Solvent as a competitive inhibitor for Candida antarctica lipase B

In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanol, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and 1-propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K(i)(I) were changed to "K(i)", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.     

Kinetic resolution of rac-alkyl alcohols via lipase-catalyzed enantioselective acylation using succinic anhydride as acylating agent

With succinic anhydride as acylating agent, three commercial lipases – Candida antarctica lipase B (CALB), Pseudomonas cepacia lipase and Pseudomonas fluorescens lipase – were employed in the kinetic resolution of a series of rac-alkyl alcohols: 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, 3-hexanol, 3-methyl-2-butanol, 6-methyl-5-heptene-2-ol, 3-methyl-2-cyclohexene-1-ol and 2-methyl-1-pentanol. The most effective tested enzyme, immobilized CALB, was able to resolve most of the alcohols with high enantioselectivity, even higher (with enantiomeric ratios up to 115 and 91, for 3-hexanol and 3-methyl-2-butanol, respectively) than when vinyl acetate was used as the acylating agent. More importantly, the unreacted alcohol and the monoester succinate produced could be easily separated by a simple aqueous base-organic solvent liquid–liquid extraction. Using succinic anhydride as acylating agent and CALB, enantiomerically pure (S)-2-pentanol with 99% ee and (R)-2-pentanol with 95% ee were prepared in gram-scale reactions.     

Several new substrates for Desulfovibrio vulgaris strain Marburg and a spontaneous mutant from it

The ability of Desulfovibrio vulgaris strain Marburg (DSM 2119) to oxidize alcohols was surveyed in the presence and absence of hydrogen-scavenging anaerobes, Acetobacterium woodii and Methanospirillum hungatei. In the presence of sulfate, D. vulgaris grew not only on ethanol, 1-propanol, and 1-butanol, but also on isobutanol, 1-pentanol, ethyleneglycol, and 1,3-propanediol. Metabolism of these alcohols was simple oxidation to the corresponding acids, except with the last two substrates: ethyleneglycol was oxidized to glycolate plus acetate, 1,3-propanediol to 3-hydroxypropionate plus acetate. Experimental evidence was obtained, suggesting that 2-methoxyethanol was not utilized by all the cells of strain marburg, but by a spontaneous mutant. 2-Methoxyethanol was oxidized to methoxyacetate by the mutant. Co-culture of strain Marburg plus A. woodii grew on ethanol, 1-propanol, 1-butanol, and 1,3-propanediol in the absence of sulfate. Co-culture of strain Marburg plus M. hungatei grew on ethanol, 1-propanol, and 1-butanol, but not on ethyleneglycol and 1,3-propanediol, Co-culture of the mutant plus A. woodii or M. hungatei did not grow on 2-methoxyethanol.     

3,3-Dimethyl-1-butanol, a parakairomone component to Aleurodicus dispersus (Hemiptera: Aleyrodidae)

C-6-based green leaf volatiles (GLVs) are signal molecules to herbivorous insects and play an important role in plant–herbivore interactions. How isomerization of GLVs affects insect’s olfactory response has been rarely tested. In laboratory and field experiments, we examined the effect of hexanol isomers on olfactory orientation of the spiraling whitefly, Aleruodicus dispersus Russell, a highly polyphagous pest. In a Y-tube oflactometer, we found that (±)-2-hexanol, 3-methyl-3-pentanol and 3,3-dimethyl-1-butanol significantly attracted female A. dispersus. The trap captures of 3,3-dimethyl-1-butanol were significantly more than that of (±)-2-hexanol and 3-methyl-3-pentanol, and its optimum concentration was 1 μ1/ml. We suggest that the anthropogenic compound 3,3-dimethyl-1-butanol can be exploited as a parakairomone (synthetic analogues of kairomone) to monitor and control adult A. dispersus.     

New chemical attractants for trapping Lacanobia subjuncta, Mamestra configurata, and Xestia c-nigrum (Lepidoptera: Noctuidae)

Significant numbers of 3 pest species of noctuid moths were captured in traps baited with acetic acid, 3-methyl-1-butanol, and 3-methyl-1-pentanol. These were Lacanobia subjuncta (Grote & Robinson); Mamestra configurata Walker, bertha armyworm; and Xestia c-nigrum (L.), spotted cutworm. The combination of acetic acid and 3-methyl-1-butanol was superior to the individual chemicals in attracting all 3 species, whereas the combination of acetic acid and 3-methyl-1-pentanol was superior to the individual chemicals in attracting X. c-nigrum. For the 3 species of moths, numbers captured were similar in traps baited with the combination of acetic acid and 3-methyl-1-butanol or acetic acid and 3-methyl-1-pentanol. Traps baited with these attractants captured both males and females at a ratio near 1:1.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

Cholesterol autoxidation: identification of the volatile fragments.

The volatile fragments of air-aged cholesterol were analysed by means of gas chromatography-mass spectrometry; The following fourteen compounds were identified: ethanol, acetic acid, acetone, 2-methylpropene, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-butanone, 2-methylpropionic acid, 2-methyl-2-butanol, 2-pentanone, 3-methyl-2-butanone, 2-methyl-1-pentene, 2-methyl-2-pentanol, and 2-methyl-4-penten-2-ol. Their formation via decomposition of initially formed sterol hydroperoxides is discussed.     

A Medium for the Isolation of Capsular Bacteria from Kefir Grains

Secondary alcohols (C₃ to C₁₀) were oxidized to the corresponding methylketones by resting mycelia of Scedosporium sp. A-4 grown on propane, but 3-pentanol and 3-hexanol were not oxidized. The oxidation of 2-propanol to acetone was inhibited by pyrazole, potassium cyanide, sodium azide and Hg^{2 +}. Alcohol dehydrogenase activity was found in the cell-free soluble fraction and this activity requires a cofactor, specifically NAD⁺. The oxidation of both 1-propanol and 2-propanol may be catalyzed by the same alcohol dehydrogenase.     

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.     

3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation

Interest in producing biofuels from renewable sources has escalated due to energy and environmental concerns. Recently, the production of higher chain alcohols from 2-keto acid pathways has shown significant progress. In this paper, we demonstrate a mutagenesis approach in developing a strain of Escherichia coli for the production of 3-methyl-1-butanol by leveraging selective pressure toward l-leucine biosynthesis and screening for increased alcohol production. Random mutagenesis and selection with 4-aza-d,l-leucine, a structural analogue to l-leucine, resulted in the development of a new strain of E. coli able to produce 4.4 g/L of 3-methyl-1-butanol. Investigation of the host’s sensitivity to 3-methyl-1-butanol directed development of a two-phase fermentation process in which titers reached 9.5 g/L of 3-methyl-1-butanol with a yield of 0.11 g/g glucose after 60 h.     

Secondary alcohols as hydrogen donors for CO2-reduction by methanogens

Out of 22 methanogens Methanobacterium formicicum, Methanobacterium bryantii M.o.H., Methanogenium marisnigri, Methanomicrobium paynteri, Methanocorpusculum parvum and the new coccoid methanogenic isolates GKZPZ and SZSXXZ were found to grow at the expense of 2-propanol and 2-butanol + CO₂. 2-Propanol was oxidized to acetone and 2-butanol was converted to 2-butanone during CO₂-reduction to methane. Growth was poor compared to that on H₂/CO₂, and in the presence of both, 2-propanol and H₂, molecular hydrogen was the preferred reductant. Acetone, formed during oxidation of 2-propanol in the absence of hydrogen, was reduced again to 2-propanol, when the culture was supplied with H₂/CO₂. Ethanol, 1-propanol, 1-butanol, 2-pentanol and cyclohexanol could not serve as hydrogen donors for methanogenesis.     

Biotransformation of β-hydroxyphenyl selenides, diphenyldiselenide and benzeneseleninic acid by whole cells of Aspergillus terreus

Aspergillus terreus CCT 3320 and A. terreus URM 3571 catalysed the biotransformations of organic β-hydroxyphenyl selenides through oxidation and methylation reactions. The kinetic resolution of (RS)-1-(phenylseleno)-2-propanol (1) via enantioselective oxidation produced (+)-(S)-1 in high enantiomeric excess (>99%) and in a yield of 50% as determined by product isolation. Oxidation of the R-enantiomer of 1, followed by elimination of the propyl moiety and subsequent methylation of the presumed intermediate, led to the formation of methylphenyl-selenide, which was isolated in a yield of 40%. Whole cells of A. terreus also biocatalysed transformations of diphenyldiselenide, benzeneseleninic acid, (RS)-1-(phenylseleno)-2-pentanol and (RS)-1-(phenylseleno)-3-methyl-2-butanol, but not of (RS)-1-(phenylseleno)-2-phenyl-methanol. This is the first report of the biomethylation of organoselenium compounds by whole cells of A. terreus.     

The substrate binding cavity of particulate methane monooxygenase from Methylosinus trichosporium OB3b expresses high enantioselectivity for n-butane and n-pentane oxidation to 2-alcohol

The particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b oxidized n-butane and n-pentane and mainly produced (R)-2-butanol and (R)-2-pentanol that comprised 78 and 89% of the product, respectively, indicating that the pro-R hydrogen of the 2-position carbon of n-butane and n-pentane is oriented toward a catalytic site within the substrate binding site of pMMO. The protein cavity adjacent to the catalytic center for pMMO has optimum volume for recognizing n-butane and n-pentane for enantioselective hydroxylation.     

Effects of Temperature on Stereochemistry of Enzymatic Reactions

A brief discussion of the theoretical basis for effects of temperature on stereoselectivity of enzyme catalysed reactions is presented. In theory, the stereoselectivity of an enzymatic reaction can either increase or decrease as the reaction temperature is raised. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus reduces 2-butanone to (R)-2-butanol at 37° C, with increased stereoselectivity at higher temperatures and in the presence of NADP analogues. In contrast, at 37°, 2-pentanone and 2-hexanone are reduced to (S)-2-pentanol and (S)-2-hexanol, respectively, but the stereoselectivity decreases at higher temperatures and in the presence of NADP analogues. Reduction of racemic 2-methylbutanal by the primary alcohol dehydrogenase from T. ethanolicus gives (S)-2-methyl-1-butanol with greater stereospecificity at 35° (51% e.e.) than at 15° (14% e.e.). Horse liver alcohol dehydrogenase shows a preference for oxidation of the (S)-enantiomers of acyclic secondary alcohols at 25°, with a decrease in stereospecificity at higher temperatures.     

Induction of Suppressed ADH Activity in Drosophila pachea Exposed to Different Alcohols

Laboratory-reared males of the cactophilic Drosophila pachea exhibit a spontaneous and sex-specific suppression of alcohol dehydrogenase (ADH) activity within 4 days after eclosion. A lack of ADH activity also is usually seen in wild-caught males, although relatively high activity is always seen in female flies. In the present study we examined the effectiveness of different alcohols and related compounds, including several found naturally in necroses of the host cactus, to induce suppressed ADH activity in wild males of D. pachea and to serve as enzyme substrates. The primary alcohols (methanol, ethanol, 1-propanol, 1-butanol, and 1-pentanol), and the secondary alcohols (2-propanol and 2-butanol), each induced activity after 24 h exposure, although to different degrees. 1,2-Propanediol was usually effective as an inducer, but 2,3-butanediol usually was ineffective. Little or no induction was seen with 1-octanol, 2-pentanol, 3-methyl-1-butanol, 3-hydroxy-2-butanone, or acetaldehyde. Although the compounds tested varied in their ability to function as ADH substrates, methanol was the only alcohol that showed no activity staining. Ethanol induction of ADH activity was apparent after 3-6 h exposure and induced activity decreased dramatically within 1 week of flies being placed in an alcohol-free environment. Ethanol exposure did not induce ADH in adult female D. pachea, or in adult males and females of D. acutilabella in which control males show reduced ADH activity compared to females. The implications of the loss of ADH activity in adult males of D. pachea, as they relate to feeding ecology and fitness, are discussed.     

Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.     

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicum l-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67 g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37 g/l 2-methyl-1-butanol and 2.76 g/l 3-methyl-1-butanol in defined medium within 48 h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.     

Rhizomorph Formation in Fungi

The effect on growth and rhizomorph formation of 12 alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butyl alcohol, tert-butyl alcohol, 1-pentanol, iso-amyl alcohol, ethylene glycol and glycerol) at different concentrations has been examined for 2 isolates of Armillaria mellea (Vahl ex Fr.) Quél. and 1 of Clitocybe geotropa (Bull. ex Fr.) Quél. The fungi were cultivated for 28 days on a synthetic, liquid glucose medium with the alcohols as supplement. The following alcohols strongly stimulated growth and rhizomorph formation: ethanol, 1-propanol and 1-butanol. A great variation was demonstrated between the isolates in relation to rhizomorph production, morphology, and ability to be stimulated by different alcohols.     

The Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas sp.

To clarify biodegradation pathways of isoalkyl substituted aromatic hydrocarbons, oxidation products of isopropylbenzene and isobutylbenzene by Ps. desmolytica S449B1 and Ps. convexa S107B1 were examined.

Oxidation products from isopropylbenzene were determined to be 3-isopropylcatechol and (+)-2-hydroxy-7-methyI-6-oxooctanoic acid. Isobutylbenzene was also oxidized to 3-isobutylcatechol and (+)-2-hydroxy-8-methyl-6-oxononanoic acid by the same strains.

From these results, the existence of an unknown reductive step in the degradation of these isoalkyl substituted aromatic hydrocarbons and the initial oxidation of these aromatic hydrocarbons by the strains were made clear. The degradation pathways of isopropylbenzene and isobutylbenzene by these strains were discussed.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

The flavor concentrate obtained by the extraction of “Katsuobushi” of bonito (Katsuwonus pelamis) with 80% ethanol and by the subsequent steam distillation of the extract was fractionated by the usual method and the resulting neutral, non-carbonyl oxygenated fraction was investigated by gas chromatography. The following components were tentatively identified: 2-pentanol and 2-methyl-1-heptanol as free alcohols, and 4 alcohols of n- and isobutanol, n-pentanol and n-dodecanol and 9 carboxylic acids of propanoic, n-butanoic, n-pentanoic, n-octanoic, n-nonanoic n-decanoic, n-dodecanoic, n-tetradecanoic and n-hexadecanoic acid as the constituents of esters. A constituent alcohol existing in the largest amount was isolated by gas chromatography and identified as 2-methyl-1-heptanol by elemental analysis, NMR, IR, and MS. A constituent acid existing in large amount was also isolated and investigated similarly, and the structure was partially estimated. 2-Methyl-1-heptanol holds a fresh woody aroma and seems to have a major effect on “Katsuobushi” flavor.