Production of essential L-branched-chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values.     

Limiting amino acids in dried microbial cells given to young turkeys

The effects of amino acid additions to diets containing methanol-grown dried microbial cells (MC) have been examined in two experiments with young turkeys. The sample of MC used was an early pelleted preparation of Methylophilus methylotrophus produced by Imperial Chemical Industries in the initial stages of the development of Pruteen. The pellets were crushed to a coarse powder prior to dietary inclusion. In the first study, turkeys fed on either 100 or 200 g MC/kg diet with supplements of methionine had similar growth rates and efficiency of food conversion as those fed on a control soya bean meal (SBM) diet containing equivalent dietary nitrogen concentrations (54 g N/kg DM). Combined additions of methionine and arginine to the MC diets had no further effect on growth performance. In the second experiment, an unsupplemented basal diet containing 150 g MC/kg diet and 55 g N/kg DM supported marginally better weight gain, efficiency of food utilisation and efficiency of carcass deposition of gross energy (GE) and N than a basal SBM diet with 53 N/kg DM. Methionine supplementation of the latter diet improved growth performance to levels approaching those in the group fed on the basal MC diet. Feeding the basal SBM and MC diets containing sub-optimal levels of dietary N (46 and 48 g N/kg DM, respectively) confirmed the slightly superior nutritional value of the MC diet. Methionine supplementation enhanced growth performance and efficiency of carcass deposition of N and GE in turkeys fed on the SBM diet. On the other hand, methionine supplementation of the corresponding basal diet containing MC induced only slight improvements in growth and efficiency of deposition of N and GE in the carcass. Combined additions of methionine and lysine to the N-restricted diets containing SBM or MC were less effective than the addition of methionine alone.     

Production of long-chain hydroxy fatty acids by microbial conversion

Hydroxy fatty acids (HFAs) are very important chemicals for versatile applications in biodegradable polymer materials and cosmetic and pharmaceutical industries. They are difficult to be synthesized via chemical routes due to the inertness of the fatty acyl chain. In contrast, these fatty acids make up a major class of natural products widespread among bacteria, yeasts, and fungi. A number of microorganisms capable of producing HFAs from fatty acids or vegetable oils have been reported. Therefore, HFAs could be produced by biotechnological strategies, especially by microbial conversion processes. Microorganisms could oxidize fatty acids either at the terminal carbon or inside the acyl chain to produce various HFAs, including α-HFAs, β-HFAs, mid-position HFAs, ω-HFAs, di-HFAs, and tri-HFAs. The enzymes and their encoded genes responsible for the hydroxylation of the carbon chain have been identified and characterized during the past few years. The involved microbes and catalytic mechanisms for the production of different types of HFAs are systematically demonstrated in this review. It provides a better view of HFA biosynthesis and lays the foundation for further industrial production.     

Production of organic acids by periplasmic enzymes present in free and immobilized cells of Zymomonas mobilis

In this work the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR)/glucono-δ-lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and different aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate affinities were observed when fructose was in the mixture with glucose, maltose, galactose, and lactose, in this order. In bioconversion runs with 0.7 mol l^?1 of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maximal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l^?1 were achieved, with conversion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0–8.0 and temperatures of 47–50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diffusion of substrates and products, maximal lactobionic acid concentration, conversion yields and specific productivity similar to those obtained with permeabilized free-cells were achieved.     

Epoxidation of propene by microbial cells immobilized on inorhanic supports

Immobilization of gas-utilizing microorganism strains (Mycobacteria, Rhodococcus, methane-utilizers) on inorganic supports based on alumina, silicates, and carbon was carried out to develop heterogeneous biocatalysts for the biotechnologic processes, including the process of propene epoxidation. Adsorption ability of these microorganisms, biocatalytic properties of resting and immobilized bacterial cells, and effect of immobilization tehniques on biocatalysis were studied. An approach of double immobilization using inorganic materials (supports and gel) was proposed as simple, universal, and available methopd to immobilize bacterial cells, resulting in a higher retention (up to 100%) of cells' enzymatic activity and enhanced stability.     

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.     

Natural microbial UV radiation filters--mycosporine-like amino acids

Ozone depletion by anthropogenic gases has increased the atmospheric transmission of solar ultraviolet-B radiation (UV-B, 280-315 nm). There is a logical link between the natural defenses of terrestrial and marine organisms against UV radiation and the prevention of UV-induced damage to human skin. UV light degrades organic molecules such as proteins and nucleic acids, giving rise to structural changes that directly affect their biological function. These compounds offer the potential for development of novel UV blockers for human use. The biological role of mycosporine-like amino acids (MAAs) and scytonemin as a defense against solar radiation in organisms, together with their structure, synthesis, distribution, regulation and effectiveness, are reviewed in this article. This review points to the role of MAAs as a natural defense against UV radiation.     

Anaerobic microbial associations degrading aromatic amino acids

Anaerobic microbial associations have been isolated that degrade aromatic amino acids to methane and carbon dioxide at high rates. Significant differences between the morphological, cytological, and physiological traits of cultures isolated from samples of adapted and unadapted sludge are shown. The effects of cultivation temperature, illumination, and presence of mineral nitrogen and bicarbonate in the medium upon adaptation of enrichment cultures to substrates and subsequent behavior of the anaerobic associations have been studied. Intermediate and final products of degradation of aminoaromatic compounds and the sequence of their formation in the cultures have been determined. We have also studied the effects of exogenous electron acceptors and additional carbon sources on the degradation of aminoaromatic compounds.     

Production of hydroxy fatty acids by microbial fatty acid-hydroxylation enzymes

Hydroxy fatty acids are widely used in chemical, food, and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of lubricants, emulsifiers, and stabilizers. They have antibiotic, anti-inflammatory, and anticancer activities and therefore can be applied for medicinal uses. Microbial fatty acid-hydroxylation enzymes, including P450, lipoxygenase, hydratase, 12-hydroxylase, and diol synthase, synthesize regio-specific hydroxy fatty acids. In this article, microbial fatty acid-hydroxylation enzymes, with a focus on region-specificity and diversity, are summarized and the production of mono-, di-, and tri-hydroxy fatty acids is introduced. Finally, the production methods of regio-specific and diverse hydroxy fatty acids, such as gene screening, protein engineering, metabolic engineering, and combinatory biosynthesis, are suggested.     

Production of solavetivone by immobilized cells of Hyoscyamus muticus

Summary The secretion of solavetivone, a phytoalexin, by the cells of Hyoscyamus muticus in response to fungal elicitation has been enhanced by gel entrapment in calcium alginate. The immobilized cells produced 53% higher product and exhibited sustained biosynthetic activity in repeated batch cycles when compared with suspended cells. Providing a non-elicited media exchange and a sufficient interval of time between repeated infection (elicitation) gave greater productivity. Apparently the cells need a period to recover from the infection.     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

Production of cellulase by immobilized whole cells of Haloarcula

Halophilic Archaea are adapted to a life in the extreme conditions and some of them are capable of growth on cellulosic waste as carbon and energy source by producing cellulase enzyme. The production of cellulase using free and immobilized cells of halophilic archaeal strain Haloarcula 2TK2 isolated from Tuzkoy Salt Mine and capable of producing cellulose was studied. The cells were cultured in a liquid medium containing 2.5 M NaCl to obtain the maximum cellulase activity and immobilized on agarose or polyacrylamide or alginate. Optimal salt dependence of free and immobilized cells of Haloarcula 2TK2 was established and the effects of pH and temperature were investigated. Immobilization to Na-alginate enhanced the enzymatic activity of the Haloarchaeal cells when compared to free cells and other polymeric supports. From the results obtained it is reasonable to infer that decomposition of plant polymers into simpler end products does occur at high salinities and cellulase producing haloarchael cells may be potentially utilized for the treatment of hypersaline waste water to remove cellulose.     

Production of NADP by immobilized cells with NAD kinase

By the radiation-copolymerization method with polyethylene glycoldimethacrylate (PGD) as a main polymerizable reagent, microbial cells of Brevibacterium ammoniagenes were immobilized with high specific activity of NAD kinase and high mechanical strength. The reagents used for the immobilization such as PGD, polyvinyl alcohol (PVA), and N,N'-methylenebisacrylamide (Bis) did not inversely affect the enzyme activity. Freezing and irradiation treatment of the cell-reagent solution did not inactivate the enzyme either, but longer freezing time or a lower irradiation dose (less than 400 krad) resulted in the unsatisfactory mechanical strength of the immobilized cells. Almost all of NAD and ATP consumed were converted into NADP within three hours reaction time. The drum reactor was found to be ideal for the reaction of immobilized cells, since it gave little mechanical stress to the immobilized cells for the effective mixing of the cells and the substrates. The immobilized cells were subjected to three hours reaction repeatedly for 30 times without any activity loss.     

Production of daunorubicin by immobilized growing Streptomyces peucetius cells

Summary Streptomyces peucetius cells were immobilized by entrapment in calcium alginate and a photosensitive synthetic polymer, and used for the production of daunorubicin (daunomycin), which is known to be an antitumour reagent. The use of cultivation media removed insoluble components in a natural medium prevented rapid decrease in daunorubicin titer after maximum production. These entrapped cells could be used at least five times for repeated daunorubicin production; the total cultivation period was 45 days.     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

Production of lysine by using immobilized livingCorynebacterium sp cells

Summary Lysine production by immobilizedCorynebacterium sp cells in alginate gel beads was investigated in flasks. ImmobilizedCorynebacterium sp cells exhibited a slightly greater lysine production than free cells and accumulated 60 g/l of L-lysine at maximum, when cultured for 120h in a medium containing 200g/l glucose as carbon source. Several factors, such as inoculum size, incubation time and alginate gel concentration were examined in order to improve lysine production by immobilized growing cells.     

Uptake of amino acids by actidione-treated yeast cells

The steady-state levels of distribution of glycine,l-aspartic acid,l-leucine and, to a lesser extent, ofl-lysine andl-methionine, in actidione-treated baker’s yeast cells are significantly altered (usually decreased) in the presence ofd-glucose,d-mannose,d-fructose, 2-deoxy-d-glucose, maltose, sucrose and, after induction,d-galactose. Stimulatory effects ofd-ribose,l-sorbose andd-xylose are not highly significant. Pronounced effects of sugars were also found anaerobically. No effect of amino acids on sugar uptake was observed. Three types of interaction appear to be present: (1) increase of energy reserves by metabolized sugars; (2) increased rate of carrier breakdown in the presence of metabolized sugars; (3) interaction at the carrier level in a “heteropolyvalent” membrane complex.     

Uptake of amino acids by actidione-treated yeast cells

The active uptake ofl-aspartic acid, glycine andl-lysine by actidione-treated cells ofSaccharomyces cerevisiae was found to be inhibited by anaerobic conditions in the absence of a source of energy, only facilitated diffusion persisting. Similarly, metabolic inhibitors (iodoacetamide, sodium fluoride and potassium sorbate) inhibited the uptake very substantially. 2,4-Dinitrophenol and sodium azide appeared to inhibit the movement of the transport carrier itself, while uranyl ions showed a complex interaction pattern, ranging from inhibition at concentrations of 10^?6–10^?4 m, to stimulation at concentrations of 3×10^?4–10^?3 m, to pronounced inhibition at higher concentrations. The uptake was pH-dependent with optima forl-aspartic acid near pH 4, for glycine near pH 5, forl-lysine near pH 6.5.