4-methylumbelliferyl Butyrate Research Articles

Selective Butyrate Esterase Probe for the Rapid Colorimetric and Fluorogenic Identification of Moraxella catarrhalis.

Clinical identification of the pathogenic bacterium Moraxella catarrhalis in cultures relies on the detection of bacterial butyrate esterase (C4-esterase) using a coumarin-based fluorogenic substrate, 4-methylumbelliferyl butyrate. However, this classical probe may give false-positive responses because of its poor stability and lack of specificity. Here, we report a new colorimetric and fluorogenic probe design employing a meso-ester-substituted boron dipyrromethene (BODIPY) dye for the specific detection of C4-esterase activity expressed by M. catarrhalis. This new probe has resistance to nonspecific hydrolysis that is far superior to the classical probe and also selectively responds to esterase with rapid colorimetric and fluorescence signal changes and large "turn-on" ratios. The probe was successfully applied to the specific detection of M. catarrhalis with high sensitivity.

Design, Synthesis, and X-ray Analysis of a Glycoconjugate Bound to Mycobacterium tuberculosis Antigen 85C

Tuberculosis (TB) is a global health threat with nearly 500 000 new cases of multidrug-resistant TB estimated to occur every year, so new drugs are desperately needed. A number of current antimycobacterial drugs work by interfering with the biosynthesis of key components of the mycolylarabinogalactan (mAG). In light of this observation, other enzymes involved in the synthesis of the mAG should also serve as targets for antimycobacterial drug development. One potential target is the Antigen 85 (Ag85) complex, a family of mycolyltransferases that are responsible for the transfer of mycolic acids from trehalose monomycolate (TMM) to the arabinogalactan. Virtual thiophenyl-arabinoside conjugates were docked to antigen Ag85C (PDB code: 1va5 ) using Glide. Compounds with good docking scores were synthesized by a Gewald synthesis followed by linking to 5-thioarabinofuranosides. The resulting thiophenyl-thioarabinofuranosides were assayed for inhibition of mycoyltransferase activity using a 4-methylumbelliferyl butyrate fluorescence assay. The conjugates showed K(i) values ranging from 18.2 to 71.0 μM. The most potent inhibitor was soaked into crystals of Mycobacterium tuberculosis antigen 85C and the structure of the complex determined. The X-ray structure shows the compound bound within the active site of the enzyme with the thiophene moiety positioned in the putative α-chain binding site of TMM and the arabinofuranoside moiety within the known carbohydrate-binding site as exhibited for the Ag85B-trehalose crystal structure. Unexpectedly, no specific hydrogen bonding interactions are being formed between the arabinofuranoside and the carbohydrate-binding site of the active site suggesting that the binding of the arabinoside within this structure is driven by shape complementarily between the arabinosyl moiety and the carbohydrate binding site.

Functional Proteomic Analysis of Rice Bran Esterases/Lipases and Characterization of a Novel Recombinant Esterase

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.

Purification and properties of digestive lipases from Chinook salmon (Oncorhynchus tshawytscha) and New Zealand hoki (Macruronus novaezelandiae)

Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8±0.1 and 5.7±0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8±0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8-8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7±0.3 vs. 0.71±0.05 s(-1) for the hoki enzyme) and lower activation energy (2.0±0.4 vs. 7.6±0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Esterase-3 polymorphism in the sugarcane borer Diatraea saccharalis (Lepidoptera, Pyralidae)

The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and alpha- and beta-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and alpha-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3F and Est-3S, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (chi² = 2.4; p < 0.01). The negative value for the Wright's fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations.

Characterization of Nasutitermes globiceps (Isoptera: Termitidae) esterases.

Esterases of Nasutitermes globiceps termites which occur on the Upper Paraná River floodplain (Brazil) were characterized. The electrophoretic pattern of the termite esterases Nasutitermes globiceps was obtained by starch gel electrophoresis. Six esterase activity zones were obtained and numbered, with esterase-1 being the most anodall one and esterase-6 the most cathodal one. Esterase-2 was detected only with substrates derived from the 4-methylumbelliferyl radical. The esterases of N. globiceps present wide substrate specificity, having been observed with substrates derived from alpha-naphthyl (acetate, propionate, and butyrate) and beta-naphthyl (acetate, butyrate) and from 4-methylumbelliferyl (acetate, propionate and butyrate). Esterase-6 is a caste-specific enzyme detected in soldiers. Only esterases 1, 3 and 5 were detected in nymphs. No genetic polymorphism has been detected thus far in the esterases of Nasutitermes globiceps. This study suggests that allozyme variation can be explored to understand Nasutitermes social structure.

Esterase-2 polymorphism in Apis mellifera

SUMMARYStarch gel electrophoresis of abdomen extracts from adult drones revealed the occurrence of electrophoretic variants of esterase-2 in Apis mellifera. We demonstrated an enzyme preference for 4-methylumbelliferyl butyrate as a substrate and a pattern of enzyme inhibition which suggests that it is an arylesterase. Tests demonstrated that esterase-2 has the highest heat stability among all A. mellifera esterases. The mean frequency of the Est-2S allele estimated for the five populations investigated was 9.22%. Electrophoretic analysis of worker pupae demonstrated that the heterozygous phenotype only presents an intermediate band between the fast and slow variants of this enzyme, suggesting a probable preferential association of the monomers for the formation of the dimer. Five new tests of two-point genetic linkage involving esterase-2 and other known markers permitted us to demonstrate the occurrence of gene linkage between the loci Est-2 and Est-1a, with a map distance estimated at 26.5 units.

Selection of optimum laboratory tests for the identification of Moraxella catarrhalis

We evaluated a variety of conventional and rapid tests and examined the erythromycin susceptibility of a collection of Moraxella catarrhalis and commensal neisseria strains in order to determine the optimum method for routine identification. One hundred and fifty three strains were tested by Gram stain, catalase, oxidase, carbohydrate degradation by two methods and the presence of esterases using indoxyl acetate, 4-methylumbelliferyl butyrate (MUB). Tween 80 and tributyrin as substrates. Erythromycin MICs and zone diameters around 1, 5 and 15 micrograms discs were determined by the NCCLS method for 151 of the strains. A combination of Gram stain, oxidase and either indoxyl acetate, spot MUB or tributyrin hydrolysis test proved to be reliable and potentially the most convenient for routine testing. MICs and zone diameters easily distinguished between the erythromycin-sensitive M. catarrhalis and the erythromycin-resistant commensal neisserias and would provide confirmation of identification if used for susceptibility testing.

Rapid identification of Branhamella catarrhalis a comparison of five rapid methods

Five methods for the rapid identification and differentiation of Branhamella catarrhalis from other Neisseria species in 86 respiratory specimens were compared. These tests included the 4-methylumbelliferyl butyrate (MUB), API quadFerm, B.CAT.Confirm, Gonochek II, and the Tributyrin disc. All five tests reliably and accurately identified 31 B. catarrhalis isolates. However, the MUB test was the least expensive, least labor intensive, and did not require overnight purity plates for performance. The MUB test provided same-day identification of B. catarrhalis isolates from the initial primary isolation culture.

Rapid identification of Branhamella catarrhalis with 4-methylumbelliferyl butyrate.

Branhamella catarrhalis can be distinguished from Neisseria spp. by the presence of butyrate esterase. This enzyme can be rapidly detected when 4-methylumbelliferyl butyrate is used as the substrate. All B. catarrhalis strains tested gave a positive fluorescence reaction within 5 min, while Neisseria spp. remained negative, even after 18 h of incubation.

A fluorigenic substrate for the rapid differentiation of Mycobacterium fortuitum from Mycobacterium chelonei on the basis of heat stable esterase activity

A fluorigenic substrate, 4-methylumbelliferyl butyrate, enables mycobacterial esterase activity to be easily and rapidly detected and quantitated. By use of this substrate, the esterase activity of Mycobacterium fortuitum was found to be significantly more heat resistant than that of M. chelonei. On this basis, a rapid, simple and inexpensivetest for the differentiation of these two species has been developed.

Esterase D Types in Human Bloodstains

A method is described for the identification of Esterase D types in dried human bloodstains using 4-methylumbelliferyl butyrate as the substrate after starch gel electrophoresis. Bloodstains up to a month old have been typed successfully.

Direct biochemical assay of the lipase activity of epidermis.

The use of synthetic fluorogenic substrate has been applied to the measurement of lipase from guinea pig epidermis. The enzyme is not inhibited by Triton X-100 in concentrations up to 2 mg/ml, has a pH optimum in the range 6.5–8.5, a Km of 1.0 × 10&lt;sup&gt;––&lt;/sup&gt;&lt;sup&gt;6&lt;/sup&gt; mol/l for 4-methyl umbelliferyl butyrate and possesses maximum activity towards esters of chain length C&lt;sub&gt;5&lt;/sub&gt;–C&lt;sub&gt;6&lt;/sub&gt;. The levels of activity observed seem sufficiently high to preclude bacterial flora as the major source. It is therefore concluded that skin possesses endogenous lipase activity.