chemical and enzymatic approaches to the synthesis of optically pure ethyl (R)-4-cyano-3-hydroxybutanoate

Ethyl (R)-4-cyano-3-hydroxybutanoate (HN) is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin (Lipitor), which is the hydroxymethylglutaryl CoA reductase inhibitor. HN is also used as a synthon in the production of L-carnitine and (R)-4-amino-3-hydroxybutanoic acid. It is necessary to have a clear understanding of the synthesis process of HN for its extensive use. This review gives an overview of different synthetic strategies of optically active HN, including chemical and enzymatic approaches. The emphasis is focused mainly on the synthetic routes using biocatalysts, such as halohydrin dehalogenase, nitrilase, carbonyl reductase, and lipase.

Role of betaine:CoA ligase (CaiC) in the activation of betaines and the transfer of coenzyme A inEscherichia coli

Characterization of the role of CaiC in the biotransformation of trimethylammonium compounds into l(-)-carnitine in Escherichia coli. The caiC gene was cloned and overexpressed in E. coli and its effect on the production of l(-)-carnitine was analysed. Betaine:CoA ligase and CoA transferase activities were analysed in cell free extracts and products were studied by electrospray mass spectrometry (ESI-MS). Substrate specificity of the caiC gene product was high, reflecting the high specialization of the carnitine pathway. Although CoA-transferase activity was also detected in vitro, the main in vivo role of CaiC was found to be the synthesis of betainyl-CoAs. Overexpression of CaiC allowed the biotransformation of crotonobetaine to l(-)-carnitine to be enhanced nearly 20-fold, the yield reaching up to 30% (with growing cells). Higher yields were obtained using resting cells (up to 60%), even when d(+)-carnitine was used as substrate. The expression of CaiC is a control step in the biotransformation of trimethylammonium compounds in E. coli. A bacterial betaine:CoA ligase has been characterized for the first time, underlining its important role for the production of l-carnitine with Escherichia coli.

Production of L-carnitine by secondary metabolism of bacteria.

The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate, are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation. Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency. Although genetically modified strains have been obtained, new strain improvement strategies are still needed, especially in Escherichia coli as a model organism for molecular biology studies. This review aims to summarize and update the state of the art in L-carnitine production using E. coli and Proteus sp, emphasizing the importance of proper reactor design and operation strategies, together with metabolic engineering aspects and the need for feed-back between wet and in silico work to optimize this biotransformation.

Salt stress effects on the central and carnitine metabolisms of Escherichia coli

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.

Role of energetic coenzyme pools in the production of l-carnitine by Escherichia coli

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into l(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into l(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD + ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD + pool indicated that it is correlated with the biotransformation process at the NAD + regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD + ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

Membrane cell retention systems for continuous production of l-carnitine using Proteus sp.

Membrane reactors with a Proteus sp. strain as the biocatalyst were used to attain continuous l-carnitine production from crotonobetaine. Hollow fiber cartridges with fibers of polysulfone provided adequate filtration rates and were reusable after long-term operation (consecutive runs of 140 h followed). Running under optimum medium composition, these systems enabled high cell densities (35–45 g dry weight/l) to be reached after 35–40 h at dilution rates ranging from 0.1 to 1.5 h −1, while cross-flow filtration units with flat-sheet membranes made of polyvinylidene difluoride (PVDF) showed lower cell concentrations (up to 25 g dry weight/l) and levels of l-carnitine production. The composition of the hollow fiber membranes probably improved filtration flow rates and definitely speed up the cleaning-up procedure because cake layers did not adhere tightly to the membranes. Furthermore, resting cell-cycles were maintained in continuous operation for 2–3 days with crotonobetaine and phosphate buffer as the only medium components, followed by cycles of cell re-activation, meaning that the system was stable for long periods of time (140–200 h). During resting cell-cycles, high cell decay rates were observed due to nutrient shortages. The hollow fiber system was able to produce high levels of l-carnitine during the growing/resting cycles with 35–50% conversion and productivities as high as 40.5 g/(l h). The optimum dilution rate was 1.0 h −1 as mass-transfer problems appeared at higher dilution rates because of high cell densities. The process described is presented as a way of recycling d-carnitine from the chemical industry, since, when dehydrated, this compound renders crotonobetaine.

Microbial and enzymatic production of l-carnitine

l-Carnitine (vitamin BT) plays a vital role in the transportation of long-chain fatty acids into mitochondrial matrix in mammals. l-Carnitine has a wide range of applications in pharmaceuticals, food products, and feed additives. Due to the increasing demand for l-carnitine in food and pharmaceutical applications, production of this compound has become prominent. However, very little has been reported on the production of l-carnitine. This review discusses the microbial and the enzymatic production of l-carnitine from different starting materials (substrates).

Production of l-carnitine from d-carnitine by dried cells of Agrobacterium sp. 525a

d-Carnitine was converted into l-carnitine with dried cells of Agrobacterium sp. 525a grown on d-carnitine medium. During the conversion reaction, NAD + concentration decreased. o-Phenanthroline inhibited the NAD + degrading activity in dried cells, and the formation of l-carnitine increased 2.8 times. About 190 mM of l-carnitine was converted from 0.5 M of d-carnitine under the best conditions.

Continuous production of l-carnitine with NADH regeneration by a nanofiltration membrane reactor with coimmobilized l-carnitine dehydrogenase and glucose dehydrogenase

l-Carnitine dehydrogenase (CDH) was partially purified from Pseudomonas putida IAM12014 for the stereospecific reduction of 3-dehydrocarnitine to l-carnitine. CDH and glucose dehydrogenase (GDH) were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of l-carnitine from 3-dehydrocarnitine with NADH regeneration. In the NFMBR, NAD was partially immobilized through rejection by the nanofiltration membrane and effectively regenerated by the conjugation reaction of CDH and GDH. Since 3-dehydrocarnitine was unstable at neutral pH, it was maintained under acidic conditions (pH 0.7) and supplied to the NFMBR separately from the other substrates, glucose and coenzyme NAD. As 50 mM 3-dehydrocarnitine in HCl solution, 0.05 mM NAD, and 100 mM glucose in 0.5 M Tris buffer (pH 8) were continuously supplied to the NFMBR with immobilized CDH (200 U/ml) and GDH (200 U/ml) at the retention time of 80 min and temperature of 25°C, the maximum conversion, reactor productivity, and NAD regeneration number were 78%, 113 g/ l/d, and 780, respectively. The half-life of the NFMBR was longer than 500 h.

Production of l-carnitine from d-carnitine by partially purified d- and l-carnitine dehydrogenase of Agrobacterium sp. 525a

d-Carnitine was converted to l-carnitine with partially purified d- and l-carnitine dehydrogenases from Agrobacterium sp. 525a. Using partially purified d- and l-carnitine dehydrogenases (pH 8.5 Tris–HCl buffer, 100 mM d-carnitine, 5 mM NAD +, 2 mM glutathione), about 64% of the d-carnitine could be converted into l-carnitine.

Studies on the Kinetics and Application of L-Carnitine Amidase for the Production of L-Carnitine

The kinetics of L-carnitine amidase from the newly isolated microorganism DSM 6320 is complex; the enzyme is competitively inhibited by the product L-carnitine as well as the unreactive enantiomer of the substrate, D-carnitine amide. The second reaction product, ammonia, does not affect the rate of hydrolysis. D-carnitine is an inhibitor, but should not be present normally. In batch hydrolysis experiments complete conversion of the L-enantiomer was achieved starting with DL-carnitine amide as substrate in concentrations up to 1000 mM. The L-carnitine obtained was of high optical purity (>99% enantiomeric excess) independent of the degree of conversion, of the specific activity of the enzyme employed, and of the method used to control the pH in the optimal range, around pH 8.5. A simple model was established describing the reaction kinetics of the enzyme. The model was used to simulate the time course of batch conversions. In continuous experiments the stability of the L-carnitine amidase was investigated ...

Synthesis of l(-)-carnitine by hydration of crotonobetaine by enterobacteria

Enterobacteria, especially Escherichia coli, Salmonella typhimurium and Proteus vulgaris, are capable of forming l(-)-carnitine by hydration of the double bond of crotonobetaine under anaerobic conditions. The carnitine hydrolyase is an inducible cytosolic enzyme which catalyses either the dehydration of l-carnitine or the hydration of crotonobetaine. In growing cultures, the addition of fumarate to a complex or minimal medium stimulated l-carnitine synthesis by diminishing the reduction of crotonobetaine to γ-butyrobetaine. However, l-carnitine synthesis was repressed after addition of nitrate or under aerobic conditions. If the carnitine hydrolyase was induced by l-carnitine or crotonobetaine, these respiratory chain electron acceptors did not impair carnitine formation by resting cells, indicating an epigenetical regulation of carnitine synthesis. Using this bacterial pathway for the biosynthesis of l-carnitine, conditions for producing a high yield are described. The method has some advantages in comparison with other biochemical or microbiological procedures for the production of l-carnitine.

Asymmetric production of L-carnitine from trans-crotonbetaine by Proteus mirabilis.

A novel enzymatic method for the production of L-carnitine (L-4-trimethylamino-3-hydroxybutyric acid) has been developed. Microorganisms that asymmetrically produce L-carnitine from trans-crotonbetane(trans-4-trimethylamino-2-butenoic acid), which was easily supplied through the chemical dehydration of DL-carnitine with an acid, were screened from stock cultures in our laboratory. Of the 686 strains of microorganisms examined, i.e., 416 bacteria, 37 actinomycetes and 233 yeasts, strains belonging to the genera, Proteus and Escherichia, were found to be efficient producers of L-carnitine from trans-crotonbetaine. Through the screening, Proteus mirabilis AJ-2772 and AJ-12137, with potent abilities, were selected as representative strains. The trans-crotonbetaine-hydrating enzyme was inducibly and intracellularly produced on either the addition of trans-crotonbetainQ or L-carnitine to the medium under partially anaerobic conditions. With intact cells of P. mirabilis AJ-2772, 40 g/l of L-carnitine chloride was produced from 62.5g/l of trans-crotonbetaine.

4708936 Process for the continuous production of L-carnitine: Hans Kulla, Pavel Lehky, Armand Squaratti, Visp, Switzerland assigned to Lonza Ltd

4708936 Process for the continuous production of L-carnitine: Hans Kulla, Pavel Lehky, Armand Squaratti, Visp, Switzerland assigned to Lonza Ltd