Masaaki Terashima

Production of Organic Acids and Amino Acids from Fish Meat by Sub-Critical Water Hydrolysis

Fish meat was easily liquefied by hydrolysis under subcritical conditions without oxidants, and aqueous phase and water‐insoluble phase containing oil and fat‐like solid were formed. Lactic acid found in the raw fish meat (about 0.03 g/g‐dry meat) was stable up to the reaction temperature 513 K (3.35 MPa). Pyroglutamic acid was produced with a yield of 0.095 kg/kg of dry meat by 30 min reaction at 553 K (6.42 MPa). Amino acids such as cystine, alanine, glycine, and leucine were produced in the temperature range 513–623 K with a maximum peak at 543 K. Amounts of cystine, alanine, glycine, and leucine produced in 5 min at 543 K (5.51 MPa) were 0.024, 0.013, 0.009, and 0.004 kg/kg of dry meat, respectively. The oil extracted with hexane contained useful fatty acids such as eicosapentanoic acid (EPA) and docosahexianoic acid (DHA). Thus, subcritical water hydrolysis would be an efficient process for recovering useful substances from organic waste such as fish waste discarded from fish market.

Expression and Purification of Functional Human α‐1‐Antitrypsin from Cultured Plant Cells

Human α‐1‐antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon‐optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice‐derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant‐derived AAT for treatments of various AAT‐deficient diseases are discussed.

Effect of osmotic pressure on human α1-antitrypsin production by plant cell culture

Abstract Human α1-antitrypsin (AAT) was produced by cell culture of genetically engineered rice cells. Although the previous study has shown that the C-terminal end of most of the recombinant AAT (rAAT) secreted into medium is processed, this work revealed that sulfhydryl protease released from disrupted cells degrades the secreted rAAT in the medium, and that the drastic change of the osmotic pressure by the medium exchange for the rAAT induction is the main cause of the cell disruption. By adjusting the osmotic pressure of the induction medium, the percentage of the active rAAT in the total rAAT increased from 12.8 to 57.4%.

Utilization of an Alternative Carbon Source for Efficient Production of Human α1-Antitrypsin by Genetically Engineered Rice Cell Culture

Human α1‐antitrypsin was produced by genetically engineered rice cells using promoter and signal peptide of a rice α‐amylase isozyme. Batch and continuous cultures were employed to investigate the effects of alternative carbon sources on the α1‐antitrypsin production. While this expression system is inducible by sugar depletion, we have found that the productivity of α1‐antitrypsin increased 2.4‐ to 3.4‐fold, compared with the control medium without carbon source, in medium containing an alternative carbon source, such as pyruvic acid and glyoxylic acid. The accumulated α1‐antitrypsin in the medium containing pyruvic acid reached 18.2−24.2 mg/g‐dry cell in 50−70 h by batch culture.

Effects of sugar concentration on recombinant human α1-antitrypsin production by genetically engineered rice cell

Productivity of recombinant human alpha(1)-antitrypsin (rAAT) with a genetically engineered rice cell using an inducible promoter has been studied by batch-wise and continuous production. A simple model explained the effect of proteases released from the disrupted cells on the rAAT degradation. Glucose concentration in the medium significantly affected the rAAT productivity in the continuous production, because the rAAT was induced by sugar depletion. When the fresh medium containing 5mM glucose was supplied to the continuous bioreactor, induction time was long and the productivity was low, indicating that the glucose concentration in the cells was high enough as to repress the promoter. When the glucose concentration in the fresh medium was reduced to 0.5mM, total amount of rAAT produced in 70h cultivation reached 6.7-7.6mg/g-dry cell, which was two times larger than the control medium without glucose.

Adsorption of Cadmium Ion and Gallium Ion to Immobilized Metallothionein Fusion Protein

A fusion protein made from maltose binding protein (pmal) and human metallothionein (MT) was expressed using E. coli. The purified recombinant protein (pmal‐MT) was immobilized on Chitopearl resin, and characteristics of pmal‐MT for metal binding were evaluated. As expected from the tertiary structure of metallothionein, the pmal‐MT ligand adsorbed 12.1 cadmium molecules per one molecule of the ligand at pH 5.2. The pmal‐MT ligand also bound 26.6 gallium molecules per one molecule of the ligand at pH 6.5. Neither cadmium ion nor gallium ion bound to a control protein bovine serum albumin (BSA). Adsorption isotherms for both ions were correlated by Langmuir‐type equations. Two types of binding sites have been elucidated on the basis of HSAB (hard and soft acid and base) theory. It was suggested that gallium ion specifically binds to amino acid residues containing oxygen and nitrogen atoms, while cadmium ion binds to specific binding sites formed by multiple cysteine residues. The pmal‐MT ligand bound these metals in the concentration range of 0.2–1.0 mM, and the bound metal ions could be eluted under relatively mild conditions (pH 2.0). The pmal‐MT Chitopearl resin was stable and could be used repeatedly without loss of binding activity. Thus, this new ligand would be useful for recovery of toxic heavy metals and/or valuable metal ions from various aqueous solutions.

Permeability of small hydrophilic solutes in small unilamellar vesicle (SUV)

Permeabilities of hydrophilic solutes with and without net charge in small unilamellar vesicles (SUV) were determined. Their permeabilities showed strong dependence on molecular weight. The permeabilities of net zero charged molecule (glucose and alanine) in SUV consisting of dioreoylphosphatidylcholine (DOPC) or soybean phosphatidylcholine (SoyPC) were larger than those in SUV consisting of dimyristoylphosphatidylcholine (DMPC). The permeabilities of net charged molecules (lysine and glutamate), on the other hand, in SUV consisting of DOPC or SoyPC were lower than those in SUV consisting of DMPC. These results suggest that the electrostatic potential barrier of lipid bilayer has significant effects on the permeabilities of hydrophilic solutes in the liposomes.

Utilization of antipeptide antibodies as affinity ligands in immunoaffinity purification

Anti-peptide antibodies against the C-terminal regions of chimeric alpha-amylase, recombinant CD2 and insulin B-chain were obtained by using peptides corresponding to the C-terminal regions as immunogens. These anti-peptide antibodies adsorbed the native proteins, as well as the antigen peptides. The proteins were purified to high purity using the anti-peptide antibodies as affinity ligands. These ligands could discriminate the target proteins having different C-terminal regions. The adsorbed proteins were specifically eluted by the eluents containing the antigen peptides.

CARBONIZATION OF ORGANIC WASTES USING SUPER-HEATED WATER VAPOR AND THEIR ADSORPTION PROPERTIES

Various organic wastes, such as waste wood chip, sake lees, used tealeaves and so on, were carbonized with the super-heated water vapor at 623 K. By the 30 90 minutes’ treatment, the organic wastes lost about 50 90 % of their original weight. The capability of gas adsorption has been evaluated. The surface areas determined by nitrogen adsorption for the carbonized materials were much smaller than that of the activated carbon Granular Shirasagi. The surface area determined by carbon dioxide adsorption, on the other hand, of the carbonated materials were almost the same order of magnitude to that of the activated carbons. These results show that the carbonized materials have micro-pores whose diameter is less than 50 nm. We have found that the waste wood and used tealeaves showed high adsorption capabilities for ammonia gas in low equilibrium pressure (< 13.3 kPa). The amount of the adsorbed ammonia for these carbonized materials were much higher that of the activated carbon Granular Shirasagi GS3 x 4/6. These results suggest that the carbonized materials from organic wastes could be utilized as adsorbents for ammonia

Mass Transfer Characteristics of a Perfusion-type gel Analyzed by Shallow Bed Method

Abstract Up-take curve of POROS HQ50 for protein depended on the flow rate of BSA solution, while that of Sepharose FF was independent of the flow rate. This result indicates that adsorption rate of the perfusion-type gel (POROS HQ50) is affected by the intra-particle convective flow. Intra-particle effective diffusivity of Sepharose FF increased with the increase in BSA concentration. Parallel diffusion model revealed that the pore diffusion was dominant in Sepharose FF. On the other hand, the intra- particle effective diffusivity in POROS HQ50 did not depend on BSA concentration. Surface diffusion was dominant in POROS HQ50, and the surface diffusivity depended on the flow rate due to the intra-particle convection.