Journal of Fermentation and Bioengineering最新文献

A halotolerant yeast strain R28 that can convert d-psicose to d-talitol was isolated from soy sauce mash and identified as Candida famata. The cells grown on l-sorbose were found to have the best conversion potential. C. famata R28 converted d-psicose to d-talitol at a faster rate in the presence of various carbohydrates such as erythritol, d-sorbitol, ribitol and glycerol in the reaction mixture. At 10% substrate concentration, the conversion ratio was above 90% using washed cells when 5% d-sorbitol was added to the reaction mixture. Moreover, for production of d-talitol by a fermentation reaction with C. famata R28, the conversion ratio was about 80% at 10% substrate concentration, and more than 98% of the substrate consumed was converted to d-talitol.

A mechanism of 2,3-butanediol (BD) stereoisomer formation was examined with respect to the BD cycle. The enzymes acetylacetoin synthase, acetylacetoin reductase (AACR), and acetylbutanediol hydrolase (ABDH), which are part of the BD cycle, were found to be present in the cell-free extract of the bacterial strain Bacillus cereus YUF-4. Two kinds of acetylbutanediol (ABD) stereoisomers were produced in the reduction of acetylacetoin (AAC) by AACR, which were identified as having 3R,4R and 3S,4R configurations by NMR spectroscopy and an enzymic method. The two ABD formations were found to be catalyzed independently by two respective enzymes: the former was catalyzed by a NADPH-dependent AACR (3R,4R-ABD forming) and the latter by a NADH-dependent AACR (3S,4R-ABD forming). The 3R,4R-ABD was converted into R,R-BD and the 3S,4R-ABD into R,S-BD by intracellular ABDH. These findings demonstrated the existence of a new BD isomer formation mechanism derived from the BD cycle.

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.

Phosphoglycerate kinases [PGK, EC 2.7.2.3] were purified as electrophoretically homogeneous proteins from four nitrifying bacteria: Nitrosomonas europaea ATCC 25978T (Ns), Nitrosomonas sp. TNO632 (TNO), Nitrosomonas sp. K1 (K1), Nitrobacter winogradskyi IFO 14297 (Nb) and nonsulfur bacterium Rhodopseudomonas palustris JCM2524 (Rp). The enzymes were all monomers with molecular masses of 44.6, 44.3, 43.7, 46.1, and 43.4 kDa, respectively. Ns-PGK and Nb-PGK had the same pH-activity curves with an optimum pH range of 8.0–8.5. The enzymes were stable in the pH range 7.0–9.0 when kept at 4°C for 48 h. The temperature optima of Ns-PGK and Nb-PGK were 50 and 40°C, respectively. Both enzymes were strongly inhibited by pCMB and SDS at 1 mM, ATP was effective, while other nucleotides did not serve as a phosphate donor. The N-terminal amino acid sequences of Ns-PGK and Nb-PGK were maximally homologous (90–95%) with TNO-PGK and Rp-PGK, respectively. However, the degree of PGK homology was as low as 45–59% between the two nitrifying bacteria genera. As previously observed, all Nitrosomonas and Nitrobacter strains constitute a monophyletic assemblage within the beta and alpha subdivisions of the proteobacteria, respectively. The differences in the N-terminal amino acid sequences of the PGKs coincided with the taxonomic differences of the bacterial genera according to the molecular phylogenetic tree.

Five methanotrophs (strains 18-2, EB1, KSWIII, KSPIII and KSPIII) and three aromatic compound oxidizers (strains KP22, KP24 and KT1) were isolated from the natural field polluted with trichloroethylene (TCE). Phylogenetic analysis based on 16S rRNA gene sequence suggested that all of the isolates belonged to the class Proteobacteria. Two of the methanotrophic isolates, strains 18-2 and EB1, were closely related to Methylocystis sp. strain M in the α subclass of Proteobacteria with sequence similarities of 98.2–98.4%, while strains KSWIII, KSPIII and KSPII were akin to Methylomonas methanica in the γ subclass of Proteobacteria with sequence similarities of 97.8–98.1%. The aromatic compounds oxidizers, strains KP22, KP24 and KT1, were assigned to the β subclass of Proteobacteria, and classified as Bordetella sp. (97.2–97.8% sequence similarity to species of the genus Bordetella), Burkholderia cepacia (99.2%) and Ralstonia eutropha (99.4%), respectively. All isolates degraded TCE when cells were grown with the appropriate substrate, i.e., methane, phenol or toluene. Detailed kinetic analyses of their TCE degradation revealed that the rates of degradation (k1) among the isolates were 10–36 ml of TCE/mg of dry cell weight/h, and the transformation capacities (Tc) were 0.01–0.13 mg of TCE/mg of dry cell weight.

The random amplified polymorphic DNA (RAPD) technique was applied to strain and species identification. Four different arbitrary primers were tested with DNAs from seven strains of four species of red tide algae of the genus Chattonella. Two of the primers showed species-dependent profiles, while the other two identified differences between strains. The results indicate that the RAPD technique is useful for distinguishing polymorphisms among species or strains.

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.

sn-glycerol-1-phosphate (G-1-P) dehydrogenase is the key enzyme for biosynthesis of the enantiomeric glycerophosphate backbone of ether phospholipids of Archaea. The gene encoding this enzyme in Methanobacterium thermoautotrophicum (egsA) was used to construct an expression plasmid pTrcG1Pdh for Escherichia coli. The G-1-P dehydrogenase activity of E. coli XL1-blue/pTrcG1Pdh was maximal 8–10 h after induction. The expressed G-1-P dehydrogenase was purified 4300-fold from the soluble fraction to homogeniety after 4300 times purification by a procedure consisting of fractionation with ammonium sulfate precipitation, and hydrophobic and ion-exchange chromatographies. The yield was about 70%. The V_max value for the forward reaction to produce G-1-P was 740 units (μmol/min)/mg, with a K_m of 0.21 mM for NADH and 0.39 mM for dihydroxyacetone phosphate. The K_m's for G-1-P and NAD⁺ in the backward reaction were 10.5 and 0.46 mM, respectively. These kinetic constants are similar to those for the enzyme from M. thermoautotrophicum. G-1-P dehydrogenase was successfully used to analyze the stereospecificity of glycerophosphate, which is an intermediate of phospholipid biosynthesis and glycerol metabolism; the rate of NADH formation was proportional to the G-1-P concentration up to 3 mM in the presence of 0.02 unit of the purified enzyme.

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.