Production of flavor esters by lipases of Staphylococcus warneri and Staphylococcus xylosus

The present paper describes the potential of Staphylococcus warneri and Staphylococcus xylosus lipases in the production of a variety of flavor esters. Both immobilized lipases produced ethyl esters from hexanoic to oleic acids with an optimum at decanoic acid. They esterified aliphatic and branched chain primary alcohols from ethanol to hexanol. Under our standard conditions, acetic, butyric, 2-methyl butyric, 3-methyl butyric, and valeric acids underwent slight esterification.     

Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.     

Acid Carboxypeptidase-like Activity Contaminating Proctase B

Ester synthesis by the purified lipase from Pseudomonas fragi 22.39 B was investigated. The lipase could synthesize esters from oleic acid and primary or secondary alcohols, but it did not react with tertiary alcohols. Also, the enzyme could use the fatty acids with straight carbon chains as substrates. The activity was enhanced by increasing the carbon number of the fatty acid, but this is not the case for alcohol. The lipase synthesized glycerides from glycerol and oleic acid. 1(3)-Monoolein and 1,3-diolein were the main products and triolein was minor. Synthesis of monoester such as butyl oleate was scarcely affected by the water content in the reaction mixture, while that of glyceride of oleic acid was much affected.     

A comparison of the fatty acid esters of estradiol and corticosterone synthesized by tissues of the rat

The biosynthesis of the fatty acid esters of the corticoid (corticosterone) and estrogen (estradiol) was compared in parallel incubations of corticosterone and estradiol with several tissues of the rat. The fatty acid composition of the esters of the two steroids was characterized in mammary and uterine tissue. In both of these tissues, the esters of estradiol were extremely heterogeneous. To the contrary, in the same tissues only one predominant ester of corticosterone, corticosterone-21-oleate, was formed. It comprised 70-80% of the total. The oleate ester of estradiol accounted for only 20% of the esters of this estrogen. In addition, fatty acid esters of an A-ring reduced metabolite of corticosterone, 5 beta-dihydrocorticosterone, was also identified. Its fatty acid composition is identical to that of corticosterone. In other experiments the fatty acid esters of both steroids were isolated from several tissues and quantified. When the amount of steroidal ester formed was compared, there was over a 100-fold difference among the various tissues in the ratio of estradiol to corticosterone ester synthesized. Thus, the rate of synthesis of the fatty acid esters of each class of steroid varies dramatically from tissue to tissue, and their fatty acid composition differs markedly as well. If the same enzyme synthesized both the estrogen and corticoid esters, then it would be expected that the relative amount of both esters synthesized in various tissues should be constant and likewise that their composition should be the same. Since neither occurred, these results suggest that the enzyme which produces the C-17 fatty acid esters of the estrogens may be different from the one which synthesizes the C-21 esters of the corticoids. The existence of separate enzyme systems for the synthesis of the fatty acid esters of these steroid hormones opens the possibility of specific physiological controls of each of these unusual steroidal metabolites.     

Short chain flavour esters synthesis by microbial lipases

Summary The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.     

Relative rates of hydrolysis by rat pancreatic lipase of esters of C2-C18 fatty acids with C1-C18 primary n-alcohols

The rate at which rat pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) hydrolyzes the esters of primary n-alcohols containing from 1 to 18 carbon atoms with fatty acids containing from 2 to 18 carbon atoms was determined. The speed of hydrolysis was influenced, apparently independently, by both the acyl and the alkyl chains. With respect to the fatty acid moiety, the esters of dodecanoic acid were usually split at the most rapid rate. Esters of butyric acid were the next most susceptible. In the case of the alcohol moiety, esters of heptyl alcohol were hydrolyzed most rapidly. On the basis of the pattern of the relative rates of hydrolysis, it is proposed that the influence of the alcohol component is a result of its orienting the ester molecule at the oil/water interface. The fatty acid effect is attributed to enzyme-substrate specificity.     

Availability of Energy in Ester and Ether Derivatives of Glycols by Growing Chicks

Biological availability of 33 esters, 17 ethers and 2 acetals of ethanediol, 1,2-propanediol, 1,3-butanediol and 1,4-butanediol was compared by mini-test with chicks. Chicks can utilize esters of ethanediol, 1,2-propanediol and 1,3-butanediol with acetic acid and fatty acids of carbon chain length from 5 to 12 with more improved palatability than that of free acids, while availability of esters of these glycols with propionic and butyric acids was low. Esters of 1,4-butanediol and ether derivatives of these glycols was not available, except ethyl ether of di-ethanediol which was partially available. Acetacetal of ethanediol was partially available but n-butyracetal was not.     

Production of PHA from starchy wastewater via organic acids

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

Traumatin- and Dinortraumatin-containing Galactolipids in Arabidopsis: THEIR FORMATION IN TISSUE-DISRUPTED LEAVES AS COUNTERPARTS OF GREEN LEAF VOLATILES*

Green leaf volatiles (GLVs) consisting of six-carbon aldehydes, alcohols, and their esters, are biosynthesized through the action of fatty acid hydroperoxide lyase (HPL), which uses fatty acid hydroperoxides as substrates. GLVs form immediately after disruption of plant leaf tissues by herbivore attacks and mechanical wounding and play a role in defense against attackers that attempt to invade through the wounds. The fates and the physiological significance of the counterparts of the HPL reaction, the 12/10-carbon oxoacids that are formed from 18/16-carbon fatty acid 13-/11-hydroperoxides, respectively, are largely unknown. In this study, we detected monogalactosyl diacylglycerols (MGDGs) containing the 12/10-carbon HPL products in disrupted leaf tissues of Arabidopsis, cabbage, tobacco, tomato, and common bean. They were identified as an MGDG containing 12-oxo-9-hydroxy-(E)-10-dodecenoic acid and 10-oxo-7-hydroxy-(E)-8-decenoic acid and an MGDG containing two 12-oxo-9-hydroxy-(E)-10-dodecenoic acids as their acyl groups. Analyses of Arabidopsis mutants lacking HPL indicated that these MGDGs were formed enzymatically through an active HPL reaction. Thus, our results suggested that in disrupted leaf tissues, MGDG-hydroperoxides were cleaved by HPL to form volatile six-carbon aldehydes and non-volatile 12/10-carbon aldehyde-containing galactolipids. Based on these results, we propose a novel oxylipin pathway that does not require the lipase reaction to form GLVs.     

Microbial Assimilation of Hydrocarbons I. Fatty Acids Derived from Normal Alkanes

Fatty acids derived from Micrococcus cerificans growing at the expense of odd- and even-carbon normal alkanes were studied. Results demonstrated that cultures grown with a variety of nonhydrocarbon substrates serving as sole carbon and energy source yielded only even-carbon fatty acids. Even-chain alkanes, dodecane through octadecane serving as sole carbon source, resulted in even-carbon fatty acids with direct correlation between carbon number of the major fatty acid species and carbon number of the alkane substrate. Odd-carbon alkanes, undecane through heptadecane serving as sole carbon source, yielded both odd- and even-carbon fatty acids. A transitional shift from even-carbon fatty acids to odd-carbon fatty acids was observed as the carbon number of the alkane substrate increased. Unsaturated fatty acids were found to comprise a significant percentage of all profiles. Analysis of unsaturated fatty acids showed all odd- and even-carbon acids analyzed were Delta(9) monounsaturated fatty acids.     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

n-Hexanal and (Z)-3-hexenal are generated from arachidonic acid and linolenic acid by a lipoxygenase in Marchantia polymorpha L.

Most terrestrial plants form green leaf volatiles (GLVs), which are mainly composed of six-carbon (C6) compounds. In our effort to study the distribution of the ability of lipoxygenase (LOX) to form GLVs, we found that a liverwort, Marchantia polymorpha, formed n-hexanal and (Z)-3-hexenal. Some LOXs execute a secondary reaction to form short chain volatiles. One of the LOXs from M. polymorpha (MpLOX7) oxygenized arachidonic and α-linolenic acids at almost equivalent efficiency and formed C6-aldehydes during its catalysis; these are likely formed from hydroperoxides of arachidonic and α-linolenic acids, with a cleavage of the bond between carbon at the base of the hydroperoxy group and carbon of double bond, which is energetically unfavorable. These lines of evidence suggest that one of the LOXs in liverwort employs an unprecedented reaction to form C6 aldehydes as by-products of its reaction with fatty acid substrates.     

Double bond requirement for the 5-lipoxygenase pathway

We showed previously that polyenoic fatty acids with double bonds at carbon 5,8,11 are good substrates for the 5-lipoxygenase and also can be converted to LTC and dihydroxy acids. In order to determine whether all three double bonds are necessary for the 5-lipoxygenase-leukotriene pathway we studied 5,8,14-eicosatrienoic and 5,11,14-eicosatrienoic acid. C14-labeled fatty acids were incubated with 10,000 X g supernatant of homogenate of rat basophilic leukemia (RBL-1) cells in the presence of Ca++ at 37 degrees C. 5,11,14-Eicosatrienoic acid was not converted by the 5-lipoxygenase pathway and 5,8,14-eicosatrienoic acid was mainly converted to 5-hydroxy-6,8,14-eicosatrienoic acid (5-HETE). This monohydroxy was identified by UV spectrometry (UV max 235 nm) and GC-mass spectrometry. Incubations with whole homogenate analyzed by HPLC and bioassay showed that no detectable LTC, LTD or LTE was formed. These data indicate that fatty acids which have double bonds at carbon 5 and carbon 8 are readily converted to the 5-hydroperoxide. However double bonds at carbon 5,8 and 11 are necessary for LTA biosynthesis. This study therefore extends the characterization of the double bond requirement of the 5-lipoxygenase-leukotriene pathway. The number of double bonds necessary at each step varies and increases with each step in the pathway.     

Lipid composition of Cunninghamella elegans cultivated on n-alkanes]

The ability to oxidize n-alkanes was studied with various species of fungi belonging to the Cunninghamella genus. These fungi are able to assimilate hydrocarbons and to accumulate up to 1.5 g/litre of biomass. The most active strain was Cunninghamella elegans (-) 1204. The amount of lipids formed, and their composition, depended on the length of the carbon chain of oxidized alkane. The content of fat in the cells increased with the length of the hydrocarbon chain. The following lipid fractions have been detected: phospholipids, monoglycerides, diglycerides, triglycerides, sterols, free fatty acids, sterol esters, and hydrocarbons. The qualitative composition of the fractions depended, to a considerable extent, on the n-alkane utilized. Investigation of the fatty-acid composition of intracellular lipids has shown that fatty acids with an even number of carbon atoms are formed from hydrocarbons with an even number of these atoms, while fatty acids both with an even and odd number of carbon atoms are synthesized from hydrocarbons with an odd number of these atoms. The relative content of the acids with the same number of carbon atoms as that of the alkane being utilized increased with the length of the carbon chain.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Formation and splitting of esters in subterminal oxidation of dodecane by Fusarium lini

Summary Extracellular oxidation products having the same number of carbon atoms as the alkane that was oxidized were isolated from a Fusarium lini culture broth grown on n-dodecane. They were secondary isomeric alcohols, corresponding isomeric ketones and isomeric esters with 12 carbon atoms.Esterase activity in cell-free extracts of the fungus which was incubated on a p-nitrophenyl-acetate substrate increased with increasing temperatures and pH-values in the ranges 20–40°C and pH 6.0 to 8.0 respectively. The activity, when incubated on p-nitrophenyl-acetate,-laurate and-palmitate substrates, decreased with decreasing fatty acid chain lengths. When incubated with isomeric esters consisting of 12 carbon atoms, it was influenced by the ester linkage position in the chain. When the alcohol chain length in the ester increased from one to six carbon atoms, the esterase activity decreased. The same effect was observed when the chain length of the acid increased from two to six carbon atoms. Minimum esterase activity was reached when both the alcohol and the acid had a chain length of six carbon atoms.The view that all ketones produced during subterminal oxidation of alkanes by Fusarium lini and perhaps other members of Moniliales are further metabolized via ester intermediates is supported. A probable non-specific esterase or lipase catalyses the hydrolysis of the isomeric esters which are formed from the ketones.     

Nitrile pathway involving acyl-CoA synthetase: overall metabolic gene organization and purification and characterization of the enzyme

Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 +/- 0.04, 0.37 +/- 0.02, and 0.22 +/- 0.02 mm, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) comprising these enzymes.