Lipase-catalyzed acylation of lily polysaccharide in ionic liquid-containing systems

A comparative study was made of immobilized Burkholderia cepacia lipase (PSL-C)-catalyzed acylation of lily polysaccharide (LP) with vinyl acetate in organic solvents, ionic liquids (ILs) and IL-containing systems. The degree of substitution (DS) of the modified LP was used to evaluate the extent of acylation and thus enzymatic activity. In this manner, an eco-friendly solvent, 2-methyltetrahydrofuran (MeTHF), was found to be the most suitable organic reaction medium. However, compared to MeTHF, enhanced enzyme activity was observed when 1-butyl-3-methylimidazolium tetrafluorobrate ([C₄MIm][BF₄]) was used as the solvent. To further enhance the DS of the modified LP product, co-solvent mixtures of [C₄MIm][BF₄] and MeTHF were investigated. Among the various MeTHF–[C₄MIm][BF₄] systems examined, 20% (v/v) MeTHF–[C₄MIm][BF₄] produced the highest DS. In this reaction medium, the optimal water activity, reaction temperature and time were 0.33, 55 °C and 18 h, respectively, producing a product DS as high as 0.67. The PSL-C enzyme exhibited a much higher stability in the IL-containing system. Additionally, PSL-C-catalyzed acylation of LP was highly regioselective, causing acylation of only C6OH.     

Enzymatic acylation of polar dipeptides: Influence of reaction media and molecular environment of functional groups

The enzymatic acylation of polar dipeptides was investigated. First, the Novozym435^®-catalyzed acylation of Lys-Ser, HCl exhibiting three potential acylable sites was carried out in organic media (2-methyl-2-butanol, oleic acid) and in an ionic liquid ([Bmim]⁺[PF₆]^?). In these reactions, the chemo-selectivity of the acylation was exclusively in favour of the N?-lysine acylation and the efficiency (substrate conversion) was demonstrated to be under control of the peptide solubility. The use of [Bmim]⁺[PF₆]^? permitted to significantly improve the dipeptide solubility, and to enhance both substrates conversion and initial rates of acylation reaction. In the three reaction media used, the O-acylated derivative of the dipeptide was never detected suggesting a weak reactivity of the serine hydroxyl group due to its molecular environment and particularly to the presence of a free carboxylic group known for its electroattractor property.Last, the acylation of a natural dipeptide (carnosine), exhibiting a very low solubility in organic solvents, was also performed. Carnosine was successfully N-acylated in 2-methyl-2-butanol, and a yield of 39% was obtained when improving the substrate solubility: a better dispersibility was obtained by application of a high pressure on the reaction medium just before starting the reaction.     

Effect of different reaction parameters on the lipase-catalyzed selective acylation of polyhydroxylated natural compounds in ionic liquids

The effect of various reaction parameters on the enzymatic acylation of plant polyhydroxylated compounds, including phenolic and flavonoid glucosides (salicin, helicin, esculin and naringin), was investigated in imidazolium-based ionic liquids (1-butyl-3-methylimidazolium tetrafluoroborate [bmim]BF₄ and 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆), using immobilized lipase B from Candida antarctica. The conversion yield, the regioselectivity and the reaction rate of the biocatalytic process strongly depended on the ionic liquid used, their water content, the incubation temperature, as well as the solubility and the concentration of substrates. For most glucosides tested, one major product (monoacylated derivative) was detected as a result of the acylation of the primary hydroxyl group of glucose moiety. The acylation rate and the regioselectivity of the process are higher in [bmim]BF₄, where the solubility of all glucosides is significantly higher than in [bmim]PF₆ or acetone. Response surface methodology (RSM) based on a five level-three variable central composite circumscribed design, was employed to evaluate the interactive effect of the molar ratio of substrates (MR), the initial concentration of glucoside (N) and the reaction time (RT), as well as for their optimization in [bmim]BF₄. At the optimal reaction conditions the maximum acylation yield was 87%. The amount of monoacylated derivatives produced in a single-step biocatalytic process reached values up to 31.6 g/l which is considerably higher than those reported for organic media.     

The effect of substrate structure on the chemoselectivity of Candida antarctica lipase B-catalyzed acylation of amino-alcohols

The selective acylation of multifunctional compounds exhibiting both alcohol and amino groups gives interesting products with many applications in food, cosmetic and pharmaceutical industries, but it is real challenge. The current work describes the different behavior shown by Candida antarctica lipase B (Novozym 435) when catalyzing the O-acylation and N-acylation of bifunctional acyl acceptors. The acylation of three amino-alcohols (alaninol, 4-amino-1-pentanol and 6-amino-1-hexanol) was studied using myristic acid as an acyl donor. To achieve this, a structure-reactivity study was performed in tert-amyl alcohol as a solvent, comparing the three amino-alcohols as acyl acceptors and a series of structurally related amines, namely (R)-sec-butylamine, 1-methoxy-2-propylamine and 1,2-diaminopropane. These substrates were designed to investigate the effect of the group located in β-position of the amino group on the acyl acceptor: the more nucleophilic the group, the more the apparent maximal velocity (V_max,app) of N-acylation increases. Moreover, the crucial role of the carbon chain length between the alcohol and amino groups on the chemoselectivity was also demonstrated. The chemoselectivity for the N-acylation was improved when the carbon chain included two carbons (alaninol) whereas the chemoselectivity for the O-acylation was improved when the carbon chain included four carbons or more (4-amino-1-pentanol and 6-amino-1-hexanol).These results provided new insights for the selective synthesis of amides or esters produced from the acylation of bifunctional substrates.     

Highly Efficient and Enzymatic Regioselective Undecylenoylation of Gastrodin in 2-Methyltetrahydrofuran-Containing Systems

Highly efficient and regioselective acylation of pharmacologically interesting gastrodin with vinyl undecylenic acid has been firstly performed through an enzymatic approach. The highest catalytic activity and regioselectivity towards the acylation of 7′-hydroxyl of gastrodin was obtained with Pseudomonas cepacia lipase. In addition, it was observed the lipase displayed higher activity in the eco-friendly solvent 2-methyltetrahydrofuran-containing systems than in other organic solvents. In the co-solvent mixture of tetrahydrofuran and 2-methyltetrahydrofuran (3/1, v/v), the reaction rate was 60.6 mM/h, substrate conversion exceeded 99%, and 7′-regioselectivity was 93%. It was also interesting that the lipase-catalyzed acylation couldn’t be influenced by the benzylic alcohol in gastrodin. However, pseudomonas cepacia lipase displayed different regioselectivity towards gastrodin and arbutin.     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.     

Controllable selective enzymatic synthesis of N-acyl and O-acylpropranolol vinyl esters and preparation of polymeric prodrug of propranolol

Controllable selective synthesis strategy of polymerizable N-acyl and O-acylpropranolol vinyl derivatives was developed by enzyme-catalyzed acylation of propranolol using divinyl dicarboxylates with different carbon chain length as acyl donor. The influence of parameters including enzyme, solvents and chain length of acyl donor on the reaction was investigated in detail. Lipase AY30 in diisopropyl ether demonstrated high selectivity towards the amino group of propranolol, while lipase M from Mucor javanicus in dioxane acylated selectively the hydroxyl group of propranolol. N-Acylpropranolol (3a–3c) and O-acylpropranolol vinyl (4a–4c) derivatives were obtained successfully, and can be used for preparing functional macromolecular prodrugs of beta-blockers drugs. N-(Vinyladipoyl)propranolol (NVAP) was copolymerized with methyl methacrylate (MMA) using AIBN as initiator. The obtained polymeric prodrug was characterized with IR, NMR and GPC. The poly(NVAP-co-MMA) has M_n of 3.23 × 10⁴, and M_w/M_n of 1.66.     

Application of organic solvent system for lipase-catalyzed regioselective benzoylation of 1-<Emphasis Type="Italic">β</Emphasis>-D-arabinofuranosylcytosine

In this paper, enzymatic regioselective acylation of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl benzoate (VB) using immobilized Candida antarctica lipase B in binary organic solvents was explored. It was found that the lipase showed high regioselectivity (> 99%) towards the 5′-OH of ara-C in the representative organic solvent mixture (hexane-pyridine). To understand the enzymatic processes and provide a fair comparison of hexane-pyridine with C₄MIm·PF₆-pyridine (the representative ionic liquid-containing system), the effect of each process variable on the reactions in hexane-pyridine was investigated. The results indicate that the optimum hexane content, initial a _w , molar ratio of VB to ara-C, and temperature were 28% (v/v), 0.11, 15, and 40°C, respectively. Under optimized conditions, the initial reaction rate in hexanepyridine (44.4 mM/h) was much higher than that in C₄MIm·PF₆-pyridine (29.4 mM/h) for each case. The maximum conversion yield, however, was increased when the reaction system was shifted from hexane-pyridine to C₄MIm·PF₆-pyridine. Further study revealed that the presence of an acidic by-product (benzoate acid, released during the acylation process) may cause rapid inactivation of the enzyme in hexane-pyridine, leading to a lower conversion rate, whereas the ionic liquid may have coating and protecting effects on the lipase during the reaction.     

Efficient synthesis of 5′-O-laurate of 1-β-d-arabinofuranosylcytosine via highly regioselective enzymatic acylation in binary solvent mixtures

Regioselective enzymatic acylations of 1-β-d-arabinofuranosylcytosine (ara-C) with vinyl laurate (VL) in binary organic solvents were explored for the preparation of 5′-O-laurate of ara-C. Among the nine kinds of enzymes, Novozym 435 showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. This lipase showed higher catalytic activity in hexane–pyridine than in other tested solvent mixtures. The most suitable VL to ara-C molar ratio, initial water activity, and reaction temperature were shown to be 15:1, 0.07, and 50 °C, respectively, under which the initial reaction rate and the maximum substrate conversion were as high as 84.0 mmol L^?1 h^?1 and 98.1%, respectively. The product of Novozym 435-catalyzed acylation was characterized by ¹³C NMR and confirmed to be 5′-O-laurate of ara-C.     

Perturbation Theory in the Catalytic Rate Constant of the Henri–Michaelis–Menten Enzymatic Reaction

The Henry–Michaelis–Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k ₂ of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k ₂ small compared to k _?1, we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E _o and?S _o , which can be comparable or much different.     

Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase-catalyzed alcoholysis

The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample, which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant (K_I) and methyl propionate and propanol dissociation constants (K_MP and K_P) have been determined in the solid/gas reactor in the presence of 2-methyl-2-butanol, and compared with values previously obtained in the absence of added component and in the presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase-catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower K_MP and K_I are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k₂, and on the other hand to a higher binding rate k₁ between substrate and free enzyme in gaseous medium.     

Mechanism of action of N-phenyl-N′-(2-chloroethyl)ureas in the colchicine-binding site at the interface between α- and β-tubulin

Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure–activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N′-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on β_ΙΙ-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-β198 rather than Cys-β239. In addition, these studies suggest that Cys-β239 together with Asn-α99, Ser-α176, Thr-α177, Leu-β246, Asn-β247, Ala-β248, Lys-β252 and Asn-β256 are implicated in the stabilization of a C-BS–CEU complex prior to the acylation of Glu-β198 by CEU. Our molecular models propose the formation of a stabilized C-BS–CEU complex before the completion of the Glu-β198 acylation; acylation triggering conformational changes of β-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.     

Highly regioselective enzymatic synthesis of 5′-O-stearate of 1-β-D-arabinofuranosylcytosine in binary organic solvent mixtures

In this paper, highly regioselective enzymatic acylations of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5′-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS–ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50°C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol·L⁻¹h⁻¹, 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13(¹³C) NMR and confirmed to be 5′-O-stearate of ara-C.     

Probing reactivity and substrate specificity of both subunits of the dimeric Mycobacterium tuberculosis FabH using alkyl-CoA disulfide inhibitors and acyl-CoA substrates

The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C₆-C₂₀ acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C₁₂ acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C₁₂ acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C₁₈-C₂₀ acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction.     

Isoamyl acetate synthesis in imidazolium-based ionic liquids using packed bed enzyme microreactor

The acylation of isoamyl alcohol with acetic anhydride catalyzed by immobilized Candida antarctica lipase B was studied in ionic liquids (ILs) based on quaternary imidazolium cations with alkyl, alkenyl, alkynyl, benzyl, alkoxyl or N-aminopropyl side chains. Among the tested ILs, the highest enzyme activity together with the highest isoamyl acetate yield were obtained in [C₇mmim][Tf₂N]. No loss of lipase B activity was observed during one-month incubation in this hydrophobic IL without the presence of substrates. Isoamyl acetate synthesis using [C₇mmim][Tf₂N] as solvent was further studied in a continuously operated miniaturized enzymatic packed bed reactor at various flow rates and temperatures. Up to 92% isoamyl acetate yield could be obtained within 15 min by using 0.5 M acetic anhydride and 1.5 M isoamyl alcohol inlet concentrations at 55 °C, corresponding to the volumetric productivity of 61 mmol l^?1 min^?1, which to the best of our knowledge is the highest reported so far for this reaction. No decrease in productivity was experienced during the subsequent runs of continuous microbioreactor operation performed within 14 consecutive days. The benefits of reactor miniaturization along with the green solvent application were therefore successfully exploited for the development of a sustainable flavour ester production.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

An expedient synthesis of fluorescent labeled ceramide-1-phosphate analogues

A synthesis for fluorescent analogs of ceramide-1-phosphate bearing 9-anthrylvinyl or 4,4-difluoro-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY) fluorophore at co-position of fatty acid residue was carried out. The key stage of the synthesis is hydrolysis of corresponding sphingomyelins catalyzed by phospholipase D from Streptomyces chromofuscus; the enzymatic yield has been raised to 50–70% by appliance of organic solvent in the incubation medium.     

Insight into the reaction mechanism of the Escherichia coli cyclopropane fatty acid synthase: Isotope exchange and kinetic isotope effects

Cyclopropanation of unsaturated lipids is an intriguing enzymatic reaction and a potential therapeutic target in Mycobacterium tuberculosis. Cyclopropane fatty acid synthase from Escherichia coli is the only in vitro model available to date for mechanistic and inhibition studies. While the overall reaction mechanism of this enzymatic process is now well accepted, some mechanistic issues are still debated. Using homogeneous E. coli enzyme we have shown that, contrary to previous report based on in vivo experiments, there is no exchange of the cylopropane methylene protons with the solvent during catalysis, as probed by ultra high resolution mass spectrometry. Using [methyl-¹⁴C]-labeled and [methyl-³H₃]-S-adenosyl-l-methionine we have measured a significant intermolecular primary tritium kinetic isotope effect (^TV/K_app = 1.8 ± 0.1) consistent with a partially rate determining deprotonation step. We conclude that both chemical steps of this enzymatic cyclopropanation, the methyl addition onto the double bond and the deprotonation step, are rate determining, a common situation in efficient enzymes.     

Enzymatic esterification of alkane-2,3-diols by the microsomes of the uropygial glands of ring-necked pheasants (Phasianus colchicus)

Extracts of uropygial glands of ring-necked pheasants (Phasianus colchicus) catalyzed diester formation from tritiated C₁₈ alkane-2,3-diol. Monoacylated diol was also tentatively identified in the enzymatic product. Subcellular fractionation showed that the esterifying activity was located mainly in the microsomal fraction. ATP and CoA were required for the esterification process, and this reaction was stimulated by Mg²⁺. Source of the acyl moieties for esterification was endogenous, and addition of exogenous fatty acid inhibited the reaction. When microsomes were treated with bovine serum albumin (BSA) in order to remove the endogenous source of acyl moieties, palmitoyl-CoA substituted for the ATP and CoA requirement. The pH optimum with ATP and CoA was between 6.0–9.0, while maximal rates of esterification were obtained with palmitoyl-CoA from pH 7.0 to 9.0. Borate ions stimulated esterification. The half maximal velocity was obtained with 2.0 × 10^?4, m diol, and 7.2 × 10^?5, m palmitoyl-CoA. Thiol reagents severely inhibited the esterification reaction with ATP and CoA, while much less inhibition was observed when palmitoyl-CoA was used. It is concluded that a microsomal acyl-CoA-diol transacylase catalyzes stepwise acylation of alkane-2,3-diols to give the diol diester which constitute the major component of the uropygial lipids of ring-necked pheasants.