Journal of Fermentation and Bioengineering最新文献

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 biomass concentration in a bubble column reactor was estimated by analyzing the conductivity change during high-density culture of Catharanthus roseus hairy roots. The accumulated biomass in the reactor was estimated from the amount of nutrients consumed, which were present in the liquid medium. Liquid medium, consisting of water and nutrients, was incorporated into the biomass, in proportion to the growth of hairy roots, consequently the ratio of the volume of liquid medium to the volume of the biomass decreased continuously. To evaluate nutrient consumption, the change in biomass volume in the reactor was introduced as a new parameter for biomass estimation. After 45 d of culture, the final concentration of hairy roots was 46.5 g dry wt./l, of which the biomass volume is about 40% of the total culture volume of the reactor. A conventional conductivity method, in which the volume change of the liquid medium was not considered, resulted in a deviation (55.76 g dry wt./l) of about 20% from the actual biomass. This procedure yielded a value of 47.1 g dry wt./l of hairy roots, which is a nearly accurate estimation of the biomass concentration. This result suggests that the volume reduction of liquid medium should be considered for biomass estimation and process control for high-density plant cell cultures.

Fungal growth inhibition by ethanol was compared with that caused by five other agents of water stress (at 25, 40 and 42.5°C), using Aspergillus oryzae. Ethanol, KCl, glycerol, glucose, sorbitol, and polyethylene glycol 400 were incorporated into media at concentrations corresponding to water activity (a_w) values in the range 1 to 0.75. Generally, as temperature increased there was a decrease in the a_w value at which optimum growth occurred. The a_w limit for growth on KCl, glycerol, glucose, sorbitol, or polyethylene glycol 400 media was about 0.85, regardless of temperature. However, the a_w limit for growth on ethanol media varied between 0.97 and 0.99 a_w and was temperature-dependent. Water stress accounted for up to 31, 18 and 6% of growth inhibition by ethanol at 25, 40, and 42.5°C, respectively. For media containing ethanol, the decrease in growth rate per unit of a_w reduction was greater as temperature increased. However, ethanol-induced water stress remained constant regardless of temperature, suggesting that other inhibitory effects of ethanol are closely temperature-dependent. Water stress may account for considerably more than 30% of growth inhibition by ethanol in cells that remain metabolically active at higher ethanol concentrations.

Peptide ligands which bound to a model monomeric protein, bovine pancreatic ribonuclease A, could be isolated from a constrained random hexapeptide phage library. Selection was successful in a low ionic strength buffer (10 mM sodium phosphate, pH 6.0), whereas it failed in TBS (50 mM Tris-Cl, 150 mM NaCl, pH 7.5). Two of the displayed amino acid sequences from among the clones isolated were AEGACEQLDYNC and AEGACLWHDQLC. Electrostatic interaction appeared to play an important role in the binding because these phages could not bind to RNase A at a high ionic strength. The results suggest that selection in low ionic strength buffers could make possible the isolation of peptide ligands against proteins of interest which do not originally interact with another peptide or protein.

Aeration of the head space in a shaken ceramic membrane flask (SCM flask) capped with a cotton plug was found to be essential for increasing cell concentration and viability of aerobic microorganisms. Ventilation through the cotton plug cap was insufficient for satisfying the oxygen demand of aerobically growing cells in the SCM flask. Dissolved oxygen concentration (DO) in the culture supernatant of Escherichia coli in batch culture using the SCM flask dropped to nearly 0 ppm when the cell concentration reached 1 g/l, while CO₂ concentration in the head space of the SCM flask increased quickly to nearly 20% owing to accumulation of the evolved CO₂. In contrast, the oxygen transfer coefficient in a SCM flask shaken at 230 rpm was as high as 220 h⁻¹, compared to that in a stirred type jar fermentor. Consequently, aeration of the head space of the SCM flask was found to be sufficient for supplying oxygen by vigorous waves created in the culture broth due to the reciprocal shaking action. Using the SCM flask, E. coli cell mass reached 84 g/l in 40 h with aeration of the head space with oxygen-enriched gas. The SCM flask with aeration to the head space enabled an increase in the concentration and productivity of viable cells to be readily achieved by continuously replenishing the culture supernatant with oxygen in conjunction with the removal of the evolved CO₂.

A Chlamydomonas sp. strain, which excretes glycerol as the major carbon compound, was isolated and designated as strain W-80. The glycerol production was much enhanced by a high concentration of CO₂, and the final concentration of the product was measured at levels up to 24.9 mM (2.29 g/l).