Joji Takahashi

Gluconic acid production at high concentrations by Aspergillus niger immobilized on a nonwoven fabric

Abstract Batch production of gluconic acid in the presence of a high concentration of glucose was investigated using free and immobilized mycelia of Aspergillus niger IAM 2094 with the aim of achieving repeatable constant production. Accumulation of 300 g/l of gluconate with a productivity of 60 g/l·h was achievable by intermittent addition of powdered glucose using filamentous-form mycelia in the presence of 150 ppm dissolved oxygen. However, this productivity became unattainable after a few repetitions. The use of pellet-form mycelia, in place of filamentous ones, did not prove effective either. However, when the mycelia were immobilized on a nonwoven fabric, a sustained level (220 g/l) of gluconate production was reproducible. Immobilized mycelia grown in a gas phase (air or oxygen) had a much longer durability than mycelia grown in a liquid culture medium. The gluconate-producing activity of immobilized mycelia grown in the presence of oxygen was much higher than that of mycelia grown in air. At 150 ppm dissolved oxygen, 220 g/l of gluconate was repeatedly produced 14 times at a constant production rate in a period of about 1,000 h.

Enzymatic Hydrolysis of Fats at High Substrate Concentrations in Biphasic Organic-Aqueous Systems

Abstract The hydrolysis of palm oil and beef tallow by lipase has been studied for practical applications in a biphasis isooactane-aqueous system using a high substrate concentration. The effective lipase concentration for the hydrolysis was found to be about 120 IU per g of substrate. The addition of twenty percent isooctane brought about the most rapid reaction and produced the highest percentage of hydrolysis. For both palm oil and beef tallow, a percentage of hydrolysis higher than 98% was achieved in the 20% isooctane system at a higher concentration of 50%. However, when the substrate concentration was higher than 50%, the final value of hydrolysis decreased as the concentration of the substrate increased. Utilization of recycled lipase was attempted using an ultrafiltration membrane reactor. Approximately 60

Ethanol stripping by pervaporation using porous PTFE membrane

% of the lipase activity was recoverable after each reaction.

Utilization of powdered pig bone as a support for immobilization of lipase

Abstract In a shell-and-tube type of module containing either porous or nonporous tubular membranes, the sweeping action of a flow inert gas in the shell side was used to strip ethanol from an aqueous ethanol solution flowing countercurrently in the tube side. A calculation of the overall mass transfer coefficient, KG, of the membrane used was made for this system. In ethanol stripping tests using a module containing polytetrafluoreethylene (PTFE) tubular membranes, the KG was found to be more affected by the liquid flow rate than the gas flow rate. Moreover, the gas side mass transfer coefficient, kG, was estimated to be about 5×10−5 mol/cm2·s·atm. The liquid side mass transfer coefficient, kL, on the other hand, was found to increase linearly with the linear velocity of the aqueous solution. Also, at an average solution temperature range of 21 to 32°C, no significant change in the KG was observed. Comparison of the KG of different tubular membranes revealed that the KG of the PTFE membrane was higher than that of polypropylene or silicone membranes under the given experimental conditions.

Production of Intracellular and Extracellular Protein from n-Butane by Pseudomonas butanovora sp. nov.

Abstract Pig bone was examined for its suitability as a support material for lipase immobilization. It was observed that pig bone (PB) particles dispersed readily in both polar and nonpolar solvents, and lipase was easily adsorbed. In particular lipase adsorbed on olive oil-soaked pig bone (OPB) particles exhibited a higher hydrolytic activity than that in lipase adsorbed on a selection of other representative supports, regardless of removing the presoaked olive oil from the particles after immobilization of lipase. The optimum pH and temperature for hydrolytic activity of OPB-adsorbed lipase were the same as those for free lipase, although thermal resistance was increased by immobilization. When OPB-adsorbed lipase was used for repeated batch reactions of olive oil hydrolysis, an activity of more than 80% of the initial activity of each run could he retained after 46 h reaction. The results suggest that PB is an excellent support material.

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

Publisher Summary The isolate of the strain capable of growing on ethane, propane, and n-butane, designated as Pseudomonas butanovora sp. nov., is remarkable for its rate of growth on n-butane and its ability to produce extracellular protein as well as intracellular protein. Pseudomonas butanovora is able to utilize a variety of organic compounds for the growth, but not sugars. Among the culture conditions tested for the accumulation of extracellular protein, the incubation temperature is found to have a considerable influence, whereas the pH of the culture system and the various nitrogen sources do not. The extracellular protein is rich in acidic amino acids, such as glutamic and aspartic acids, and the contents of these amino acids are about 1.5 times as much as those in the intracellular proteins. In contrast, the intracellular protein is rich in arginine, an alkaline amino acid, and the content is over twice as much as that in the extracellular protein.

Effect of substrate presoaking treatment of support materials on the activity of immobilized glucoamylase

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.

CIRCULATION TIMES OF VARIOUS FLUIDS IN STIRRED FERMENTORS

The activity of immobilized glucoamylase was remarkably increased by presoaking treatment of the supports in soluble starch solution. Pig bone (PB) particles-100 showed the largest substrate presoaking effect among some representative support materials, increasing the activity of immobilized glucoamylase by 10 times. The improvement in the activity was due to the increase in the specific activity of immobilized proteins. In order to get sufficient substrate presoaking effect, a rapid crosslinking treatment of the enzyme and the substrate-presoaked support was required. The glucoamylase immobilized on PB sheet was very stable and gave a high starch hydrolysis of DE95 (dextrose equivalent) for about 1 month in continuous process.

Substrate presoaking effect on immobilization of protease on pig bone particles.

ABSTRACT Circulation times for various fluids were measured in stirred vessels with and without aeration, and the effects of rheological properties of fluids and geometrical factors of vessels on the distribution of circulation times were investigated. Distributions of circulation times under various conditions were approximately fitted with distribution curves of the logarithmic normal probability, and the distribution curves were less affected by rheological properties of fluids with increasing vessel size. In the turbulent regime in geometrically similar vessels of various sizes, the operating condition required to achieve the same value of mean circulation time is that the values of P v /D 2 must be equal to one another. In the case of different liquid depths in a given vessel, the operating condition for obtaining the same value of mean circulation time is that the values of P v /H 2 must be equal. The distribution curves for fully turbulent fluids in a vessel having a constant diameter and liquid depth were identical regardless of impeller size. As an example of application of this work, the rates of leakage of intracellular substances from suspended mold mycelia are discussed by taking into account the basic findings for circulation time.

Kinetics of Enzymatic Hydrolysis of Lipids in Biphasic Organic-Aqueous Systems

Abstract When pig bone (PB) particles and other support materials were presoaked in casein solution and crosslinked with glutaraldehyde, the activity of immobilized protease, was two or more times greater than those of the proteases immobilized on non-treated supports. Chitopearl was an exception. The highest activity, 25000 U/g-support, was obtained with the presoaked PB particles. The effect of presoaking the supports in the substrate solution on the immobilized protease was also observed with other protein substrates. The effect of the presoaking treatment increased with an increase in the molecular weight of the protein. The increase in the activity of the immobilized enzyme was due to increases in both the amount of adsorbed protease and specific activity. Furthermore, the stability of protease immobilized on PB was remarkably improved by transforming the PB particles into a sheet.