Isolation and properties of a <Emphasis Type="Italic">levo-</Emphasis>lactonase from <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> ECU2002: a robust biocatalyst for production of chiral lactones

A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration, acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50°C and 7.5, respectively, for γ-butyrolactone hydrolysis. The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which α-hydroxy-γ-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to γ-butyrolactone. The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including β-butyrolactone, α-hydroxy-γ-butyrolactone, α-hydroxy-β,β-dimethyl-γ-butyrolactone (pantolactone), and β-hydroxy-γ-butyrolactone, affording (+)-hydroxy acids in high (94.8∼98.2%) enantiomeric excesses (ee) and good conversions (38.2∼44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording the product in 94∼97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized.     

Isolation and identification of potent allelopathic substances in rattail fescue

Aqueous methanol extracts of rattail fescue (Vulpia myuros) inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), timothy (Phleum pratense), Digitaria sanguinalis and Lolium multiflorum. Increasing the extract concentration increased the inhibition, suggesting that rattail fescue may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of rattail fescue was purified and two main inhibitory substances were isolated and identified by spectral data as (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol. Both substances inhibited root and shoot growth of cress at concentrations greater than 0.3 μM. The concentrations required for 50% growth inhibition on root and shoot growth of cress, lettuce, alfalfa, timothy, D. sanguinalis and L. multiflorum were 2.7–19.7 μM for (−)-3-hydroxy-β-ionone, and 2.1–34.5 μM for (+)-3-oxo-α-ionol. The concentration of (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol, respectively, in rattail fescue was 7.8 and 3.7 μg g⁻¹ fresh weight. Considering the endogenous level and the inhibitory activity, (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol may work as allelopathic substances in rattail fescue through the growth inhibition of neighboring plant species.     

Change in compactness of inclusion bodies of recombinant β-galactosidase expressed in the <Emphasis Type="Italic">araBAD</Emphasis> promoter system of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We investigated the relevance of the relationship between the compactness of β-galactosidase inclusion bodies (β-gal IBs) and their enhanced enzymatic activity with or without the addition of D-fucose (inducer analog) or methyl α-D-glucopyranoside (α-MG, catabolite repressor) after induction in the araBAD promoter system of Escherichia coli. Experiments conducted to evaluate the solubilization of β-gal IBs in guanidine hydrochloride as well as their trypsin degradation and temperature stability revealed that β-gal IBs expressed in response to the addition of D-fucose or α-MG had a looser structure. Additionally, β-gal IBs expressed when D-fucose or α-MG was added were more quickly solubilized in guanidine hydrochloride or degraded by trypsin-treatment than those produced when these compounds were not added. Moreover, the activity of β-gal IBs expressed when D-fucose or α-MG were added was less stable at various temperatures. Consequently, we deduced that the looser structure of β-gal IBs resulted in enhanced enzymatic activity of β-gal IBs upon addition of D-fucose or α-MG after induction.     

Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56

Sesquiterpenes are particularly interesting as flavorings and fragrances or as pharmaceuticals. Regio- or stereoselective functionalizations of terpenes are one of the main goals of synthetic organic chemistry, which are possible through radical reactions but are not selective enough to introduce the desired chiral alcohol function into those compounds. Cytochrome P450 monooxygenases are versatile biocatalysts and are capable of performing selective oxidations of organic molecules. We were able to demonstrate that CYP109D1 from Sorangium cellulosum So ce56 functions as a biocatalyst for the highly regioselective hydroxylation of norisoprenoids, α- and β-ionone, which are important aroma compounds of floral scents. The substrates α- and β-ionone were regioselectively hydroxylated to 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively, which was confirmed by ¹H NMR and ¹³C NMR. The results of docking α- and β-ionone into a homology model of CYP109D1 gave a rational explanation for the regio-selectivity of the hydroxylation. Kinetic studies revealed that α- and β-ionone can be hydroxylated with nearly identical V _max and K _m values. This is the first comprehensive investigation of the regioselective hydroxylation of norisoprenoids by CYP109D1.     

Enzyme analyses demonstrate that β-methylbutyric acid is converted to β-hydroxy-β-methylbutyric acid via the leucine catabolic pathway by Galactomyces reessii

Galactomyces reessii accomplishes the enzymatic transformation of β-methylbutyric acid (isovaleric acid) to β-hydroxy-β-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas β-methylbutyric acid hydroxylase, α-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, β-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to β-hydroxy-β-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to β-hydroxy-β-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into β-hydroxy-β-methylbutyric acid, thus demonstrating that β-methylbutyric acid is part of the leucine catabolic pathway. The rate of β-methylbutyric acid conversion to β-hydroxy-β-methylbutyric acid with cell-free extract was 0.013 μmol β-hydroxy-β-methylbutyric acid (mg protein)^–1 h^–1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 μmol β-hydroxy-β-methylbutyric acid (g cells)^–1 h^–1] was also observed with β-methylbutyric acid. The results indicate that β-methylbutyric acid is transformed to β-hydroxy-β-methylbutyric acid through the leucine catabolic pathway. Received: 18 July 1997 / Accepted: 12 November 1997     

Preparation of optically active β-hydroxy-α-amino acid by immobilized <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with serine hydroxymethyl transferase activity

In this research, an improved method for preparation of optically pure β-hydroxy-α-amino acids, catalyzed by serine hydroxymethyl transferase with threonine aldolase activity, is reported. Using recombinant serine hydroxymethyl transferase (SHMT), an enzymatic resolution process was established. A series of new substrates, β-phenylserine, β-(nitrophenyl) serine and β-(methylsulfonylphenyl) serine were used in the resolution process catalyzed by immobilized Escherichia coli cells with SHMT activity. It was observed that the K _m for l-threonine was 28-fold higher than that for l-allo-threonine, suggesting that this enzyme can be classified as a low-specificity l-allo-threonine aldolase. The results also shows that SHMT activity with β-phenylserine as substrate was about 1.48-fold and 1.25-fold higher than that with β-(methylsulfonylphenyl) serine and β-(nitrophenyl) serine as substrate, respectively. Reaction conditions were optimized by using 200 mmol/l β-hydroxy-α-amino acid, and 0.1 g/ml of immobilized SHMT cells at pH 7.5 and 45°C. Under these conditions, the immobilized cells were continuously used 10 times, yielding an average conversion rate of 60.4%. Bead activity did not change significantly the first five times they were used, and the average conversion rate during the first five instances was 84.1%. The immobilized cells exhibited favourable operational stability.     

Polar steroids from <Emphasis Type="Italic">Solaster endeca</Emphasis> starfish and the physiological activity of polar steroids from three starfish species

Four polyhydroxylated steroids, new (20R)-5α-cholestan-3β,6α,8,15α,24,26-hexaol (I) and known (20R,25S)-5α-cholestan-3β,6α,8,15β,16β,26-hexaol, (20R,25S)-5α-cholestan-3β,6α,15β,16β,26-pentaol, and marthasterone sulfate were isolated from the Solaster endeca starfish inhabiting the Sea of Okhotsk and characterized. Steroid (I) contains a 24,26-dihydroxylated side chain, which is unique for starfish polyols. The isolated steroids and related metabolites from two starfish species of the Evasterias genus (in total, 15 compounds) were weakly cytotoxic in a human HeLa cell culture and some of them were inhibitors of non-specific esterase from mouse Ehrlich carcinoma. The effects of these compounds on the p53 protein activity were studied in a yeast two-hybrid test system and both inhibitors and stimulators of this activity were found among them.     

Threonine aldolases—screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids

Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon–carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of β-hydroxy-α-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral β-hydroxy-α-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a l-threo-phenylserine derivative or l-threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of l-threonine or l-allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of β-hydroxy-α-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.     

Three new polyhydroxysteroids from the tropical starfish <Emphasis Type="Italic">Asteropsis carinifera</Emphasis>

Thirteen steroidal compounds including three new polyhydroxysteroids, (24R,25S)-24-methyl-5α-cholestane-3β,6α,8,15β,16β,26-hexaol, (22E,24R,25S)-24-methyl-5α-cholest-22-ene-3β,6α,8,15β,16β,26-hexaol, and (22E,24R,25S)-24-methyl-5α-cholest-22-ene-3β,4β,6α,8,15β,16β,26-heptaol, have been isolated along with ten previously known polyhydroxysteroids from the tropical starfish Asteropsis carinifera collected near the coast of Vietnam. The structures of the new compounds were elucidated by spectroscopic methods (mainly 2D NMR and ESI mass spectrometry).     

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5′-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of l-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at −20°C and 4°C. It was observed that the K _m for l-allo-threonine was 38-fold higher than that for l-threonine, suggesting this enzyme can be classified as a specific l-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6–7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible β-hydroxy-α-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of β-hydroxy-α-amino acids.     

Testa is involved in the control of storage mobilisation in seeds of <Emphasis Type="Italic">Sesbania virgata</Emphasis> (Cav.) Pers., a tropical legume tree from of the Atlantic Forest

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) contain galactomannan as a cell wall storage polysaccharide in the endosperm. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the role of the testa (seed coat) on galactomannan degradation during and after germination. Seeds were imbibed in water, with and without the testa, and used to evaluate the effect of this tissue on storage mobilisation, as well as its possible role in the galactomannan hydrolases activities. Immunocytochemistry and immunodotblots were used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. Endo-β-mannanase and α-galactosidase activities were found in the testa and latter in the endosperm during galactomannan degradation. The former enzyme was immunologically detected in the testa, mainly during germination. The absence of the testa during imbibition led to the anticipation of protein mobilisation and increased of the α-galactosidase activity and galactomannan degradation. Thus, the testa appears to play a role during storage mobilisation in the legume seed of S. virgata probably by participating in the control of the production, modification and/or storage of the hydrolases.     

Generation of aroma compounds from Ditaxis heterantha by Saccharomyces cerevisiae

Ditaxis heterantha, a plant of the Euphorbiaceae family, is growing wild in the semiarid regions of Mexico. The seed endosperm contains yellow pigments (carotenoids). By high-pressure liquid chromatography the total pigment (TP) was separated into seven fractions: two of them, heterathin (F4) and ditaxin (F5), characterized as apocarotenoids, represent 80% of TP. Both molecules have double bonds, which seem to be the target for degradation and aroma formation. In this work, TP, F4, and F5 were supplied to nine cultures able to degrade lutein. From these strains, only one (identified as Saccharomyces cerevisiae) was able to produce aromas from either TP or F4. Using TP as substrate, the produced aromas were 4-oxo-isophorone (1), isophorone (2), cinnamic aldehyde (6), 3-hydroxy-β-cyclocitral (7), safranal (8), geranyl (9), 3-oxo-α-ionone (10), 3-oxo-α-ionol (11), 3-oxo-7,8-dihydro-α-ionone (12), and eugenol (13). Of these aromas, only seven were produced from F4: (1), (2), (7), (8), (10), (11), and (12). In both cases, safranal was the main degradation product (30%). The enzymatic activity responsible for this effect was found in the cytosolic fraction and detected only when S. cerevisiae was grown in the presence of TP or F4.     

Genotoxic activity of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> essential oil in <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> diploid cells

Eucalyptus globulus essential oil was evaluated for its genotoxic potential using a somatic segregation assay and a diploid strain of the fungus Aspergillus nidulans, heterozygous for nutritional and conidia color markers. The main compounds of the current essential oil sample were eucalyptol (49.0 %), α-pinene (8.9), β-pinene (1.5), globulol (6.9), α-eudesmol (1.12), spathulenol (1.42), γ-cadinene (1.45), trans-β-elemenone (1.23) and aromandendrene (2.3), totaling 74 % of oil. Oil at 0.12 and 0.25 μL/mL was found to increase the mitotic instability of the original diploid strain and the number of diploid mitotic recombinants of A. nidulans. The genotoxicity of the oil was associated with the induction of mitotic crossing-over or with oil-broken chromosomes.     

Biotransformation of β-ionone by engineered cytochrome P450 BM-3

Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min⁻¹). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min⁻¹). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.     

A broader role for AmyR in <Emphasis Type="Italic">Aspergillus niger</Emphasis>: regulation of the utilisation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose or <Emphasis Type="SmallCaps">d</Emphasis>-galactose containing oligo- and polysaccharides

AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.     

A new isoflavone glycoside from <Emphasis Type="Italic">Trifolium pratense</Emphasis> L.

The isoflavonoid composition of red clover (Trifolium pratense L.) has been studied. The following compounds have been detected: cyclopolyol (+)-pinitol, not found in clover before; known isoflavones formononetin, prunetin, genistein, and prunetin-4′-O-β-D-glucopyranoside; isoflavone monogalactosides formononetin-7-O-β-D-galactopyranoside, inermin-3-O-β-D-galactopyranoside, and genistein-7-O-β-D-galactopyranoside; and a novel compound, prunetin-4′-α-D-glucopyranoside. The structures of these compounds have been proven by UV, IR, 1H NMR, 13C NMR, mass, and circular dichroism spectra. (+)-Pinitol is known to possess biological activity.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.     

Bioconversion of lutein to products with aroma

A residual mud sample from the marigold flower dehydration process was screened and 19 putative colonies were isolated for their ability to degrade lutein in a chemically defined medium supplemented with marigold flower flour as a carbon source. Among the colonies isolated, two generated volatile compounds in fermentation and one was chosen for further study for its ability to produce a strong tobacco smell. This colony contained two microorganisms, identified as Geotrichum sp. and Bacillus sp. The aroma production requires the presence of both microorganisms and lutein. Using gas chromatography coupled to mass spectrometry (GC/MS), four compounds were identified: 7,8-dihydro- β-ionol, β-ionone, 7,8-dihydro-β-ionone, and 3-hydroxy-β-ionone, in proportions of 84.2%, 9.4%, 3.5%, and 2.9%, respectively. Received: 30 November 1999 / Received revision: 18 April 2000 / Accepted: 1 May 2000     

Enhancement of α- and β-Galactosidase Activity in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> by Different Metal Ions

The hydrolysis of oligosaccharides and lactose is of great importance to the food industry. Normally, oligosaccharides like raffinose, stachyose, and verbascose which are rich in different plants like soy bean are considered indigestible by the human gut. Moreover, many humans suffer from lactose intolerance due to the absence of effective enzyme that can digest lactose. α-Galactosidase can digest oligosaccharides like raffinose, while β-galactosidases can hydrolyze lactose. Therefore, selection of microorganisms safe for human use and capable of producing high levels of enzymes becomes an attractive task. The objective of this study was to investigate the enhancement of α- and β-galactosidase activity in Lactobacillus reuteri by different metal ions. Ten millimolar of Na⁺, K⁺, Fe²⁺, and Mg²⁺ and 1 mM of Mn²⁺ were added separately to the growth culture of six strains of L. reuteri (CF2-7F, DSM20016, MF14-C, MM2-3, MM7, and SD2112). Results showed that L. reuteri CF2-7F had the highest α- and β-galactosidase activity when grown in the medium with added Mn²⁺ ions (22.7 and 19.3 Gal U/ml, respectively). 0.0274% of Mn²⁺ ions lead to 27, 18% enhancement of α- and β-galactosidase activity over the control group, and therefore, it could be added to the growth culture of CF2-7F to produce enhanced levels of α- and β-galactosidase activity. The addition of Fe²⁺ led to a significant (P < 0.01) decrease in the activity of both enzymes for most strains. This study shows that modified culture medium with that 0.0274% Mn²⁺ can be used to promote the production for α- and β-galactosidase in L. reuteri CF2-7F, which may lead to enhancement of α- and β-galactosidase activity and have a good potential to be used in the food industry.     

In vitro cytokine stimulation assay for glycolipid biosurfactant from <Emphasis Type="Italic">Rhodococcus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>: role of monocyte adhesion

Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1β and IL-6 production when applied as an ultrasonic emulsion to the adherent human peripheral blood monocyte culture. However, a lack of cytokine-stimulating activity was registered with the GLB applied as a hydrophobic film coating in 24-well culture plates, indicating that it may have been due to its inhibitory effect on monocyte adhesion. The mode of GLB application may therefore play an important role in in vitro assay of immunostimulatory activity of this compound as well as other bacterial glycolipids. Additionally, GLB from R. ruber displayed no cytotoxicity against human lymphocytes and therefore could be proposed as a potential immunomodulating and antitumor agent.