Where to get ethyl acetate




















These results show that Eat1 homologues are better catalysts than Sce Atf1 for in vivo ethyl acetate production in E. The higher yield obtained with lower gene expression levels indicates that Wan Eat1 was more active than its K.

The ethyl acetate yields increased with rising inducer concentrations Fig. Determining the optimal inducer concentrations thus resulted in significantly improved ethyl acetate yields. The highest ethyl acetate yield was achieved in E. It produced 0. Selecting the best AAT gene and optimising, its expression level diminished the metabolic bottleneck present in ethyl acetate production, but pyruvate still accumulated Fig. This indicated that the conversion efficiency of Eat1 was still insufficient to handle the EMP metabolic pathway flux.

Removal of the mitochondrial pre-sequences of K. We tested if this elevated stability also led to more ethyl acetate production. Optimisation of gene expression levels for Kma trEat1 F and K resulted in a lower accumulation of pyruvate compared to the unprocessed version Fig. However, this did not result in a higher ethyl acetate yield; only the acetate yield increased. Comparison of truncated eat1 genes under various gene expression levels induced by 0.

Succinate and formate were detected but concentrations are not shown. Kma K. Nevertheless, the optimum inducer concentration shifted to 0. Ethyl acetate production was also higher at 0. At the same time, the acetate yields increased for induction levels above 0. However, at higher IPTG concentrations these differences were absent.

The acetate yield in the strain producing Wan trEat1 N also increased relative to the strain producing Wan Eat1.

Figure 5 d, f. As discussed above, a limiting Eat1 efficiency resulted in the accumulation of ethanol. Pyruvate was produced by E. The higher stability of the truncated Eat1 versions did indeed result in a decreased pyruvate yield Fig. Instead, the acetate yield increased, likely due to the esterase and thioesterase side activities of Eat1. In all serum bottle experiments described above, glucose consumption was incomplete, most likely caused by the accumulation of organic acids, especially formate, and the associated pH decreased due to a limited buffering capacity of the medium.

To avoid limitations caused by medium acidification, additional cultivations were performed in pH-controlled reactors under anaerobic conditions. To limit the accumulation of formate even further, Na 2 SeO 3 was added to stimulate the conversion of formate into H 2 and CO 2 by Fhl. A constant flow of nitrogen gas was applied to keep the culture conditions anoxic. This resulted in stripping of ethyl acetate, H 2 and CO 2 from the broth and the concentrations of these compounds in the exhaust gas were therefore analysed.

We cultivated E. Gene expression was induced with the optimal IPTG concentration of each strain based on the findings of previous experiments Figs. In contrast to the shake-flask experiments, glucose was fully consumed at the end of the batch fermentations and ethyl acetate production proceeded until glucose was depleted Fig.

Formate was converted into CO 2 and H 2 by E. There was no correlation between the conversion efficiency, the strain, or reactor vessel. Between Ethyl acetate production in pH-controlled bioreactors with continuous gas stripping. Two examples of controlled batch fermentations are shown.

Strains were grown under anaerobic conditions in minimal medium containing 55 mM glucose. The cumulative mass of ethyl acetate, CO 2 and H 2 removed by gas stripping was divided by the culture volume of the reactor and in case of ethyl acetate added to concentrations measured in the liquid.

Effect of pH-control and continuous ethyl acetate stripping on product yield and volumetric productivity. The numbers above the bars represent the carbon recovery of the fermentations. Formate and CO 2 yields were lumped together to compensate for the variation in H 2 formation. Experiments were performed as biological duplicates or triplicates; error bars represent the standard deviation.

Eat1 unprocessed Eat1, trEat1 truncated Eat1. Consistently, all strains cultivated in pH-controlled bioreactors showed improved performance compared to the serum bottle cultivations.

Once the unprocessed Kma Eat1 was induced with an optimal 0. A yield of 0. A similar yield was obtained in strains producing Kma trEat1 K in the presence of 0. The best producers tested in pH-controlled bioreactors were E. They formed Generally, ethyl acetate yields were between 1. The highest yield was obtained by the strain producing Wan trEat1 N, reaching 0.

The yields of ethanol and pyruvate decreased with increasing ethyl acetate yields Fig. For the strains producing unprocessed Wan Eat1 and Wan trEat1 N pyruvate accumulation was almost entirely abolished Figs.

The ethyl acetate yield for Wan Eat1 was consequently higher compared to the strains producing the Kma Eat1 variants. Not only did the trEat1 variants require lower induction levels and accumulated less by-products, glucose was also depleted faster. As a result, the volumetric productivity of ethyl acetate Q EA was higher. A similar trend was present in E. The hydrolysis of ethyl acetate by the side activity of Eat1 might be restricted by efficiently removing all ethyl acetate by gas stripping.

But due to low gas flow rates, ethyl acetate still accumulated in the liquid during the fermentation. At times of maximum productivities, liquid ethyl acetate concentrations ranged from 2. We describe the engineering of E.

In all cultivations of the metabolically streamlined E. This redox-neutral accumulation of pyruvate and ethanol indicated that the in vivo activity of Eat1 was insufficient to cope with the supply of acetyl-CoA and ethanol. Screening of E. The expression of Sce atf1 evoked acetate production, which may be related to its thioesterase activity [ 35 ].

Alternatively, Atf1 may act as an esterase, but this has not been determined. It is unknown whether ethanol inhibits the hydrolytic activity of Sce Atf1 in the same way as was demonstrated for Eat1 [ 17 ]. It was observed before that Atf1 exhibits low affinity for the catalysis of ethyl acetate despite external ethanol addition [ 15 ].

Thus, the inefficient ethyl acetate production by Sce Atf1 may have been caused by differences in substrate specificity or fermentation conditions. However, Atf1 in S. Nevertheless, the results of this study disqualified it as catalyst for effective ethyl acetate production under the tested conditions. Next to plasmid maintenance also inducer compounds are commonly imposing an additional burden to the cells [ 7 , 31 ].

Moreover, inclusion bodies may form if translation rates are too high and can been a bottleneck in the heterologous expression of AATs [ 53 , 56 ]. It is possible that lower IPTG concentrations increased the amount of correctly folded protein and led to higher ethyl acetate production.

This may result from an overall higher efficiency of the Wan Eat1 variant, compared to Kma Eat1. Consistently, Wan Eat1 and its truncated variant were most efficient.

Optimising the Eat1 efficiency by selecting a better Eat1 variant and improving the expression indeed led to a significant decrease in pyruvate accumulation. Performing similar optimisations on truncated variants, had similar effects, but interestingly also led to lower induction levels for similar or better results.

Proper cleavage likely improved protein stability, which was reflected by the lower required inducer concentration. Whilst especially the transfer to pH-controlled reactor systems boosted general performance of the presented ethyl acetate production process, production of other dissimilatory products, like succinate, ethanol and acetate, needs to be further minimised. As fermentations were performed under anaerobic conditions, ethanol and acetate could not be assimilated for additional ethyl acetate formation but remained as by-products of the fermentation.

The disruption of ackA did not block acetate synthesis completely. The predominant acetate-forming route under anaerobic conditions is the conversion of acetyl-CoA to acetyl-P and further to acetate [ 52 ]. Two genes are involved in this pathway, phosphotransacetylase pta and acetate kinase ackA , whilst other enzymes with similar catalytic activities, such as propionate kinase are able to perform the same reaction [ 14 ].

Disrupting pta and additional acid kinases might block acetate production completely. Acetate accumulation by the ackA knockout strain, and to some extent ethanol accumulation, may also result from the hydrolytic side activities of Eat1.

It has been shown that this esterase and thioesterase activity is prevented above a critical ethanol concentration [ 17 ]. Below this critical concentration, there was no net ethyl acetate synthesis and ethanol and acetate were produced instead. Under the tested conditions, Atf1 exhibited more esterase and thioesterase activities, barely producing ethyl acetate. The tested eat1 variants of W. Better understanding of the protein structure and catalytic mechanisms is needed to streamline the desired catalytic activities even further.

A build-up of high formate levels could be detrimental to cell growth and function and might have inhibited the serum bottle fermentations [ 49 ]. In batch bioreactors, this problem might be tackled by applying pH control. Moreover, converting formate to CO 2 and H 2 via the Fhl complex would allow for co-production of ethyl acetate and H 2 , the latter also being a valuable biofuel [ 5 ].

The reason for the high variability is not clear, but it may be due to the complex transcriptional regulation of the 15 genes that are required to form an active Fhl complex [ 57 , 3 , 39 ]. The issue might be prevented in the future by constitutively overexpressing fhlA , the transcriptional activator of the Fhl system to improve H 2 production [ 39 , 55 ]. Addition of nickel may also be explored as this compound is required in the functional Fhl system [ 32 ].

In situ product removal via gas stripping has already been applied in some yeast production systems [ 43 , 29 ]. Primarily, it improves downstream processing or can be used to prevent product inhibition during fermentations [ 45 , 19 ]. Whilst no critical concentrations of ethyl acetate were reached in the performed fermentations, gas stripping could benefit the fermentations by limiting ethyl acetate hydrolysis.

However, temporary accumulation and hydrolysis of ethyl acetate in the medium could not be avoided by the applied stripping rates, particularly for efficient ethyl acetate producers such as Kma trEat1 K or Wan trEat1 N Therefore, performances of the respective strains may still improve when higher stripping rates are applied.

Whilst the reduction of degradation of the product by gas stripping improves the performance in the current research, it primarily aims at preventing product toxicity [ 19 ]. Currently the reached titres of ethyl acetate are well below toxic levels for E. Whether the volumetric productivities can also compete with those reached by aerobic systems, is another factor that needs to be evaluated in the future. We demonstrated that E. The combined effects of several rounds of metabolic, protein and process engineering resulted in an up to The highest ethyl acetate yield was achieved with E.

This strain formed 0. The strains and plasmids used in this study are listed in Tables 1 and 2 , respectively.

All K. Routinely , E. The serum bottles were made anaerobic by flushing with nitrogen. A second overnight cultivation under same conditions was performed after 1—2 mL of the LB culture was transferred to 50 mL modified M9 medium in a mL Erlenmeyer flask. The anaerobic serum bottles were inoculated to an initial OD of 0. Ethyl acetate production in serum bottles was measured only in the liquid phase.

Anaerobic fermentations were performed in 1. The medium was supplemented with vitamins and trace elements [ 46 ]. Inocula were prepared by transferring 0. The reactors were inoculated to an initial OD of 0. The cumulative mass of compound C m C,gas , mol stripped up to each time point t n , h was calculated using Eq.

F gas,out was calculated assuming N 2 as an inert gas and knowing the total volumetric gas flow into the reactor F gas,in and the volumetric fractions of N 2 in the corresponding in- and outflows X N2,in , X N2,out at time point t using Eq.

The cumulative mass of stripped ethyl acetate after Eq. The resulting value is an apparent ester concentration at time t n which would be found in the culture medium if no stripping was applied. The compounds included in the calculation were glucose, ethyl acetate, ethanol, acetate, succinate, pyruvate, formate and CO 2.

Biomass formation was included in the calculation assuming a biomass composition of CH 2 O 0. The HPLC was operated at 0. Propionic acid 50 mM was used as an internal standard. Ethyl acetate and ethanol in liquid samples were measured by an Agilent B gas chromatograph equipped with a flame ionisation detector GC-FID and an Agilent autosampler. Samples were analysed by injecting 0. Technical Divisions Collaborate with scientists in your field of chemistry and stay current in your area of specialization.

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This is the second of four articles about key solvents—Ed. Ethyl acetate is one of the simplest carboxylate esters. Former Molecule of the Week methyl formate is the simplest. The colorless liquid has a sweet, fruity odor that most people find pleasant. As you might expect, ethyl acetate was first synthesized from ethanol and acetic acid.

The reaction was the classic acid-catalyzed Fischer esterification, which dates back to This is still the most widely used commercial synthesis. Please contact your local Brenntag representative if you have any questions about this information. Ethyl Acetate. Products Ethyl Acetate. Characteristics Appearance clear colorless liquid Odor mild pleasant fruity odor Solubilities soluble in water Boiling point Coatings and Construction : It is used as a solvent in inks and paints and in furniture and heavy equipment coatings.

Pharmaceutical : It is used as a solvent to extract ingredient from its source. Quick Answers. What is ethyl acetate? Where can I buy ethyl acetate? How can I buy ethyl acetate?



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