RESUMEN

Paenibacillus polymyxa ATCC 12321 produced more acetic acid and less butanediol from xylose than from glucose. The product yields from xylose were ethanol (0.72 mol/mol sugar), (R,R)-2,3-butanediol (0.31 mol/mol sugar), and acetate (0.38 mol/mol sugar) while those from glucose were ethanol (0.74 mol/mol sugar), (R,R)-2,3-butanediol (0.46 mol/mol sugar), and acetate (0.05 mol/mol sugar). Higher acetate kinase activity and lower acetate uptake ability were found in xylose-grown cells than in glucose-grown cells. Furthermore, phosphoketolase activity was higher in xylose-grown cells than in glucose-grown cells. In fed-batch culture on xylose, glucose feeding raised the butanediol yield to 0.56 mol/mol sugar and reduced acetate accumulation to 0.04 mol/mol sugar.

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To reduce the solid waste in soy sauce refuse (SSR) that is a by-product of soy sauce fermentation, both biological and chemical treatment were examined. When anaerobic digestion of SSR was carried out at the amounts of 25 g/l under 30% (v/v) inocula of thermophilic methanogenic sludge at 50 degrees C, 120 mM of CH4 was produced and 50% (w/v) of mixed-liquor suspended solids in SSR was decreased after 35 days. Although in this culture 30 days' cultivation was required to get 100 mM of CH4, when the same amounts of SSR (25 g/l) were repeatedly added to the culture, the time requirement to get 100 mM of CH4 could be reduced to 20 days and 15 days at second and third batch cultures, respectively. When SSR was treated with 5% (w/v) NaOH for 24 h, the supernatant contained 98% (w/v) of protein that were alkaline-solubilized and insoluble in the intact refuse, and the residual pellet contained insoluble fiber.

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The removal of organic matter from a coastal mud sediment was carried out by a methane fermentation process under anaerobic conditions. In a batch acidogenic fermentation, the addition of vitamins containing thiamine, nicotinic acid and biotin dramatically enhanced acetate production from the mud sediment (200 g wet wt l(-1) artificial sea water), yielding 77 mM acetate after 6 days, which corresponded to 77% of the organic matter in the mud sediment, measured on the basis of chemical oxygen demand. Thereafter, the two-fold diluted, post-acidogenic fermentation liquor (PAF liquor) was continuously treated at 2.4x original dilution rate day(-1) for 30 days, using an upflow anaerobic sludge blanket methanogenic reactor containing the acclimated methanogenic sludge from the mud sediment. Acetate, 42 mM in the PAF liquor, was converted to methane at a maximum methane production rate of 96 mmol l(-1) day(-1); and 87.5% of the acetate and 88.7% of the total organic carbon in the PAF liquor were removed. Moreover, an efficient treatment of the mud sediment was carried out by a semi-continuous, two-stage reactor system, where the culture broth was circulated between acidogenic and methanogenic reactors. This two-stage reactor system gave a stable operation at 4-day intervals for one treatment period, yielding 112 mmol methane from the wet mud in the PAF liquor (278 g l(-1)).

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We found that the asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate (EHB) by Paracoccus denitrificans could be induced by nitrate addition under anaerobic conditions. However, the addition of electron donors such as glucose, ethanol, and methanol together with nitrate did not stimulate EHB production from EAA. When the reaction was carried out under optimum conditions (cell concentration, 10 g-d.w.l(-1); EAA, 150 mM; NO3-, 100 mM), after 8 h of reaction 49 mM of EHB with an optical purity of 98.9% e.e. was produced. Furthermore, a fed-batch reaction was carried out with an intermittent addition of nitrate and EAA to reduce substrate inhibition. The linear reduction of EAA to EHB proceeded for 40 h after initiation of the reaction, and finally 124 mM of EHB with an optical purity of 88.7% e.e. was produced after 104 h.

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By the addition of 150 mM acetate into a batch culture at an initial pH of 6.8, the production of 2,3-butanediol (BDL) by Paenibacillus polymyxa reached 248 mM, yielding 0.87 mol.mol(-1) glucose, where the ratio of acetate consumed to glucose consumed (A/C ratio) was calculated as 0.35 mol acetate mol(-1) glucose. Therefore, a fed-batch culture was carried out by feeding glucose and acetate at a ratio of 0.35 mol acetate mol(-1) glucose. In the fed-batch culture performed at pH 6.8, BDL production reached 637 mM, yielding 0.81 mol.mol(-1) glucose, although the A/C ratio was only 0.18 mol acetate mol(-1) glucose. By decreasing pH to 6.3 in the fed-batch culture, BDL production reached 566 mM, yielding 0.88 mol.mol(-1) glucose and the A/C ratio was 0.32 mol acetate mol(-1) glucose. The optical purity of BDL, which was expressed as enantiomeric excess, was retained at more than 98% of the (R, R)-stereoisomer at the end of culture, which was comparable to that without acetate addition.

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The influence of the oxygen and glucose supply on primary metabolism (fermentation, respiration, and anabolism) and astaxanthin production in the yeast Phaffia rhodozyma was investigated. When P. rhodozyma grew under fermentative conditions with limited oxygen or high concentrations of glucose, the astaxanthin production rate decreased remarkably. On the other hand, when the yeast grew under aerobic conditions, the astaxanthin production rate increased with increasing oxygen uptake. A kinetic analysis showed that the respiration rate correlated positively with the astaxanthin production rate, whereas there was a negative correlation with the ethanol production rate. The influence of glucose concentration at a fixed nitrogen concentration with a high level of oxygen was then investigated. The results showed that astaxanthin production was enhanced by an initial high carbon/nitrogen ratio (C/N ratio) present in the medium, but cell growth was inhibited by a high glucose concentration. A stoichiometric analysis suggested that astaxanthin production was enhanced by decreasing the amount of NADPH required for anabolism, which could be achieved by the repression of protein biosynthesis with a high C/N ratio. Based on these results, we performed a two-stage fed-batch culture, in which cell growth was enhanced by a low C/N ratio in the first stage and astaxanthin production was enhanced by a high C/N ratio in the second stage. In this culture system, the highest astaxanthin production, 16.0 mg per liter, was obtained.

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We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

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In a green alga, Haematococcus pluvialis, a morphological change of vegetative cells into cyst cells was rapidly induced by the addition of acetate or acetate plus Fe to the vegetative growth phase. Accompanied by cyst formation, algal astaxanthin formation was more enhanced by the addition of acetate plus Fe than by the addition of acetate alone. Encystment and enhanced carotenoid biosynthesis were inhibited by either actinomycin D or cycloheximide. However, after cyst formation was induced by the addition of acetate alone, carotenoid formation could be enhanced with the subsequent addition of Fe even in the presence of the inhibitors. The Fe -enhanced carotenogenesis was inhibited by potassium iodide, a scavenger for hydroxyl radical, suggesting that hydroxyl radical formed by an iron-catalyzed Fenton reaction may be required for enhanced carotenoid biosynthesis. Moreover, it was demonstrated that four active oxygen species, singlet oxygen, superoxide anion radical, hydrogen peroxide, and peroxy radical, were capable of replacing Fe in its role in the enhanced carotenoid formation in the acetate-induced cyst. From these results, it was concluded that oxidative stress is involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells.

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Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane. Pyridoxal 5'-phosphate can prevent this modification efficiently, whereas pyridoxal 5'-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond. The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme. Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the alpha-H of substrates. The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to alpha-H labilization.

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Holotryptophanase inactivated by oxidation of cysteinyl residues showed a different absorption spectrum from the native enzyme. At pH 8.0, the native enzyme preferentially existed as a 337-nm species (active form), whereas in the inactive enzyme a 420-nm species (inactive form) was dominant. During the reactivation of the enzyme by reduction with dithiothreitol, an increase at 337 nm and a decrease at 420 nm were observed with concomitant increase in enzymatic activity, which was accompanied by the appearance of two cysteinyl residues per monomer. Specific S-cyanylation of cysteinyl residues by nitrothiocyanobenzoic-acid-inactivated apotryptophanase with the modification of one cysteinyl residue per monomer, whereas holotryptophanase was highly resistant to inactivation with nitrothiocyanobenzoic acid. The essential role of the active-site-bound pyridoxal 5'-phosphate in protection against inactivation was confirmed by the agreement of the K1/2 (protection) of 5.0 microM for pyridoxal 5'-phosphate with Km of 2.0 microM in enzyme catalysis. The inactivation by nitrothiocyanobenzoic acid caused a similar shift in the equilibrium between the 337-nm species and 420-nm species, i.e. decrease of the 337-nm species and increase of the 420-nm species. From the pH dependence of the equilibrium between these two species, pKa of 7.9 and 7.4 was obtained for the inactive and the dithiothreitol-activated enzyme, respectively, indicating that cysteinyl residue(s) participated in lowering the pKa of the interconversion between the 337-nm species (active form) and 420-nm species (inactive form). The possible role of cysteinyl residues in the function of tryptophanase is discussed.

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