Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing l-amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize l-amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various l-amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from l-methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm^?2h^?1were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in l-amino acid oxidase [l-amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Characterization of α-maltotetraohydrolase produced by Pseudomonas sp. MS300 isolated from the deepest site of the Mariana Trench

We have isolated a Pseudomonas-like amylase producer, the strain MS300, which displayed a large halo on starch medium, from the deepest site of the Mariana Trench. The strain MS300 produced two major and two minor α-maltotetraohydrolases (G4-amylase). The two major G4-amylases share the same molecular weight of 55 000 but had different pI values, 5.0 and 4.7, respectively. The optimum temperature for activity of both major G4-amylases is 40°C, and the optimum pH is 6.8 for one and 8.9 for the other. MS300 produced more amylase under high hydrostatic pressure than under atmospheric pressure. Strain MS300 may be active in the deep sea at a depth of 10 897 m. Received: December 11, 1997 / Accepted: April 16, 1998     

Bioreduction of α-chloroacetophenone by whole cells of marine fungi

The asymmetric reduction of 2-chloro-1-phenylethanone (1) by seven strains of marine fungi was evaluated and afforded (S)-(-)-2-chloro-1-phenylethanol with, in the best case, an enantiomeric excess of 50% and an isolated yield of 60%. The ability of marine fungi to catalyse the reduction was directly dependent on growth in artificial sea water-based medium containing a high concentration of Cl⁻ (1.2 M). When fungi were grown in the absence of artificial sea water, no reduction of 1 by whole cells was observed. The biocatalytic reduction of 1 was more efficient at neutral rather than acidic pH values and in the absence of glucose as co-substrate.     

The Production of α-Ketoglutaric Acid from Salicylic Acid by Pseudomonas sp.†

Metabolites from salicylic acid by microorganisms were investigated. About eighty strains of bacteria which were able to utilize salicylic acid as a sole source of carbon were isolated from soil and other natural sources.

Among these bacteria, several strains produced a large amount of keto acids in the culture fluid during the cultivation. The acid was isolated from the culture fluid of strain K 102 in crystalline form. The crystal was identified as α-ketoglutaric acid by physicochemical methods. From the taxonomical studies, the isolated bacterial strains K 102 and K 362 were assumed to be Pseudomonas sp.     

Syntheses and highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions using chiral oxacalix[2]arene[2]bisbinaphthes

The triazine-based bisbinaphthyl crown ethers oxacalix[2]arene[2]bisbinaphthes R-1, R-2, R-3 and S-1, S-2, S-3 were synthesized. The interactions of these compounds with various α-aminocarboxylic acid anions were studied. The crown ethers were found to carry out highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions. It is observed that within a certain concentration range, one enantiomer of the chiral α-aminocarboxylic acid anions can increase the fluorescence intensity of the crown ethers by fivefold to sixfold, whereas the other enantiomer scarcely enhances the fluorescence. Such unusually high enantioselective responses make these crown ethers very attractive as fluorescent sensors in determining the enantiomeric composition of α-aminocarboxylic acid anions.     

α-Chymotrypsin-catalyzed enantioselective amidation of chiral amines using an N-protected amino acid carbamoylmethyl ester as acyl donor

-Chymotrypsin catalyzed the highly enantioselective amidation of chiral amines such as 1-(1-naphthyl)ethylamine using N-benzyloxycarbonyl-(S)-phenylalanine carbamoylmethyl ester as acyl donor (E = 25–660) in acetonitrile with low water content.     

Alkaline inulinase production by a newly isolated bacterium Marinimicrobium sp. LS–A18 and inulin hydrolysis by the enzyme

To date, all of microbial inulinases reported showed optimal activity at pH values ranging from 3.5 to 7.0. A bacterial strain, Marinimicrobium sp. LS-A18, showing high extracellular inulinolytic activity was isolated from a marine solar saltern of the Yellow Sea in China. Maximum enzyme activity was obtained at 55°C and pH 9.0, respectively. The inulinase activity was induced by inulin, but not by the other carbon sources employed. Under the optimal medium and culture condition, the highest inulinase activity, 14.6 U/ml, was obtained after 96 h of incubation at shake flask level. The optimal medium for inulinase production was MHI medium containing 4% inulin, 1% peptone and 5% NaCl, while the optimal culture condition for inulinase production were pH 7.5, temperature 37°C, agitation speed 210 rpm, medium volume 40 ml in 250 ml shake flask, and incubation time 96 h. A large amount of monosaccharides was released after inulin hydrolysis by the inulinase from strain LS-A18. This is the first report on alkaline inulinase production from microorganism.     

Study of the experimental conditions for the lipase production by a newly isolated strain of Pseudomonas aeruginosa for the enantioselective hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate

A lipase producing bacterium has been isolated from the soil to enantiospecifically hydrolyze the (±)-methyl trans-3(4-methoxyphenyl) glycidate (MPGM), an intermediate in the synthesis of cardiovascular drug, diltiazem. This hydrolysis provided the desired (−)-MPGM in 44% yield with 99% enantiomeric excess. The organism was identified and confirmed as Pseudomonas aeruginosa by 16S rRNA sequencing. The various physiochemical parameters have been optimized for the maximum production of lipase in shake flask. Beef extract was found to be the best nitrogen source for lipase production. The optimized cultivation conditions were 30°C with an initial medium pH 8 in shake flask. Both inoculum age and inoculum concentration have positive effect on the lipase production and (±)-MPGM (3 mM) was found to be the optimal inducer.An erratum to this article can be found at     

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L⁻¹ of dry cell weight (OD₆₀₀ = 120) within 24 h. A plasmid DNA yield of 100 mg L⁻¹ (1.7 mg g⁻¹ cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L⁻¹) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L⁻¹ was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10–12 g L⁻¹ (OD₆₀₀ = 25–30) and plasmid yields of 5–8 mg L⁻¹ (approximately 0.7 mg g⁻¹ cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (μ) from 0.69 h⁻¹ to 0.13 h⁻¹ and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. Received 22 March 1996/ Accepted in revised form 20 September 1996     

Expression of the Alcaligenes eutrophus poly(β-hydroxybutyric acid)-synthetic pathway in Pseudomonas sp.

The 12.5-kb EcoRI restriction fragment PP1 of Alcaligenes eutrophus strain H16, which encodes for -ketothiolase, NADP-dependent acetoacetyl-CoA reductase and poly(-hydroxybutyric acid)-synthase was mobilized to six different species of the genus Pseudomonas belonging to the rRNA homology group I. Pseudomonas aeruginosa, P. fluorescens, P. putida, P. oleovorans, P. stutzeri and P. syringae, which are unable to synthesize and accumulate poly(-hydroxybutyric acid), PHB, were employed as recipients. Whereas the A. eutrophus PHB-synthetic enzymes were only marginally expressed in P. stutzeri, they were readily expressed in the other species. For example, the specific activity of PHB-synthase was 1.8 U/g protein in transconjugants of P. stutzeri but was between 21 and 77 U/mg protein in transconjugants of the other species. All recombinant strains harboring plasmid pVK101::PP1 except those of P. stutzeri accumulated PHB; the PHB content of the cells grown on gluconate under nitrogen limitation varied between 8 and 24.3% of the cellular dry mass.Abbreviations PHB poly(-hydroxybutyric acid) - PHA poly(hydroxyalkanoic acid)     

Production of 2-O-α-d-glucopyranosyl l-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp.

Transglycosylation to produce a 2-O--d-glucopyranosyl l-ascorbic acid (AA-2G) was studied using cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. A series of maltooligosaccharides substituted 2-O-derivatives of l-ascorbic acid (AA) were analyzed by HPLC. The maltooligosaccharides were hydrolyzed by glucoamylase to give AA-2G. CGTase also produced AA-2G using dextrin as a glycosyl donor and AA as an acceptor. CGTase utilized -, -, and -CDs, amylose, soluble starch and corn starch as glycosyl donors but not glucose.     

Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells

The effects of three fatty acids on cytotoxic aggregate formation of Ca²⁺-depleted bovine α-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 °C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovine α-lactalbumin made lethal to tumor cells), a complex of the partially unfolded α-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of α-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.     

Monoamine Transamination Catalyzed by ω-Amino Acid; Pyruvate Aminotransferase of Pseudomonas sp. F-126

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F-126 catalyzes stoichiometricalJy a transamination between various amines and pyruvate. Most of alkyl and aromatic monoamines served as an amino donor. The enzyme activity was affected by carbon number of straight-chain alkylmonoamines with a maximum activity at 5-carbon unit, n-amylamine. Michaelis constants for n-butylamine and pyruvate were calculated to be 66.6 mm and 5.5 mm respectively. The enzyme was active in the alkaline range with a maximum at pH 10.5 ~ 11.0, though not any activity was observed at the pH below 8.0. The optimum temperature for the reaction was at 60°C.     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

Preparation of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid and its derivatives from racemates by enantioselective degradation with a newly isolated bacterial strain <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ECU0401

An enantioselective mandelate-degrading bacterium, Alcaligenes sp. ECU0401, was newly isolated from soil. By fed-batch culture, (R)-(-)-mandelic acid was successfully prepared in a 5-L fermenter with 32.8% isolated yield and >99.9% enantiomeric excesses (e.e.) from totally 3.04% (w/v) of racemic mandelic acid after 99 h of biotransformation. The optimal reaction pH and temperature were 6.5 and 30 degrees C, respectively. Using the resting cell as a biocatalyst for asymmetric degradation of racemic mandelic acid and chloro-substituted derivatives thereof, (R)-(-)-mandelic acid, (R)-(-)-o-chloromandelic acid, (S)-(+)-m-chloromandelic acid and (S)-(+)-p-chloromandelic acid were recovered with high analytic yields and excellent enantiomeric excesses (e.e. > 99.9%). (R)-(-)-Mandelic acid could also be obtained after 12 h of biotransformation with 41.5% isolated yield and >99.9% e.e.     

Use of α-aminoadipic acid for the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. l-alpha-Amino[6-(14)C]adipic acid has been prepared from the dl-amino acid by oxidation of the l-isomer with l-amino acid oxidase to alpha-oxo[6-(14)C]adipic acid and by transamination of the latter with l-glutamic acid in an extract of a Cephalosporium sp. prepared by ultrasonic treatment of the mycelium. 2. The optical configuration of small amounts of (14)C-labelled alpha-aminoadipic acid from the mycelium of the Cephalosporium sp. has been determined by treatment with l-amino acid oxidase and measurement of the proportion of radioactivity subsequently retained on a column of a strong cation-exchange resin. 3. alpha-Aminoadipic acid which had been labelled in the mycelium from [1-(14)C]acetate appeared to contain more than 99% of the l-isomer. 4. l-alpha-Amino[(14)C]adipic acid (sodium salt) was taken up much more rapidly than the d-isomer, or alpha-oxo[6-(14)C]adipic acid, by suspensions of washed mycelium of the Cephalosporium sp. in water. The pool of intracellular alpha-aminoadipic acid was expandable. 5. Intracellular products found to be labelled with (14)C from l-alpha-amino[(14)C]adipic acid were delta-aminovaleric acid, saccharopine, lysine, protein, compounds which behaved like penicillin N, cephalosporin C and deacetylcephalosporin C respectively on paper chromatography and electrophoresis, and a peptide whose amino acid residues include alpha-aminoadipic acid, cysteine and valine. 6. l-alpha-Amino[(14)C]adipic acid acted as a precursor of the delta-(d-alpha-aminoadipoyl) side chains of extracellular penicillin N and cephalosporin C. 7. (14)C from d-alpha-amino[(14)C]adipic acid was incorporated into penicillin N and cephalosporin C, but the incorporation was accompanied by a relatively high dilution of specific radioactivity and some l-alpha-amino[(14)C]adipic acid was found in the intracellular pool. 8. These findings are discussed in relation to the origin of the d- configuration of the alpha-aminoadipoyl side chain of the antibiotics.     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.