Journal of Fermentation and Bioengineering最新文献

The acclimation behavior of activated sludge against nitrification inhibitors was explored by continuously adding thiourea and/or aniline to unacclimated sludge. Thiourea, which is difficult to biodegrade, severely inhibited the ammonia oxidation step, whereas aniline inhibited both the ammonia and nitrite oxidation steps and its biodegradation by nitrifying sludge was possible after a short acclimation period. Since thiourea-acclimated sludge showed higher tolerance than unacclimated sludge, the recovery of complete nitrification after being in contact with thiourea over a long period was attributed not only to the acquisition of biodegrading ability but also to the tolerance acquired by the sludge. Once both biodegrading ability and tolerance were acquired, they remained even after stopping the addition of thiourea for several weeks. In contrast, aniline-acclimated sludge was suspected to acquire only biodegrading ability.

The roles of histidine residues in the catalysis of the transformation of 2-oxoglutarate into ethylene via the ethylene-forming enzyme (EFE) from Pseudomonas syringae were studied using site-directed mutagenesis with substitution of glutamine for ten individual histidine residues. The mutant enzymes, which were expressed in Escherichia coli, were purified to homogeneity, and assayed in vitro for K_m, k_cat and thermostability. The relative k_cat of two mutated EFEs, H305Q and H335Q, were 40% and 60%, respectively. However, a mutation at either His-189 or His-233 caused a total loss of activity, implying that these residues play important roles in the binding of iron. The k_cat values for other mutant enzymes were 11-to 55-fold less than that for the wild-type enzyme. For six partially inactive mutated EFEs (but not for H305Q or H335Q), the first order rate constants for heat inactivation at 30°C were 11-to 24-fold higher than for the wild-type enzyme. It is noteworthy that the value of the first order rate constant for heat inactivation of H268Q was identical to that of H335Q. The substitution of H268 resulted in a drastic decrease of the k_cat value (relative k_cat was 1.8%). This suggests that the substitution at His-268 may cause the disruption of the active site of the EFE. Heat inactivation studies with the puridied mutant enzymes revealed that some mutant enzymes, such as H168Q and H116Q, were more thermolabile than the wild-type enzyme.

Chlorella pyrenoidosa Chick catalyzed the reduction of methyl 3-oxopentanoate (Me 3-oxoPen) to the corresponding (S)-(+)-3-hydroxy ester in 60% enantiomeric excess (ee). An improvement of the ee of (S)-(+)-3-hydroxy ester was achieved by increasing substrate concentration and by the addition of 1% metal salts, namely, NaCl and KCl (75–78% ee). No shift of ee toward the (R)-(−)-3-hydroxy ester was observed following this addition. The addition of allyl alcohol brought about a decrease in both the chemical yields and the ee of (S)-(+)-3-hydroxy ester. Differences in the ability to reduce me 3-oxoPen were observed among three strains of Chlorella, of which C. pyrenoidosa Chick yields the (S)-(+)-3-hydroxy ester but C. regularis and C. vulgaris Beijerinck yield (R)-(−)-enantiomer. In particular, C. regularis transformed Me 3-oxoPen to (R)-(−)-3-hydroxy ester in 79% ee, with a 10% yield.

Analysis of fatty acid composition in wild-type cells of Hansenula polymorpha strain CBS 1976 revealed the presence of 18 : 1 (Δ9), 18 : 2 (Δ9,12) and 18 : 3 (Δ9,12,15) unsaturated fatty acids (UFAs), indicating that the α-linolenic desaturation pathway operates in this yeast. H. polymorpha cells also showed ability for uptake and incorporation of exogenous UFAs. By ethyl methanesulfonate mutagenesis, nine unsaturated fatty acid auxotrophic mutants of H. polymorpha were isolated. These mutants exhibited the growth arrest phenotype on nutrient medium and on nutrient medium supplemented with saturated fatty acids, but grew on media supplemented with various UFAs. Genetic analysis revealed that single recessive nuclear mutation conferred Ufa auxotrophy on these mutants. Fatty acid analysis by gas chromatography showed the accumulation of 18 : 0 but a decrease in the amount of 18 : 1 and 18 : 2 in mutant cells compared with the wild-type cells. Integrated physiological and genetical data suggested that mutations in all mutants occurred in one gene and probably led to defects in Δ9-desaturation pathway.

The heat resistance of Brevibacterium sp. JCM6894 was examined as a function of externally added NaCl concentrations. About a 5-log cycle reduction of the viable cell numbers was observed to result from heat treatment for 30 min at 47°C in the absence of NaCl. When the cells were heated in the buffer containing 2 M NaCl, the viability was maintained within less than 1-log cycle reduction after incubation for 30 min at 56°C. During the heat treatment for 30 min at 47°C in the presence of 2 M NaCl, Na⁺ and K⁺ ions in the cells increased and decreased by 13 and 26 μg ions per mg of cell protein, respectively. Under this condition, the amount of free amino acids in the cells changed little except for glutamate and hydroxyproline, which were reduced by 72 and 43 nmol per mg cell protein, respectively. These results indicate that the salt stress itself and Na⁺ ions existing in the cytoplasm are more important factors than in vivo protein synthesis for preventing the thermal death of the resting cells of this strain.

A β-xylosidase was purified from the culture filtrate of the thermophilic fungus Thermoascus sp. by ultrafiltration, ethanol precipitation, and chromatography with DEAE-Toyopearl 650M, Mono Q $\text{HR5}\text{5}$, and Phenyl Superose $\text{HR5}\text{5}$. The purified β-xylosidase was found to be homogeneous on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was estimated to be 107 kDA by gel filtration chromatography (Superdex 200 HR) and 100 kDa by SDS-PAGE. The optimum activity of the enzyme was observed at pH 4.5 and 55°C. The enzyme was stable up to 60°C at pH 4.5 for 1 h. The enzyme exhibited hydrolytic activity on phenyl β-d-xyloside and xylan (birch wood). Fifteen of the amino acid residues in the amino terminal region of the Thermoascus sp. β-xylosidase were homologous with residues in the equivalent region of the maturation protein β-glucosidase from Aspergillus aculeatus.

We studied the effect of serial transfers on solventogenesis capacity and spore-forming ability by Clostridium acetobutylicum ATCC 824 in two culture media. In reinforced clostridial medium (RCM), an abrupt decrease of the ability to ferment glucose and produce solvent occurred after the 10th to 12th transfer and spore formation failed after the 25th transfer. When 1.5 g/l of acetic and butyric acids were added to the RCM, however, the capacity to produce a normal level of solvents was not lost, whereas spore-forming ability was lost after the 23rd transfer. The shift from solventogenesis to acidogenesis did not occur in chemically defined medium (CDM) even by the 40th transfer. The dramatic decrease of solventogenesis in the degenerate culture was coupled with acetate/butyrate CoA transferase and acetoacetate decarboxylase deficient activities.

The moss Pohlia flexuosa was obtained as the protonema from its matured capsules. It was grown by shaking in modified Murashige-Skoog's medium, and the dry weight obtained was 40 mg per 20 ml medium after 15 d. The mature protonema removed Hg²⁺ from aqueous solutions. The higher the initial Hg²⁺ concentration, the higher the amount of Hg²⁺ removed, and the concentration factor, which is an indicator of the ability to remove heavy metals, was 2000 ml/g of dry moss at 50 μg/ml of HgCl₂. The optimal pH and temperature for Hg²⁺ removal were 7.0 and 25°C, respectively. Sodium azide and dinitrophenol at 0.1 mM inhibited Hg²⁺ removal up to a relative removal of 81 and 77%, respectively. The importance of two processes, bioaccumulation and adsorption, in the removal of Hg²⁺ by this moss is discussed.