Purification and characterization of organic solvent and detergent tolerant lipase from thermotolerant Bacillus sp. RN2.

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9-11 and temperature range of 45-60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

Cloning of lipoxygenase genes from a cyanobacterium, Nostoc punctiforme, and its expression in Eschelichia coli.

Oxylipin metabolism represents one of the important hormonal and defensive mechanisms employed by plants, algae, or animals. It begins mostly with the reaction of lipoxygenases (LOXs), which catalyze the oxygenation of polyunsaturated fatty acids to form the corresponding hydroperoxides. At present, little information about LOXs in cyanobacteria has been reported. Herein, we report the first isolation of two LOX genes (NpLOX1 and NpLOX2) from a cyanobacterium, Nostoc punctiforme ATCC29133. Incubations of recombinant NpLOX1 and NpLOX2 proteins expressed in Eschelichia coli with linoleic acid resulted in the predominant formation of linoleic acid 13-S-hydroperoxide. Other C18 and C20 fatty acids could also be substrates for NpLOX enzymes. Phylogenetic analysis of NpLOX sequences showed that the NpLOX enzymes shared a high homology with LOX sequence of a bacterial pathogen, Pseudomonas aeruginosa, and these bacterial LOXs formed a subfamily distinct from those of plants, algae, and mammals.

Biosynthesis of fatty acid derived aldehydes is induced upon mechanical wounding and its products show fungicidal activities in cucumber.

Fatty acid 9/13-hydroperoxide lyase (9/13-HPL) in cucumber is an enzyme that can cleave either 9- or 13-hydroperoxides of polyunsaturated fatty acids to form C9- or C6-aldehydes, respectively, as products. In order to reveal the physiological function of 9/13-HPL, its expression profiles were analyzed, and it was found that 9/13-HPL expression was developmentally regulated and high in the hypocotyls, female flowers and mature fruits. However, its transcript as well as its activity was only induced by mechanical wounding in mature leaves. To analyze the biosynthesis of HPL-derived aldehydes in more detail we isolated and characterized the yet missing 9-lipoxygenase (LOX) that is mainly expressed in hypocotyls, cotyledons and flowers and that may provide HPL with fatty acid 9-hydroperoxides as substrates. As in the case with C6-aldehydes in most plant species, C9-aldehydes were also formed rapidly after disruption of the tissues. C9-aldehydes had fungicidal activities against fungal pathogens, Botrytis cinerea and Fusarium oxysporum. Because the concentration needed to cause toxic effect on the pathogens was almost equivalent to that found in disrupted tissues, the C9-aldehydes thus formed could be helpful to sterilize the wounds since they are less volatile in comparison to C6-aldehydes.

Rice fatty acid alpha-dioxygenase is induced by pathogen attack and heavy metal stress: activation through jasmonate signaling.

Plant fatty acid alpha-dioxygenases (DOXs) catalyze the stereospecific conversion of fatty acids into the corresponding (R)-2-hydroperoxy fatty acids. In several plant species the corresponding gene was shown to be induced by pathogen infection, herbivore attack and environmental stresses. The precise signaling pathway accountable for the induction remains unidentified. In the present study, the effects of bacterial infection, oxidative- and heavy metal-stresses, and plant signaling molecules such as jasmonate, salicylic acid (SA), and ethylene (ET) on expression of a fatty acid alpha-DOX (OsDOX) gene in rice seedlings were investigated. The rice blight bacteria, Xanthomonas oryzae, elicited the accumulation of OsDOX transcripts in the leaves in both the incompatible and compatible interactions. Treating the seedling with CuSO4 also significantly enhanced the OsDOX expression. The degree of induction was shown to be mostly parallel to the level of endogenous jasmonic acid (JA) in the leaves. In contrast, SA was little effective and ET down-regulated not only the OsDOX expression but also the endogenous level of JA in rice seedlings. These results suggested that the OsDOX gene expression by a variety of stress-related stimuli was activated through jasmonate signaling and was negatively regulated by ET.

Stereochemical correlation between 10-hydroperoxyoctadecadienoic acid and 1-octen-3-ol in Lentinula edodes and Tricholoma matsutake mushrooms.

Linoleic acid (LA) incubated with a homogenate of Lentinula edodes or Tricholoma matsutake mushroom significantly increased the amount of (R)-1-octen-3-ol. The alcohol was identified as (S)-10-HODE with 90-87% and >99% enantiomeric excess (ee), respectively. During the incubation of LA with these homogenates in the presence of glutathione-glutathione peroxidase (GSH-GPx), which can reduce hydroperoxy fatty acids to the corresponding hydroxy acids, the formation of (R)-1-octen-3-ol was significantly inhibited, whereas the amount of 10-hydroxy-(8E,12Z)-8,12-octadecadienoic acid (10-HODE) was significantly increased. The acid was identified as (S)-10-HODE with 92-88% ee and >99% ee, respectively. The decrease in the amount of alcohol was approximately the same as the increase in amount of HODE in both mushrooms. These results indicate a stereochemical correlation between (R)-1-octen-3-ol and (S)-10-hydroperoxy-(8E,12Z)-8,12-octadecadienoic acid [(S)-10-HPODE] in both mushrooms.

Concise synthesis of (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,13Z)-7,10,13-hexadecatrienal, and (8Z,11Z)-8,11-heptadecadienal, components of the essential oil of marine green alga Ulva pertusa.

The long-chain aldehydes, (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,13Z)-7,10,13-hexadecatrienal, and (8Z,11Z)-8,11-heptadecadienal, were concisely synthesized by using Grignard coupling, catalytic hydrogenation with the Lindlar catalyst, and oxidation with Dess-Martin periodinane as the key steps. Particularly, (8Z,11Z,14Z)-8,11,14-heptadecatrienal and (7Z,10Z,13Z)-7,10,13-hexadecatrienal both possessed a seaweed-like odor.

A tomato lipase homologous to DAD1 (LeLID1) is induced in post-germinative growing stage and encodes a triacylglycerol lipase.

A tomato lipase gene homologous to Arabidopsis DAD1 (lipase homologous to DAD1; LeLID1) was cloned and characterized. The corresponding transcript increased rapidly during germination of the seeds and reached a maximum level at four days after germination. Thereafter, it decreased rapidly. Little expression could be found in flowers or fruits. Immunoblot analyses showed that the gene products could be found in the cotyledons and hypocotyls, but not in the roots. In the cotyledons most LeLID1 could be recovered in a soluble fraction. The recombinant LeLID1 protein showed maximum lipase activity at pH 8.0. It showed high activity against triacylglycerols (TAGs) with long acyl chains, but little activity with phosphatidylcholine or monogalactosyldiacylglycerol. TAGs composed of short acyl chains could not be a substrate for the enzyme. A possible involvement of LeLID1 in fat mobilization during seed germination is discussed.

Linoleic acid 10-hydroperoxide as an intermediate during formation of 1-octen-3-ol from linoleic acid in Lentinus decadetes.

In order to confirm the biosynthetic pathway to 1-octen-3-ol from linoleic acid, a crude enzyme solution was prepared from the edible mushroom, Lentinus decadetes. When the reaction was performed in the presence of glutathione peroxidase, which can reduce organic hydroperoxide to the corresponding hydroxide, the amount of 1-octen-3-ol formed from linoleic acid was decreased. At the same time, an accumulation of linoleic acid 10-hydroxide could be detected. The 10-hydroperoxide therefore seems to be an intermediate on the biosynthetic pathway.

2,4-Decadienals are produced via (R)-11-HPITE from arachidonic acid in marine green alga Ulva conglobata.

Marine green alga Ulva conglobata was investigated for the biogeneration of oxygenated products from exogenously added arachidonic acid (ARA). A crude enzyme from the alga afforded the detectable amount of a hydroperoxyicosatetraenoic acid (HPITE), which was identified as (R)-11-HPITE by HPLC and GC-MS. Headspace-SPME method indicated that ARA was selectively used to form 2,4-decadienals. These results showed that 2,4-decadienals are produced via (R)-11-HPITE from ARA exclusively.

Hydroperoxy-arachidonic acid mediated n-hexanal and (Z)-3- and (E)-2-nonenal formation in Laminaria angustata.

In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.

C6-aldehyde formation by fatty acid hydroperoxide lyase in the brown alga Laminaria angustata.

Some marine algae can form volatile aldehydes such as n-hexanal, hexenals, and nonenals. In higher plants it is well established that these short-chain aldehydes are formed from C18 fatty acids via actions of lipoxygenase and fatty acid hydroperoxide lyase, however, the biosynthetic pathway in marine algae has not been fully established yet. A brown alga, Laminaria angustata, forms relatively higher amounts of C6- and C9-aldehydes. When linoleic acid was added to a homogenate prepared from the fronds of this algae, formation of n-hexanal was observed. When glutathione peroxidase was added to the reaction mixture concomitant with glutathione, the formation of n-hexanal from linoleic acid was inhibited, and oxygenated fatty acids accumulated. By chemical analyses one of the major oxygenated fatty acids was shown to be (S)-13-hydroxy-(Z, E)-9, 11-octadecadienoic acid. Therefore, it is assumed that n-hexanal is formed from linoleic acid via a sequential action of lipoxygenase and fatty acid hydroperoxide lyase (HPL), by an almost similar pathway as the counterpart found in higher plants HPL partially purified from the fronds has a rather strict substrate specificity, and only 13-hydroperoxide of linoleic acid, and 15-hydroperoxide of arachidonic acid are the essentially suitable substrates for the enzyme. By surveying various species of marine algae including Phaeophyta, Rhodophyta and Chlorophyta it was shown that almost all the marine algae have HPL activity. Thus, a wide distribution of the enzyme is expected.

Biomimetic conversion of (3S)-(-)-neodictyoprolenol to optically pure (1S,2R)-(-)-dictyopterene B, marine algal sex pheromone.

Both enantiomers of (3S)-(-)- and (3R)-(+)-Neodictyoprolenol [(3S,5Z,8Z)-(-)-1,5,8-undecatrien-3-ol] were successfully converted to the algal sex pheromone, (1S,2R)-(-)-dictyopterene B and (1R,2S)-(+)-dictyopterene B in high enantiomeric purities (e. e. > 99%), respectively, by the biomimetic reaction involving phosphorylation and elimination under a mild condition.

Kinetics of barley FA hydroperoxide lyase are modulated by salts and detergents.

The cDNA from barley coding FA hydroperoxide lyase (HPL) was cloned. A recombinant protein derived from the cDNA was expressed in Escherichia coli as an active enzyme. Thus far, there have been no reports on HPL in monocotyledonous plants. The recombinant protein was shown to be most active to linolenic acid 13-hydroperoxide, followed by linoleic acid 13-hydroperoxide. 9-Hydroperoxides of the FA could not be substrates for the recombinant HPL. The activity was dramatically enhanced in the presence of a detergent and/or a salt in the reaction mixture. At the same time, the kinetics of the reaction, including inactivation and the Vmax value of the HPL, were also greatly modulated, depending on the concentration of a monovalent cation and/or a detergent in the reaction mixture. These results suggest that these effectors induced a conformational change in barley HPL, resulting in an improvement in substrate binding and in enzyme activity.

Enantioselective formation of (R)-9-HPODE and (R)-9-HPOTrE in marine green alga Ulva conglobata.

When linoleic and linolenic acid were incubated with a crude enzyme of marine green alga Ulva conglobata, the corresponding (R)-9-hydroperoxy-(10E, 12Z)-10, 12-octadecadienoic acid [(R)-9-HPODE] and (R)-9-hydroperoxy-(10E, 12Z, 15Z)-10, 12, 15-octadecatrienoic acid [(R)-9-HPOTrE] were formed with a high enantiomeric excess (>99%), respectively.

Catalytic properties of rice alpha-oxygenase. A comparison with mammalian prostaglandin H synthases.

Long-chain fatty acids can be metabolized to C(n)(-1) aldehydes by alpha-oxidation in plants. The reaction mechanism of the enzyme has not been elucidated. In this study, a complete nucleotide sequence of fatty acid alpha-oxygenase gene in rice plants (Oryza sativa) was isolated. The deduced amino acid sequence showed some similarity with those of mammalian prostaglandin H synthases (PGHSs). The gene was expressed in Escherichia coli and purified to apparently homogeneous state. It showed the highest activity with linoleic acid and predominantly formed 2-hydroperoxide of the fatty acid (C(n)), which is then spontaneously decarboxylated to form corresponding C(n)(-1) aldehyde. With linoleic or linoleic acids as a substrate, rice alpha-oxygenase formed no product having a lambda(max) at approximately 234 nm, which indicated that the enzyme could not oxygenize the pentadiene system in the substrate. The spectroscopic feature of the purified enzyme in its ferrous state is similar to that of mammalian PGHS, whereas that of dithionite-reduced state showed significant difference. Site-directed mutagenesis revealed that His-158, Tyr-380, and Ser-558 were essential for the alpha-oxygenase activity. These residues are conserved in PGHS and known as a heme ligand, a source of a radical species to initiate oxygenation reaction and a residue involved in substrate binding, respectively. This finding suggested that the initial step of the oxygenation reaction in alpha-oxygenase has a high similarity with that of PGHS. The rice alpha-oxygenase activity was inhibited by imidazole but hardly inhibited by nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen, and flurbiprofen, which are known as typical PGHS inhibitors. In addition, peroxidase activity could not be detected with alpha-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate. From these findings, the catalytic resemblance between alpha-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities.

Long-chain aldehyde-forming activity in tobacco leaves.

Long-chain aldehydes (LCA), such as pentadecanal (PD), (8Z,11Z)-heptadecadienal (HDD) and (8Z,11Z,14Z)-heptadecatrienal (HDT), were identified in the essential oils obtained from fresh green tobacco leaves (Nicotiana tabacum cv. BY2 and N. tabacum cv. MC). PD, HDD and HDT were found to be produced enzymatically from palmitic acid (PA), linoleic acid and alpha-linolenic acid, respectively. LCA-forming activity for PA changed during growth of tobacco leaves and was highest at flowering time.