Butyryl Coenzyme A serves as a substrate for Butyryl-CoA:acetate-CoA transferase (But, EC 2.8.3.8). This enzyme catalyzes the transfer of the CoA group from butyryl-CoA to acetate. [1]
Butyryl Coenzyme A serves as a substrate for Butyryl-CoA:acetate CoA transferase (BCoAT, EC 2.8.3.8). This enzyme catalyzes the conversion of butyryl-CoA to butyrate.[2]

- The activity of butyryl-CoA:acetate-CoA transferase (But) was measured using a citrate synthase-coupled assay. The enzyme uses butyryl-CoA and acetate as substrates. The generated acetyl-CoA is condensed with oxaloacetate by citrate synthase, liberating free CoA, which reacts with 5,5'-dithio-bis-(2-nitrobenzoate) to form a yellow thiophenolate anion. Reaction rates were monitored by measuring absorbance at 412 nm at 39°C. [1]
- Among 12 putative but genes identified from swine intestinal butyrate-producing bacterial strains, eight genes encoded proteins with strong But enzyme activity, with specific activities ranging from 7,004 μM·min⁻¹·mg⁻¹ (strain 27-5-10) to 27,819 μM·min⁻¹·mg⁻¹ (strain 831b). When propionyl-CoA was used as a substrate, these highly active genes exhibited similar activity. [1]
- Highly active But proteins contain a conserved amino acid motif LQLGIGG. The glycine residue in this motif was conserved in all highly active sequences, while proteins with low But activity contained at least one substitution in this motif. [1]

---

- The purified BCoAT enzyme from Clostridium tyrobutyricum BEY8 exhibited specific activity towards butyryl-CoA, with a value of 26.2 ± 0.09 U/mg protein. However, this enzyme showed no detectable activity towards capryl-CoA.[2]
- The purified CoAT enzyme from Ruminococcaceae bacterium CPB6 (CPB6-CoAT) also showed activity towards butyryl-CoA, with a specific activity of 10.8 ± 0.02 U/mg protein. Notably, its activity towards capryl-CoA (27.6 ± 0.15 U/mg protein) was approximately 2.6-fold higher than that towards butyryl-CoA, indicating that CPB6-CoAT prefers capryl-CoA as a substrate.[2]
- Enzyme kinetic studies showed that BEY8-BCoAT had a Km value of 370 ± 4.1 μM, a kcat of 13.9 ± 0.7 min⁻¹, and a catalytic efficiency (kcat/Km) of 37.7 ± 0.2 mM⁻¹·min⁻¹ towards butyryl-CoA.[2]
- CPB6-CoAT exhibited Km, kcat, and kcat/Km values of 537 ± 10 μM, 5.81 ± 1.5 min⁻¹, and 10.8 ± 0.2 mM⁻¹·min⁻¹, respectively, towards butyryl-CoA. Towards capryl-CoA, the values were 359 ± 5.3 μM, 14.7 ± 0.9 min⁻¹, and 41.1 ± 0.2 mM⁻¹·min⁻¹, respectively. The catalytic efficiency of CPB6-CoAT towards capryl-CoA was 3.8-fold higher than that towards butyryl-CoA.[2]

- In swine proximal colon contents, RNA-based but gene libraries revealed greater OTU diversity than DNA-based libraries (Shannon diversity index with Wilcoxon paired test, P < 0.03; Shannon evenness with Wilcoxon paired test, P < 0.03), indicating that the actively transcribing butyrate-producing community differs from the most abundant members. [1]
- A total of 92 unique but gene OTUs (97% similarity) were detected from DNA and RNA extracted from the colonic contents of six pigs. Fourteen OTUs were detected in RNA libraries from every pig, four OTUs were detected in all DNA libraries, and three OTUs (OTU4, OTU14, OTU23) were detected in every library regardless of nucleic acid type. [1]

- Citrate synthase-coupled assay for butyryl-CoA transferase activity: The reaction system contains the substrates butyryl-CoA and acetate. The butyryl-CoA:acetate-CoA transferase catalyzes the transfer of CoA, producing butyrate and acetyl-CoA. The generated acetyl-CoA is condensed with oxaloacetate by citrate synthase, releasing free CoA. The released CoA reacts with 5,5'-dithio-bis-(2-nitrobenzoate) to form a yellow thiophenolate anion. Reaction rates were measured by monitoring absorbance at 412 nm at 39°C. Crude cell lysates were used for the assay and diluted with sterile water as necessary to achieve the linear range for the reaction rate. The reaction was repeated in the absence of acetate to confirm that the measured rate was not due to CoA-hydrolase activity. [1]

---

- Citrate Synthase-Coupled Assay for CoAT Activity: The reaction was carried out at 25°C in a total volume of 1 mL. The reaction mixture contained: 100 mM potassium phosphate buffer (pH 7.0), 200 mM sodium acetate, 1 mM 5,5'-dithiobis(2-nitrobenzoate) (DTNB), 1 mM oxaloacetate, 8.4 nk citrate synthase, and 0.5 mM CoA derivative (butyryl-CoA or capryl-CoA). The reaction was initiated by the addition of enzyme (final concentration up to 20 ng/mL). The free CoA released (equimolar to the amount of acetyl-CoA formed) reacted with DTNB to form a yellow thiophenolate anion. The reaction rate was determined by monitoring the change in absorbance at 412 nm. One unit of activity (U) is defined as the amount of enzyme required to produce 1 μmol of acetyl-CoA per minute under these conditions.[2]
- Enzyme Kinetic Parameter Determination: Kinetic parameters were determined using the same coupled spectrophotometric assay as described above. The concentrations of butyryl-CoA or capryl-CoA were varied from 0.5 to 5 mM. The Km and Vmax values were calculated using the Lineweaver-Burk transformation of the Michaelis-Menten equation. The catalytic constant (kcat) was defined as the number of substrate molecules converted per enzyme molecule per second. All measurements were performed in triplicate for each biological replication.[2]

- Gene cloning and protein expression: Candidate but genes were cloned into the pET-TOPO-101 vector and transformed into TOP10 E. coli chemically competent cells. Positive clones with full-length gene inserts were confirmed by sequencing. Cloned DNAs were transformed into E. coli BL21 Star competent cells for protein expression. Cultures (100 ml) were grown for 12 h in LB containing 50 μg/ml carbenicillin. Expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After an additional 6 h of growth, cultures were harvested by centrifugation, washed, and resuspended in sterile phosphate-buffered saline (PBS). Cells were lysed by two passages through a French press. Lysates were centrifuged to remove remaining unlysed cells, and the supernatant was used for activity assays. [1]

- Sample collection and processing: Proximal colon contents (10 cm distal from the cecum) from pigs were immediately placed in RNAlater and quickly homogenized to preserve nucleic acid integrity. Samples were subsequently frozen at -80°C until extraction (within 1 month). DNAs and RNAs were extracted from proximal colon contents of six pigs fed a standard diet using DNA and RNA extraction kits. The iScript Select kit was used to generate cDNA from the RNA using random hexamer primers. [1]

[1]. Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine. Appl Environ Microbiol. 2016 Oct 27;82(22):6788-6798.

[2]. Butyryl/Caproyl-CoA:Acetate CoA-transferase: cloning, expression and characterization of the key enzyme involved in medium-chain fatty acid biosynthesis. Biosci Rep. 2021 Aug 27;41(8):BSR20211135.

- Background: Butyrate is essential for colonic homeostasis and is the preferred energy source for colonocytes. Butyrate reduces local oxygen concentrations, causing epithelial hypoxia and limiting the growth of facultative aerobic pathogens like Salmonella. Additionally, butyrate alters host gene expression to promote immune tolerance to the colonic microbiota and improve colonic epithelial barrier function. [1]
- Butyrate synthesis pathway: The most common pathway for butyrate production in colonic environments involves the condensation of two molecules of acetyl-CoA, followed by reduction to butyryl-CoA. After butyryl-CoA is generated, two different enzymes are responsible for the final conversion to butyrate: butyrate kinase (Buk) and butyryl-CoA:acetate-CoA transferase (But), with the But protein being the most common in the colonic environment. [1]
- Phylogenetic analysis: The genes encoding highly functional But proteins are phylogenetically separated from potential paralogues, but this separation is not absolute. The vast majority of OTUs detected by the funbut primers (99.4%) clustered with but sequences encoding highly active enzymes, while only 0.6% of sequences fell outside this main functional clade. [1]

---

- Background and Mechanism of Action: In the reverse β-oxidation pathway, butyrate production typically begins with the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA, which is then converted to butyryl-CoA. Finally, butyryl-CoA undergoes CoA exchange with exogenous acetate via butyryl-CoA:acetate CoA transferase (BCoAT), generating acetyl-CoA and butyrate. This enzyme is considered a biomarker for identifying butyrate-producing bacteria.[2]
- Substrate Specificity Differences: BCoAT from the butyrate-producing bacterium Clostridium tyrobutyricum showed activity only towards butyryl-CoA and no activity towards capryl-CoA, indicating that this enzyme is only involved in chain elongation of C2-C4, not in the elongation from C4 to C6 or C8. In contrast, CoAT from the caproate-producing bacterium Ruminococcaceae bacterium CPB6 exhibited higher affinity and catalytic efficiency towards capryl-CoA, confirming the existence of a specific capryl-CoA:acetate CoA transferase (CCoAT).[2]
- Conserved Motif and Site-Directed Mutagenesis: Multiple sequence alignment revealed a highly conserved motif, GGQXDFXXGAXX (GGQLDFVLGAYL for CPB6-CoAT, located at amino acids 342-353). Site-directed mutagenesis experiments showed that substituting Asp³⁴⁶ with His led to approximately 76% loss of BCoAT activity and 72% loss of CCoAT activity. Substituting Ala³⁵¹ with Pro resulted in approximately 50% loss of BCoAT activity and 55% loss of CCoAT activity. These results indicate that this conserved motif is the active center of CPB6-CoAT, and the Asp³⁴⁶ and Ala³⁵¹ residues significantly impact enzymatic activity.[2]

C25H44LI2N7O19P3S

885.52

1107039-35-9

102282-28-0

Solid Powder

[Li+].[Li+].CCCC(=O)SCCNC(=O)CCNC(=O)[C@@H](C(C)(C)COP(=O)([O-])OP(=O)([O-])OC[C@@H]1[C@H]([C@H]([C@@H](O1)N2C=NC3=C(N=CN=C32)N)O)OP(=O)(O)O)O.O.O

Butyryl-Coenzyme A (lithium hydrate); 1107039-35-9; Butyryl CoA lithium hydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~56.46 mM; with heating and sonication)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)