The ilvBNC operon of Corynebacterium glutamicum encodes acetohydroxy acid synthase and isomero-reductase, which are key enzymes of L-isoleucine, L-valine and L-leucine syntheses. In this study we identified the transcript initiation site of ilvBNC operon 292 nucleotides in front of the first structural gene, and detected the formation of a short transcript from the leader region in addition to the full length transcript of the operon. This identifies the control of ilvBNC transcription by an attenuation mechanism involving antitermination. Mutations in the leader region were made and their effect on the operon expression in ilvB'lacZ fusions was quantified, Although a presumed leader-peptide-coding region is only one nucleotide away from the transcript initiation site determined, there is clear evidence to support the formation of this leader peptide: (i) the substitution of initiation codon ATG of the peptide by AGG reduced lacZ expression of the appropriate fusion construct to 19%; (ii) the replacement of three subsequent Val codons by Ala codons resulted in the loss of Val-dependent expression; and (iii) a leader peptide LacZ fusion resulted in active beta -galactosidase. Based on these results, it is concluded that transcription of ilvBNC is controlled by a translational-coupled attenuation mechanism. The absence of a ribosome binding site for leader peptide formation means that additional mechanisms may contribute to the transcription control at the decoding initiation step in the leader peptide formation.
The D-Xylose reductase (XR) gene (xyrA) of Candida tropicalis IFO 0618 was expressed in Escherichia coli JM109. The enzymatic properties of each recombinant XR such as the Km value for D-xylose and NADPH, the substrate specificity for other sugars and the optimal pH were essentially the same as those of the corresponding enzyme of C. tropicalis. The recombinant XR was more heat-stable than C. tropicalis XR at 60 degrees C. E. coli, expressing the xyrA gene, successfully converted D-xylose to xylitol. When D-xylose (50 g/l) and D-glucose (5 g/l) were added to IPTG-induced cells, 13.3 g/l of xylitol was produced during 20 h of cultivation.
Nitrous oxide (N2O) is emitted from wastewater treatment processes. It is known as a greenhouse gas that contributes to global warming (over 200 times more per molecule than carbon dioxide) and to the destruction of the ozone layer. It is therefore of great importance to develop technology that can suppress N2O emission. The effects of an anoxic period on N2O emission and nitrogen removal were investigated in an actual domestic wastewater treatment plant. When operated with intermittent aeration, most of the N2O was emitted into the atmosphere during the aerobic period. N2O emission from the intermittent process was estimated to be 0.43–1.89 g N2O person−1 year−1. Maintaining a dissolved oxygen (DO) concentration of over 0.5 mg l−1 during the aerobic period resulted in the complete conversion of the influent NH4-N to NO3-N and a 60-min anoxic period was sufficient for denitrification to be completed. The findings show that an optimum combination of aerobic and anoxic conditions and their suitable control are very important for improving nitrogen removal efficiency and controlling N2O emission.
A method based on the use of whole lyophilized microbial cells has been developed for resolution of 2-alkanols. Firstly, the esterification of racemic 2-octanol with butyric acid was studied. Rhizopus oryzae CBS 112-07 grown on Tween 80 was selected as a suitable biocatalyst. Appropriate choice of solvent, temperature and reactant concentration allowed for highly selective esterification (>97% enantiomeric excess of R-ester, 41% molar conversion). Direct esterification of 2-octanol with butyric acid was compared with interesterification using different acylating reagents. The best results were obtained with butyric acid and tributyrin. Esterification of other racemic 2-alkanols (2-butanol, 2-pentanol, 2-hexanol, 2-heptanol) showed that chain length played a crucial role in enantioselectivity, since lower enatiomeric excesses were observed using short chain alcohols.
Alginate lyase III of Sphingomonas sp. A1 cleaved the glycosidic linkage of polymannuronate and heteropolymeric region composed of mannuronate and guluronate, but was inert on polyguluronate. The enzyme was observed to act endolytically, interact with tetrasaccharide in alginate, and form di- and trisaccharides as final products. This result suggests that the enzyme recognizes the unit of tetrasaccharide in alginate and cleaves the middle linkage of the tetrasaccharide.
The adsorption parameter, Amax (maximum protein adsorbed/g substrate) and KA (adsorption equilibrium constant) of Trichoderma reesei C-5 cellulases were 58.8 mg protein/g cellulose and 11.6 × 104l/mol respectively. The activation energies for the adsorption rate constants of cellobiohydrolases and endoglucanases of C-5 were 30% and 11% lower respectively than that in the parent T. reesei QM9414 enzymes indicating the greater binding ability of the former. This was also reflected in its increased saccharification efficiency.
A simple polymerase chain reaction (PCR) method for identification of the four species of Saccharomyces sensu stricto was developed. The method is based on amplification of the RPL2 region using three primers designed to detect the structural differences between the RPL2 flanking regions of S. cerevisiae and S. bayanus. A 1.1-kb DNA fragment was amplified in both S. cerevisiae and S. paradoxus, although the amount of amplified DNA in S. paradoxus was small compared to that in S. cerevisiae. A 1.9-kb DNA fragment was amplified specifically in S. bayanus. Both 1.1-kb and 1.9-kb DNA fragments were amplified in S. pastorianus, which is thought to be a hybrid species of S. cerevisiae and S. bayanus. Thus, using the three primers, the four species of Saccharomyces sensu stricto could be easily distinguished by the size and amount of the PCR product.
When low molecular weight alginate (guluronic acid (G)-oligomers, M.W.≦2,000) was added to Catharanthus roseus cell culture, intracellularly stored ajmalicine was released into the broth. Addition of agarose and high molecular weight alginate did not promote the release of ajmalicine. C. roseus protoplasts were immobilized in guluronic acid rich (G-rich) alginate gel beads (artificial cell wall having a kind of elicitor function) and used for ajmalicine production. Immobilized protoplasts could be cultivated in a shake flask at low osmotic pressure, without disruption. The extracellular ajmalicine production by the protoplasts immobilized in alginate was much higher than that by the cells immobilized in alginate and protoplasts immobilized in agarose. On the 7 d of cultivation, cell wall regeneration in the immobilized protoplasts was detected under a fluorescence microscope. This implies that prevention of cell wall regeneration is a prerequisite for long term process with protoplasts. When 30 mM CaCl2 was added to the broth, active protoplasts were maintained for 15 d with neither cell wall regeneration nor inhibition of indole alkaloid production. The specific productivities of various indole alkaloids (ajmalicine, catharanthine and tryptamine) by the immobilized C. roseus protoplasts in the presence of CaCl2 in the broth were much higher than those of the immobilized cells.
The cbhI gene, coding for a major cellobiohydrolase (CBHI) of Aspergillus aculeatus, was cloned and sequenced. The gene consists of 1620-bp and encodes a protein containing 540 amino acids with a calculated molecular mass of 56,723 Da. CBHI, composed of an N-terminal catalytic domain belonging to family 7 of the glycosyl hydrolases, and a C-terminal cellulose-binding domain (CBD) belonging to family I of the CBDs, showed high similarity with other fungal CBHIs, especially with that of Penicillium janthinellum. The cbhI gene transcription start points in A. aculeatus were defined by primer extension, and the putative promoter sequence was analyzed. This sequence was found to be closely related to the consensus sequences of various fungal genes. Transcription analysis by ribonuclease protection assay revealed that the cbhI gene is induced by low-molecular-weight cellooligosaccharide and repressed by glucose. The results emphasize the possibility that in the A. aculeatus cellulase system, cellobiose is the true inducer and the role of the cbhI gene lies within the cascade regulating cellulase induction.
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.
l-Lyxose was prepared from ribitol by a new method comprising a potent microbial oxidation reaction to convert ribitol to l-ribulose, epimerization of the l-ribulose to l-xylulose, and isomerization of the l-xylulose to produce l-lyxose. The complete transformation of ribitol to l-ribulose was achieved using washed cells of Acetobacter aceti IFO 3281 at high substrate concentrations ranging from 5–20%. The l-ribulose produced was then used as the substrate for the production of l-lyxose using immobilized l-rhamnose isomerase (l-RI) of Pseudomonas sp. strain LL172 and immobilized d-tagatose 3-epimerase (d-TE) of recombinant Escherichia coli JM 105. At equilibrium, the yield of l-lyxose from l-ribulose was determined to be about 60%, and the product could be isolated easily from the reaction mixture after degradation of ketoses using Pseudomonas sp. 172a. Following various product purification steps, about 5.0 g l-lyxose crystals were recovered from 10.0 g ribitol in a flask reaction. The crystallized product was finally identified by HPLC, IR spectrum, NMR, and optical rotation measurements.
The variation in the colony size distribution with time of microalga Botryococcus braunii, which produces extracellular polysaccharides as well as high levels of liquid hydrocarbon, was measured in bubble column photobioreactors. The effect of average light intensity on the size of colonies was examined by fixing films impervious to light on the column wall and measuring the light intensity within the photobioreactors. The colony size distribution of this alga was found to fit the log-normal distribution. Under identical hydrodynamic conditions, the average colony size shifted to an equilibrium size, which was determined depending on the average light intensity within the photobioreactors.
α-l-Arabinofuranosidase from Streptomyces diastatochromogenes 065 released only the terminal arabinose of arabinoxylo-oligosaccharides. The enzyme hydrolyzed methyl arabinofuranobiosides to arabinose and methyl arabinofuranoside in the order of (1→2)>(1→3)>(1→5)-linkages. The enzyme preferentially hydrolyzed the (1→3)-linkage over the (1→5)-linkage of methyl arabinofuranotrioside.
Dark fermentation in the marine green alga, Chlorococcum littorale, was investigated with emphasis on ethanol production. Under dark anaerobic conditions, 27% of cellular starch was consumed within 24 h at 25°C, the cellular starch decomposition being accelerated at higher temperatures. Ethanol, acetate, hydrogen and carbon dioxide were obtained as fermentation products. The maximum productivity of ethanol was 450 μmol/g-dry wt. at 30°C. The fermentation pathway for cellular starch was proposed from the yields of the end-products and the determined enzyme activities. Ethanol was formed from pyruvate by pyruvate decarboxylase and alcohol dehydrogenase. the change in fermentation pattern that varied with cell concentration in the reaction vials suggested that the hydrogen partial pressure affected the consumption mode of reducing equivalents under dark fermentation. Ethanol productivity was improved by adding methyl viologen, while hydrogen production decreased.
The constitutive l-aminoacylase, which is used for optical resolution of dl-α-aminosuberic acid (dl-Asu), has been purified and characterized from Pseudomonas maltophila B1. The crude enzyme showed a specific activity of 0.062 units/mg for N-acetyl(Ac)-l-Asu. This value is very high compared with those from Aspergillus melleus, porcine kidney, and Bacillus stearothermophilus. Molecular masses of 108 kDa for the native enzyme and 50 kDa for the subunit were determined, indicating a dimer. The enzyme activity was optimal at pH 8.0 and at 55°C. The enzyme hydrolyzed N-acyl derivatives of various neutral l-amino acids and acidic l-amino acids, l-glutamate and l-Asu. The enzyme also had dipeptidase activity. The Km values for N-Ac-l-alanine and N-Ac-dl-Asu were determined at 2.32 and 12.7 mM, respectively. The apoenzyme was activated using Zn2+, Ca2+, and Co2+. Glyoxylate, dl-lactate, phenylboronic acid (PBA), butaneboronic acid (BBA), diethylpyrocar-bonate (DEP), and phenylglyoxal (PGO) inhibited enzyme activity.
The EST2 gene, encoding an isoamyl acetate-hydrolyzing esterase, was disrupted in a diploid strain of Saccharomyces cerevisiae UT-1 (MATa/MATα ura3/ura3 trp1/trp1 EST2/EST2), which is derived from the industrial sake yeast Kyokai no. 701 (strain K-701), by using two disruption plasmids (pDest2U, est2::URA3; and pDest2T, est2::TRP1) sequentially. Genomic Southern blot analysis revealed that both loci of the EST2 gene on the chromosome of strain UT-1 were disrupted. The resultant mutants were named UTUT-1 and UTUT-2 (a/MATα ura3/ura3 trp1/trp1 est2::URA3/est2::TRP1). Deficiency in Est2p esterase was also confirmed by activity staining of the gel after native-polyacrylamide gel electrophoresis of cell extracts of the two mutant strains. Small scale sake brewing was carried out using these sake yeasts and the strains they were derived from, and their brewing properties were compared. The fermentation profiles of the four strains (strains K-701, UT-1, UTUT-1, and UTUT-2) were largely similar. The components of the resulting sake were also similar except for the acetate ester concentration, although strains UTUT-1 and UTUT-2 produced approximately 2-times more isoamyl acetate than the wild type K-701. These resuts strongly suggest that the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in the sake mash. Strains UTUT-1 and UTUT-2, deficient in Est2p esterase, are suitable for sake brewing.
A 7-aminocephalosporanic acid (7-ACA) deacetylating enzyme was purified to homogeneity from Rhodotorula glutinis 38B1, whose resting cells have been previously reported as useful for the conversion of 7-ACA derivatives [Sakai et al., Appl. Environ. Microbiol., 62, 2667–2672, 1996]. The purified enzyme was a dimer comprised of identical subunits with a molecular mass of 82 kDa. The purified enzyme used cephalosporin C and several 7-ACA derivatives with low Km and high kcat values as substrates, as well as some acetyl esters with relatively long-chain alcohols. Based on this substrate specificity, the enzyme is classified as cephalosporin-C deacetylase (EC 3.1.1.41). The enzyme was most active at 35°C and pH 5.5, and was inhibited by several serine enzyme inhibitors. The purified enzyme was glycosylated on the addition of an asparagine-linked “hybrid type” oligosaccharide, and most of the enzyme activity was found in the purified cell wall fraction. The enzyme localization and kinetic properties explain the high efficiency of 7-ACA deacetylation in a resting-cell reaction.
The effects of electric current on the fermentation characteristics of yeast were investigated. When 10 mA direct current (DC) or 100 mA alternating current (AC) was applied to the culture broth, significant increases in cell growth and alcohol production rates occurred. The contents of higher alcohols, esters and organic acids in the culture broths to which AC and DC were applied, were different from those in the control culture (no current application). Several compounds such as acetaldehyde and acetic acid, were formed from ethanol as a result of electrode reaction.
A non-self-transmissible multiple-copy plasmid, pEC3, isolated from the phytopathogenic bacterium, Erwinia carotovora subsp. carotovora, can be mobilized with the help of a self-transmissible plasmid. When donors carrying a cya or a crp mutation were used, the mobilization efficiencies of pEC3 were markedly decreased. The covalently closed circular conformation of plasmid DNA containing both oriT and mob genes of pEC3 was found to be relaxed after the addition of cyclic AMP (cAMP). The relaxed DNA contained a specific single-strand DNA nick within the oriT region. RNA transcription of the mob operon was also increased by the addition of cAMP. These results indicate that the expression of the mob operon of pEC3 is positively regulated by cAMP and that the mob gene products are involved in nicking within the oriT region.
The plasmid-encoded arsenical resistance operon of plasmid pKW301 confers a high level resistance to arsenicals and antimonials on cells of Escherichia coli. Although it has been reported that ArsR of pKW301, a homologue of negative regulatory proteins, was previously shown to exhibit only weak homology to other ArsR proteins, it was identified as a DNA-binding protein by its behavior in gel mobility shift assays. In this study, ArsR exhibited a specific affinity for the ars promoter region. Gel mobility shift assay in the presence of sodium arsenite and potassium antimonial tartrate indicated that these compounds were inducers of expression of the ars operon. The specific binding site, as determined by DNase I footprint analysis, spans nucleotides 28 and 31 of the coding and noncoding strand, respectively, in the ars promoter region. ArsD, a second putative repressor protein encoded by arsD, was also identified as a DNA-binding protein by gel mobility shift assay.
