Production of amino acids by free-living heterotrophic nitrogen-fixing bacteria

Summary Interest of microbial production of amino acids has been increased greatly since development of biotechnological methods. These methods represent a perspective way applied in a future large-scale manufacture of inexpensive amino acids. In this context, the isolation of producing organisms that may be exploited in the desing of alternative methods for the production of amino acids could be of primary importance.In this review we will describe the liberation of amino acids (methionine, lysine, arginine, tryptophane and glutamic acid) byAzotobacter andAzospirillum during growth in culture media with different carbon sources under diazotrophic and adiazotrophic conditions. These organisms may be useful in developing new methods for the industrial production of amino acids.     

Production of D-phenylglycine-related amino acids by immobilized microbial cells

Bacterial dihydropyrimidinase was shown to catalyze the hydrolytic cleavage of various 5-substituted hydantoins to the corresponding N-carbamyl-D-amino acids under alkaline conditions. Therefore, an enzymatic method for preparing the D-forms of phenylglycine-related amino acids was developed using immobilized bacterial cells with high enzyme activity. Alkalophilic bacteria were a good enzyme source for this process. The process is simple and economical for use in the production of various amino acids with the D-configuration.     

Net uptake of dissolved free amino acids by four scleractinian corals

High pressure liquid chromatography was employed to provide the first definitive proof of the net uptake of dissolved free amino acids (DFFA) at nanomolar levels by four scleractinian corals (Montastrea annularis, Madracis mirabilis, Agaricia fragilìs, and Favia fragum). During 2 h incubations all species exhibited simultaneous net uptake of eight amino acids. For M. annularis and F. fragum uptake of some dissolved amino acids occurred at concentrations lower than those found in reef waters. Microbial activity or adsorption of DFAA to exposed coral skeletons during these experiments did not appear to be important. Although it seems unlikely that DFAA uptake can provide a significant energy source for corals under ambient condìtions, it may be important in the acquisition and retention of nitrogen by these animals.     

Thiophosphorylation of free amino acids and enzyme protein by thiophosphoramidate ions

In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of ³¹P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of ³¹P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His²⁶) or lysine (Lys³³) side-chain.     

Immobilization and mineralization of dissolved free amino acids by stream-bed biofilms

1. Radiolabelled (¹⁴C) amino acids were used to investigate the influence of sediment size as well as dissolved free amino acid (DFAA) concentration and composition on immobilization and mineralization of DFAAs by biofilms from a first-order stream. 2. Over time (240 min), biofilms on stony substrata immobilized a DFAA mixture more effectively than those on sandy substrata, however proportional mineralization of immobilized DFAAs was higher for sandy substrata (36 v 20%). 3. Using stony substrata, the DFAA mixture was immobilized more rapidly than glycine alone at ‘near-natural’ amino acid concentrations (c. 37 μgl^?1), as well as enriched concentrations (1 and 100 mg 1-^?1). Instantaneous rates of glycine immobilization and mineralization were not saturated at glycine enrichments of up to 980 mgl^?1. 4. With both the amino acid mixture and glycine alone, proportional mineralization of the immobilized amino acids increased on enrichment to Img 1-^?1 (DFAA mixture: from 25 to 37%; glycine alone: from 50 to 54%), but then fell on further enrichment to 100mgl^?1 (DFAA mixture: 11%; glycine alone: 7%). 5. Results are discussed in terms of the potential trophic utility of immobilized DFAAs as well as the apparent roles of biotic and abiotic immobilization mechanisms. Immobilization and mineralization responses to variables investigated in this study give an insight into potential variability of carbon immobilization and retention in stream-bed sediments. This is fundamental to an understanding of how DOC may become available to higher trophic levels.     

Uptake of free amino acids by the diatom,Melosira mediocris

Individual ¹⁴C-labelled amino acids are rapidly removed from dilute solution in artificial sea water (0.2 mol 1^–1) by suspensions of Meliosira medocris. The rate of disappearance of radioactivity corresponds closely to removal of primary amines as determined by measurement of the rate of decrease of fluorescamine-positive material. Net removal of naturally occurring free amino acids from the sea water habitat from which the alga was isolated is demonstrated using high performance liquid chromatography. Removal of amino acids from natural sources makes a significant contribution to the carbon requirements of the alga as well as supplying significant amounts of amino nitrogen.     

Extraction of free amino acids from tomato leaves

Conditions for extraction of free amino acids from tomato leaves were examined. Two methods of sample preservation were also tested. Best results were obtained when samples were preserved by lyophilization and extracted by Soxhlet for 6 h at 40°C at a ratio of 1 g plant material/80 mL 80% (v/v) ethanol.     

Production of organic acids and amino acids from fish meat by sub-critical water hydrolysis

Fish meat was easily liquefied by hydrolysis under subcritical conditions without oxidants, and aqueous phase and water-insoluble phase containing oil and fat-like solid were formed. Lactic acid found in the raw fish meat (about 0.03 g/g-dry meat) was stable up to the reaction temperature 513 K (3.35 MPa). Pyroglutamic acid was produced with a yield of 0.095 kg/kg of dry meat by 30 min reaction at 553 K (6.42 MPa). Amino acids such as cystine, alanine, glycine, and leucine were produced in the temperature range 513-623 K with a maximum peak at 543 K. Amounts of cystine, alanine, glycine, and leucine produced in 5 min at 543 K (5.51 MPa) were 0.024, 0.013, 0. 009, and 0.004 kg/kg of dry meat, respectively. The oil extracted with hexane contained useful fatty acids such as eicosapentanoic acid (EPA) and docosahexianoic acid (DHA). Thus, subcritical water hydrolysis would be an efficient process for recovering useful substances from organic waste such as fish waste discarded from fish market.     

Production of carbon monoxide from aromatic amino acids by Morganella morganii

Morganella morganii produced CO when cultured in a medium containing casamino acids or peptone as the sole carbon source. Although the production of CO was distinctly enhanced by the addition of hemin to the medium, the amounts of CO produced in the absence of hemin were nearly proportional to the amounts of peptone added to the culture media. Examination of 20 amino acids for their ability to produce CO by resting cells revealed that phenylalanine, tyrosine, histidine and tryptophan were the sources of CO. Oxygen and hemin were necessary for CO production from the amino acids except tryptophan which produced CO in the absence of hemin. When cells were incubated for 4 h at 30° C in the mixture containing 40 mol tyrosine and 1 mol hemin, about 15 mol CO was produced; the activity of CO production was about 1.2 mol CO/h · mg cell nitrogen. Phenylpyruvic acid, p-hydroxyphenylpyruvic acid and imidazolepyruvic acid also produced CO in the presence of hemin, while indolepyruvic acid produced CO regardless of the presence or absence of hemin. The production of CO by the 2-oxo acids proceeded spontaneously and did not require the presence of M. morganii cells.