Involvement of Manganese in Conversion of Phenylalanine to Benzaldehyde by Lactic Acid Bacteria

We examined the involvement of Mn(II) in the conversion of phenylalanine to benzaldehyde in cell extracts of lactic acid bacteria. Experiments performed with Lactobacillus plantarum demonstrated that Mn(II), present at high levels in this strain, is involved in benzaldehyde formation by catalyzing the conversion of phenylpyruvic acid. Experiments performed with various lactic acid bacterial strains belonging to different genera revealed that benzaldehyde formation in a strain was related to a high Mn(II) level.     

Conversion of Phenylalanine to Benzaldehyde Initiated by an Aminotransferase in Lactobacillus plantarum

The production of benzaldehyde from phenylalanine has been studied in various microorganisms, and several metabolic pathways have been proposed in the literature for the formation of this aromatic flavor compound. In this study, we describe benzaldehyde formation from phenylalanine by using a cell extract of Lactobacillus plantarum. Phenylalanine was initially converted to phenylpyruvic acid by an aminotransferase in the cell extract, and the keto acid was further transformed to benzaldehyde. However, control experiments with boiled cell extract revealed that the subsequent conversion of phenylpyruvic acid was a chemical oxidation step. It was observed that several cations could replace the extract in the conversion of phenylpyruvic acid to benzaldehyde. Addition of Cu(II) ions to phenylpyruvic acid resulted not only in the formation of benzaldehyde, but also in the generation of phenylacetic acid, mandelic acid, and phenylglyoxylic acid. These compounds have been considered intermediates in the biological conversion of phenylalanine. The chemical conversion step of phenylpyruvic acid was dependent on temperature, pH, the availability of cations, and the presence of oxygen.     

Screening, identification and kinetic characterization of a bacterium for Mn(II) uptake and oxidation

Following sample collection and screening at a number of Mn-associated mine sites in Northern Australia, a microbial strain was selected for its enhanced rate of Mn uptake. The strain was identified by phylogenetic analysis as a Rhizobium sp. Kinetic studies of Mn(II) uptake and oxidation by this strain in glucose-based media established that the uptake of Mn(II) was much greater than the conversion of Mn(II) to Mn oxide. Chemical analysis and scanning electron microscopy confirmed the production of significant amounts of polysaccharides by this strain. These polysaccharides may play a role both in enhancing Mn(II) accumulation and in minimizing Mn oxide production.     

Manganese, superoxide dismutase, and oxygen tolerance in some lactic acid bacteria

A previous study of the aerotolerant bacterium Lactobacillus plantarum, which lacks superoxide dismutase (SOD), demonstrated that it possesses a novel substitute for this defensive enzyme. Thus, L. plantarum contains 20 to 25 mM Mn(II),m in a dialyzable form, which is able to scavenge O2- apparently as effectively as do the micromolar levels of SOD present in most other organisms. This report describes a survey of the lactic acid bacteria. The substitution of millimolar levels of Mn(II) for micromolar levels of SOD is a common occurrence in this group of microorganisms, which contained either SOD or high levels of Mn(II), but not both. Two strains were found which had neither high levels of Mn(II) nor SOD, and they were, as was expected, very oxygen intolerant. Lactic acid bacteria containing SOD grew better aerobically than anaerobically, whereas the organisms containing Mn(II) in place of SOD showed aerobic growth which was best, at best, equal to anaerobic growth. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) increases the rate of O2- production in these organisms. Lactobacillus strains containing high intracellular Mn(II) were more resistant to the oxygen-dependent toxicity of plumbagin than were strains containing lower levels of Mn(II). The results support the conclusion that a high internal level of Mn(II) provides these organisms with an important defence against endogenous O2-.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Formation of hydride-Meisenheimer complexes of picric acid (2,4, 6-trinitrophenol) and 2,4-dinitrophenol during mineralization of picric acid by Nocardioides sp. strain CB 22-2

There are only a few examples of microbial conversion of picric acid (2,4,6-trinitrophenol). None of the organisms that have been described previously is able to use this compound as a sole source of carbon, nitrogen, and energy at high rates. In this study we isolated and characterized a strain, strain CB 22-2, that was able to use picric acid as a sole source of carbon and energy at concentrations up to 40 mM and at rates of 1.6 mmol. h(-1). g (dry weight) of cells(-1) in continuous cultures and 920 micromol. h(-1). g (dry weight) of cells(-1) in flasks. In addition, this strain was able to use picric acid as a sole source of nitrogen at comparable rates in a nitrogen-free medium. Biochemical characterization and 16S ribosomal DNA analysis revealed that strain CB 22-2 is a Nocardioides sp. strain. High-pressure liquid chromatography and UV-visible light data, the low residual chemical oxygen demand, and the stoichiometric release of 2.9 +/- 0.1 mol of nitrite per mol of picric acid provided strong evidence that complete mineralization of picric acid occurred. During transformation, the metabolites detected in the culture supernatant were the [H-]-Meisenheimer complexes of picric acid and 2,4-dinitrophenol (H--DNP), as well as 2,4-dinitrophenol. Experiments performed with crude extracts revealed that H--DNP formation indeed is a physiologically relevant step in picric acid metabolism.     

Manganese(IV) oxide production by Acremonium sp. strain KR21-2 and extracellular Mn(II) oxidase activity

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.     

Yeast-mediated preparation of l-PAC in an organic solvent

The yeast-mediated acyloin condensation of benzaldehyde and pyruvic acid to form l-PAC occurs in a petroleum spirit solvent system at room temperature with moderate conversion (30%) and high enantioselectivity (86%ee) after 24 h. The addition of a small amount of ethanol (0.5% mL) to the reaction mixture inhibits the formation of the side product benzyl alcohol and increases the conversion to l-PAC. Conducting the reaction using 13C labeled pyruvate indicated that the pyruvate was incorporated into the l-PAC and that the excess pyruvate was converted into ethanol. Conducting the reaction at 5 degrees C results in similar conversion but higher enantioselectivity.     

The scavenging of superoxide radical by manganous complexes: in vitro

Dialyzable manganese has been shown to be present in millimolar concentrations within cells of Lactobacillus plantarum and related lactic acid bacteria. This unusual accumulation of Mn appears to serve the same function as Superoxide dismutase (SOD), conferring hyperbaric oxygen and Superoxide tolerance on these SOD-free organisms. The form of the Mn in the lactic acid bacteria and the mechanisms whereby it protects the cell from oxygen damage are unknown. This report examines the mechanisms by which Mn catalytically scavenges O₂^?, both in the xanthine oxidase/cytochrome c SOD assay and in a number of in vitro systems relevant to the in vivo situation. In all the reaction mixtures examined, Mn(II) is first oxidized by O₂^? to Mn(III), and H₂O₂ is formed. In pyrophosphate buffer the Mn(III) thus formed is re-reduced to Mn(II) by a second O₂^?, making the reaction a true metal-catalyzed dismutation like that catalyzed by SOD. Alternatively, if the reaction takes place in orthophosphate or a number of other buffers, the Mn(III) is preferentially reduced largely by reductants other than O₂^?, such as thiols, urate, hydroquinone, or H₂O₂. H₂O₂, a common product of the lactic acid bacteria, reacted rapidly with Mn(III) to form O₂, apparently without intermediate O₂ release. Free hexaquo Mn(II) ions were shown by electron spin resonance spectroscopy and activity assays in noncomplexing buffers to be poorly reactive with O₂^?. In contrast, Mn(II) formed complexes having a high catalytic activity in scavenging O₂^? with a number of organic acids, including malate, pyruvate, propionate, succinate, and lactate, with the Mn-lactate complex showing the greatest activity.     

Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; K_m, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.     

Multicopper oxidase involvement in both Mn(II) and Mn(III) oxidation during bacterial formation of MnO2

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO₂ formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria indicates that multicopper oxidases (MCOs) are required for MnO₂ formation. However, MCOs catalyze one-electron oxidations, whereas the conversion of Mn(II) to MnO₂ is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O₂ and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO₂ also depends on O₂ and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, which is indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO₂ formation.     

Direct fermentative production of lactic acid on cassava and other starch substrates

Lactobacillus amylovorus utilized raw corn, rice and wheat starch medium to produce lactic acid with a productivity of 10.1, 7.9 and 7.8 g lactic acid/L, but had lower productivities of 4.8 g/L and 4.2 g/L on cassava and potato starch in basal medium respectively. When peptone (1%) is added to basal medium with cassava starch as substrate, conversion rate increased from 43% conversion to 70% conversion (7.7 g lactic acid/L). The availability of some components of protein in corn starch is assumed to be the reason for high lactic acid production as compared to that of cassava starch.     

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.     

Direct fermentation of various pure and crude starchy substrates to L(+) lactic acid using Lactobacillus amylophilus GV6

Lactobacillus amylophilus GV6 fermented a variety of pure and natural starches directly to L(+) lactic acid. Starch to lactic acid conversion efficiency was more than 90% by strain GV6 at low substrate concentrations with all starches. The strain GV6 produced high yields of lactic acid per g of substrate utilized with pure starches such as soluble starch, corn starch, and potato starch, yielding 92–96% at low substrate concentrations in 2 days and 78–89% at high substrate (10%) concentrations in 4–6 days. Strain GV6 also produced high yields of lactic acid per g of substrate utilized with crude starchy substrates such as wheat flour, sorghum flour, cassava flour, rice flour and barley flour yielding 90–93% at low substrate concentrations in 2 days and 80% or more at high substrate concentrations in 6–7 days. Lactic acid yields by L. amylophilus GV6 with pure starches were comparable when low cost crude starchy substrates were used. Lactic acid productivity by strain GV6 is higher than for any other previously reported strains of L. amylophilus.     

Comparative evaluation of manganese peroxidase- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids by different methods

The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP in the presence of Mn(II) ions but the rate of their oxidation was not directly related to degree of their unsaturation. As it has been shown by monitoring oxygen consumption and conjugated dienes formation the linoleic acid was the most easily oxidizable fatty acid for MnP/Mn(II) and chelated Mn(III). However, when the lipid peroxidation (LPO) activity was monitored by TBARS formation the linolenic acid gave the highest results. High accumulation of TBARS was also recorded during peroxidation of linoleic acid initiated by MnP/Mn(II). Action of Mn(III)-tartrate on the PUFAs mimics action of MnP in the presence of Mn(II) indicating that Mn(III) ions are involved in LPO initiation. Although in our experiments Mn(III) tartrate gave faster than MnP/Mn(II) initial oxidation of the unsaturated fatty acids with consumption of O₂ and formation of conjugated dienes the process was not productive and did not support further development of LPO. The higher effectiveness of MnP/Mn(II)-initiated LPO system depends on the turnover of manganese provided by MnP. It is proposed that the oxygen consumption assay is the best express method for evaluation of MnP- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids.     

A numerical taxonomic study of lactic acid bacteria from vacuum-packed beef, pork, lamb and bacon

S haw B. G. & H arding C harmaigne D. 1984. A numerical taxonomic study of lactic acid bacteria from vacuum packed beef, pork, lamb and bacon. Journal of Applied Bacteriology 56 , 25–40.
A numerical taxonomic study using 79 unit characters has been performed on 100 isolates of lactic acid bacteria from refrigerated vacuum-packed beef, pork, lamb and bacon. Three clusters were observed at 78% S which contained all the strains apart from three unidentifiable streptobacteria, one Leuconostoc , and one strain of Pediococcus pentosaceus . One cluster (III) consisted of only one strain of Leuc. paramesenteroides and six unidentifiable Leuconostoc strains. The two largest clusters (I and II) were both composed entirely of streptobacteria. Cluster I contained 31 strains (G + C content 33–2–36–9 moles %) which were not identifiable with any described species. Cluster II contained 57 strains (G + C content 40–7–43–7 moles %) which were provisionally identified with Lactobacillus sake or Lact. bavaricus according to the lactic acid isomer produced. The division of nearly all the streptobacteria into two clearly defined clusters has resolved problems which have existed in the classification of lactic acid bacteria from vacuum-packed meat.     

Optimization of probiotic and lactic acid production by Lactobacillus plantarum in submerged bioreactor systems

Biomass and lactic acid production by a Lactobacillus plantarum strain isolated from Serrano cheese, a microorganism traditionally used in foods and recognized as a potent probiotic, was optimized. Optimization procedures were carried out in submerged batch bioreactors using cheese whey as the main carbon source. Sequential experimental Plackett–Burman designs followed by central composite design (CCD) were used to assess the influence of temperature, pH, stirring, aeration rate, and concentrations of lactose, peptone, and yeast extract on biomass and lactic acid production. Results showed that temperature, pH, aeration rate, lactose, and peptone were the most influential variables for biomass formation. Under optimized conditions, the CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g l⁻¹ (dw) of L. plantarum. At the central point of the CCD, a biomass of 10.2 g l⁻¹ (dw), with conversion rates of 0.10 g of cell g⁻¹ lactose and 1.08 g lactic acid g⁻¹ lactose (w/w), was obtained. These results provide useful information about the optimal cultivation conditions for growing L. plantarum in batch bioreactors in order to boost biomass to be used as industrial probiotic and to obtain high yields of conversion of lactose to lactic acid.     

Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes

Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett-Burman design and response surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40g/L of lactic acid was obtained ( approximately 96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO(3), w/v).     

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.     

Construction of a constitutively expressed homo-fermentative pathway in Lactobacillus brevis

Lactobacillus brevis is a promising lactic acid producing strain that simultaneously utilizes glucose and xylose from lignocellulosic hydrolysate without carbon catabolic repression and inhibition. The production of by-products acetic acid and ethanol has been the major drawback of this strain. Two genes, pfkA (fructose-6-phosphate kinase [PFK]) and fbaA (fructose-1,6-biphosphate aldolase [FBA]), that encode the key enzymes of the EMP/glycolytic pathway from Lactobacillus rhamnosus, were fused to the downstream of the strong promoter P32 and expressed in L. brevis s3f4 as a strategy to minimize the formation of by-products. By expressing the two enzymes, a homo-fermentative pathway for lactic acid production was constructed. The lactic acid yields achieved from glucose in the transformants were 1.12 and 1.16 mol/mol, which is higher than that of the native strain (0.74 mol/mol). However, the lactic acid yield from xylose in the transformants stayed the same as that of the native strain. Enzyme assay indicated that the activity of the foreign protein FBA in the transformants was much higher than that of the native strains, but was ten times lower than that in L. rhamnosus. This result was consistent with the metabolic flux analysis, which indicated that the conversion efficiency of the expressed PFK and FBA was somewhat low. Less than 20 % of the carbons accumulated in the form of fructose-6-phosphate were converted into glyceraldehyde-3-phosphate (GAP) by the expressed PFK and FBA. Metabolic flux analysis also indicated that the enzyme phosphoketolase (XPK) played an important role in splitting the carbon flow from the pentose phosphate pathway to the phosphoketolase pathway. This study suggested that the lactic acid yield of L. brevis could be improved by constructing a homo-fermentative pathway.