Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 μmol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 μmol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40°C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 × g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.     

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enantiopure derivatives of 1,2-alkanediols: substrate requirements of lipase B from Candida antarctica

Efficient methods for kinetic resolution of 1-phenoxy-2-butanol, 1-phenylmethoxy-2-butanol, and 1-phenoxy-2-pentanol were developed using lipase B from Candida antarctica as catalyst. Resolutions were performed in order to investigate the substrate requirements needed to obtain a high E-value. The effect of the substrate structure on E is different for transesterifications in organic media as compared to hydrolysis. The influence of different acyl donors on the E-value was also investigated.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.     

Ferulic acid esterase from Humicola Insolens catalyzes enantioselective transesterification of secondary alcohols

Ferulic acid esterase (FAE) from Humicola insolens was found to catalyze transesterifications of secondary alcohols with high enantioselectivity. In all cases the enzyme showed R enantiopreference.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C(2) to C(4)n-Alkane-Grown Bacteria

Nineteen new C(2) to C(4)n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C(2) to C(4)n-alkanes. Cell suspensions of these C(2) to C(4)n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60 degrees C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Amino Acid Sequences of Glycopeptides from Cucumisin

Oxidation of short-chain iso-alkanes (isobutane, isopentane, 2-methylpentane, and 3-methylpentane) was studied with propane-grown resting mycelia of Scedosporium sp. A-4. Isobutane was oxidized to terf-butanol, but both isobutane and tert-butanol were not used for growth. Isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-Methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2-pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-Methylpentane was not oxidized. Oxidation of branched alcohols was also studied.     

A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols.

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.     

Biodiesel production from triolein and short chain alcohols through biocatalysis

Oleic acid alkyl esters (biodiesel) were synthesised by biocatalysis in solvent-free conditions. Different commercial immobilised lipases, namely Candida antarctica B, Rizhomucor miehei, and Pseudomonas cepacia, were tested towards the reaction between triolein and butanol to produce butyl oleate. Pseudomonas cepacia lipase resulted to be the most active enzyme reaching 100% of conversion after 6h. Different operative conditions such as reaction temperature, water activity, and reagent stoichiometric ratio were investigated and optimised. These conditions were then used to investigate the effect of linear and branched short chain alcohols. Methanol and 2-butanol were the worst alcohols: the former, probably, due to its low miscibility with the oil and the latter because secondary alcohols usually are less reactive than primary alcohols. Conversely, linear and branched primary alcohols with short alkyl chains (C(2)--C(4)) showed high reaction rate and conversion. A mixture of linear and branched short chain alcohols that mimics the residual of ethanol distillation (fusel oil) was successfully used for oleic acid ester synthesis. These compounds are important in biodiesel mixtures since they improve low temperature properties.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methane- and methanol-grown microbes.

Cultures of methane- or methanol-utilizing microbes, including obligate (both types I and II) and facultative methylotrophic bacteria, obligate methanol utilizers, and methanol-grown yeasts were isolated from lake water of Warinanco Park, Linden, N.J., and lake and soil samples of Bayway Refinery, Linden, N.J. Resting-cell suspensions of these, and of other known C1-utilizing microbes, oxidized secondary alcohols to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Succinate-grown cells of facultative methylotrophs did not oxidize secondary alcohols. Among the secondary alcohols, 2-butanol was oxidized at the highest rate. The optimal conditions for in vivo methyl ketone formation were compared among five different types of C1-utilizing microbes. Some enzymatic degradation of 2-butanone was observed. The product, 2-butanone, did not inhibit the oxidation of 2-butanol. The rate of the 2-butanone production was linear for the first 4 h of incubation for all five cultures tested. A yeast culture had the highest production rate. The optimum temperature for the production of 2-butanone was 35 degrees C for all the bacteria tested. The yeast culture had a higher temperature optimum (40 degrees C), and there was a reasonably high 2-butanone production rate even at 45 degrees C. Metal-chelating agents inhibit the production of 2-butanone, suggesting the involvement of metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble extract of sonically disrupted cells. The cell-free system requires a cofactor, specifically nicotinamide adenine dinucleotide, for its activity. This is the first report of a nicotinamide adenine dinucleotide-dependent, secondary alcohol-specific enzyme.     

R-Enantioselective galactosylation of secondary alcohols using b-galactosidase

Significant R-enantioselection occurred for b-galactosyl transfer to the secondary alcohols, 2-butanol, 2-octanol, 3-methyl-2-butanol, 1-phenylethanol, 1,2-propanediol, 1-methoxy-2-propanol and 3-hydroxybutanoates, with b-galac-tosidase from Escherichia coli. The highest R-enantioselection of 98% e.e. was for the b-galactosylation of 1-phenylethanol using b-galactosidase from E. coli.     

Ab initio construction of protein tertiary structures using a hierarchical approach

We present a hierarchical method to predict protein tertiary structure models from sequence. We start with complete enumeration of conformations using a simple tetrahedral lattice model. We then build conformations with increasing detail, and at each step select a subset of conformations using empirical energy functions with increasing complexity. After enumeration on lattice, we select a subset of low energy conformations using a statistical residue-residue contact energy function, and generate all-atom models using predicted secondary structure. A combined knowledge-based atomic level energy function is then used to select subsets of the all-atom models. The final predictions are generated using a consensus distance geometry procedure. We test the feasibility of the procedure on a set of 12 small proteins covering a wide range of protein topologies. A rigorous double-blind test of our method was made under the auspices of the CASP3 experiment, where we did ab initio structure predictions for 12 proteins using this approach. The performance of our methodology at CASP3 is reasonably good and completely consistent with our initial tests.     

Incorporating knowledge-based biases into an energy-based side-chain modeling method: application to comparative modeling of protein structure

The performance of the self-consistent mean field theory (SCMFT) method for side-chain modeling, employing rotamer energies calculated with the flexible rotamer model (FRM), is evaluated in the context of comparative modeling of protein structure. Predictions were carried out on a test set of 56 model backbones of varying accuracy, to allow side-chain prediction accuracy to be analyzed as a function of backbone accuracy. A progressive decrease in the accuracy of prediction was observed as backbone accuracy decreased. However, even for very low backbone accuracy, prediction was substantially higher than random, indicating that the FRM can, in part, compensate for the errors in the modeled tertiary environment. It was also investigated whether the introduction in the FRM-SCMFT method of knowledge-based biases, derived from a backbone-dependent rotamer library, could enhance its performance. A bias derived from the backbone-dependent rotamer conformations alone did not improve prediction accuracy. However, a bias derived from the backbone-dependent rotamer probabilities improved prediction accuracy considerably. This bias was incorporated through two different strategies. In one (the indirect strategy), rotamer probabilities were used to reject unlikely rotamers a priori, thus restricting prediction by FRM-SCMFT to a subset containing only the most probable rotamers in the library. In the other (the direct strategy), rotamer energies were transformed into pseudo-energies that were added to the average potential energies of the respective rotamers, thereby creating hybrid energy-based/knowledge-based average rotamer energies, which were used by the FRM-SCMFT method for prediction. For all degrees of backbone accuracy, an optimal strength of the knowledge-based bias existed for both strategies for which predictions were more accurate than pure energy-based predictions, and also than pure knowledge-based predictions. Hybrid knowledge-based/energy-based methods were obtained from both strategies and compared with the SCWRL method, a hybrid method based on the same backbone-dependent rotamer library. The accuracy of the indirect method was approximately the same as that of the SCWRL method, but that of the direct method was significantly higher.     

Odorant binding protein based biomimetic sensors for detection of alcohols associated with Salmonella contamination in packaged beef

Detection of food-borne bacteria present in the food products is critical to prevent the spread of infectious diseases. Intelligent quality sensors are being developed for detecting bacterial pathogens such as Salmonella in beef. One of our research thrusts was to develop novel sensing materials sensitive to specific indicator alcohols at low concentrations. Present work focuses on developing olfactory sensors mimicking insect odorant binding protein to detect alcohols in low concentrations at room temperature. A quartz crystal microbalance (QCM) based sensor in conjunction with synthetic peptide was developed to detect volatile organic compounds indicative to Salmonella contamination in packaged beef. The peptide sequence used as sensing materials was derived from the amino acids sequence of Drosophila odorant binding protein, LUSH. The sensors were used to detect alcohols: 3-methyl-1-butanol and 1-hexanol. The sensors were sensitive to alcohols with estimated lower detection limits of <5 ppm. Thus, the LUSH-derived QCM sensors exhibited potential to detect alcohols at low ppm concentrations.