Isao Takata

Immobilization of enzymes and microbial cells using carrageenan as matrix

Conditions for the gelation of k ‐carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k ‐Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water‐miscible organic solvents. By using this property of k ‐carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor‐made to various shapes such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.

Screening of matrix suitable for immobilization of microbial cells

To find a suitable matrix for immobilization of microbial cells, synthetic and natural polymers were screened. As a result,kappa-carrageenan,iota-carrageenan, furcellaran, sodium alginate, ethyl succinylated cellulose, succinylated zein, and 2-methyl-5-vinyl-pyridine-methylacrylate-methacrylic acid copolymer were studied. These polymers were induced to gel under mild conditions.Streptomyces phaeochromogenes cells having glucose isomerase activity were successfully immobilized in these polymer matrices. If a gelinducing reagent were added to a substrate solution, these gel matrices could be stabilized. The microbial cells did not leak out from the gel lattice. When these immobilized cells were treated with hardening reagents such as glutaraldehyde or tannins, the gel matrices were strengthened, and the glucose isomerase activity became stable for a long period even in the absence of gel-inducing reagents. Among these polymer matrices tested,kappa -carrageenan was most suitable for immobilization of microbial cells.

Immobilization of Brevibacterium flavum with carrageenan and its application for continuous production of l-malic acid

Abstract Extensive experiments were carried out to improve the productivity of l -malic acid from fumaric acid using Brevibacterium flavum immobilized with carrageenan. The most favourable preparation for the continuous production of l -malic acid was obtained when 16 g of B. flavum cells was entrapped in 100 ml 3.4% carrageenan gel. However, the immobilized cells produced an unwanted by-product, succinic acid. Treatment of the immobilized cells with 0.6% bile extract suppressed the side reaction and gave the highest operational stability of fumarase activity. By the immobilization of intact cells, the optimal temperature of the enzyme reaction shifted to 10°C higher, the optimal pH became broader, and the operational stability of fumarase activity increased. The effect of temperature on the stability of fumarase activity in the immobilized cell column was investigated under conditions of continuous enzyme reaction. The decay of fumarase activity during continuous enzyme reaction was expressed by an exponential relationship. The productivity of the immobilized B. flavum using carrageenan was as high as 5.2 times that of the conventional immobilized B. ammoniagenes using polyacrylamide .

Effect of a novel anti-rheumatic drug, TA-383, on type II collagen-induced arthritis

The effects of Ta-383 (0.016, 0.08, 0.4, 2 and 10 mg/kg) and anti-rheumatic drugs (lobenzarit 10 and 50 mg/kg, dexamethasone 0.25 mg/kg) were evaluated on type II collagen-induced arthritis in DBA/1J mice. The arthritis score was markedly suppressed in the groups treated with dexamethasone and TA-383. Serum anti-type II collagen IgG was significantly suppressed in the groups treated with dexamethasone and 0.4 mg/kg TA-383. Histopathological evaluation of the knee joints revealed suppression of the inflammatory changes in the groups treated with dexamethasone and TA-383. These findings suggest that the histopathological examination of the joints of the animal model is useful for the evaluation of anti-rheumatic drugs, and that TA-383 has suppressive effects on type II collagen-induced arthritis, an animal model for human rheumatoid arthritis.

Continuous production of L-malic acid by immobilized cells

Abstract l-Malic acid is used extensively in the pharmaceutical industry and as a food additive. It is now produced on an industrial scale by the enzymatic conversion of fumaric acid using immobilized cells of Brevibacterium flavum . Recent improvements to this system, especially the use of x -carrageenan supports, have resulted in a continuous process capable of yielding 30 tonnes of l-malic acid per month.

Screening of microorganisms having high fumarase activity and their immobilization with carrageenan

SummaryTo develop an efficient method for continuous production of L-malic acid from fumaric acid using immobilized microbial cells, screening of microorganisms having high fumarase activity was carried out and cultural conditions of selected microorganisms were investigated. As a result of screening microorganisms belonging to the genera Brevibacterium, Proteus, Pseudomonas, and Sarcina were found to produce fumarase in high levels. Among these microorganisms Brevibacterium ammoniagenes, B. flavum, Proteus vulgaris, and Pseudomonas fluorescens were further selected for their high fumarase levels in the cultivation on several media. These 4 microorganisms were entrapped into a k-carrageenan gel lattice, and the resultant immobilized B. flavum showed the highest fumarase activity and operational stability.Cultural conditions for the fumarase formation and the operational stability of fumarase activity of immobilized B. flavum are detailed. Productivity for L-malic acid using immobilized B. flavum with k-carrageenan was 2.3 fold of that using immobilized B. ammoniagenes with polyacrylamide.

Enhanced expression of interleukin 6 in rat and murine arthritis models.

Interleukin 6 (IL-6) is a multifunctional cytokine and plays an important role in host defense mechanisms. Enhanced production of IL-6 has been reported in polyclonal B-cell abnormalities and autoimmune diseases such as rheumatoid arthritis (RA). To investigate the role of IL-6 inflammatory joint diseases, serum IL-6 levels of three animal models of RA, namely type II collagen (CII)-induced murine, rat arthritis and adjuvant-induced rat arthritis, were monitored. In these models, serum IL-6 increased with the development of arthritis. Serum IL-6 was not elevated by immunization with a non-arthritogenic immunogen such as bovine type I collagen (CI) and bovine serum albumin (BSA) to DBA/1J mice. The serum IL-6 level was correlated well with the severity of adjuvant-induced arthritis. The elevated IL-6 in sera may be associated with the overproduction of IL-6 at the arthritis paws, because higher IL-6 activity was detected in the homogenates of arthritic paws as compared with the control paws. Synovial fibroblasts were isolated from the arthritis knee joints of DBA/1J mice. These cells expressed type I interleukin 1 (IL-1) receptor constitutively and produced large amounts of IL-6 in response to IL-1 in vitro. Enhanced production of IL-1 was also detected at the arthritis paws. These results suggest that the elevated IL-6 in sera may be associated with the overproduced IL-6 in response to the increased IL-1 at the arthritic joints. Serum IL-6 may be a useful parameter for monitoring disease activity.

[16] Immobilization of cells in carrageenan

Publisher Summary There are advantages and disadvantages to the immobilization methods of microbial cells, and the ideal general methods applicable to the immobilization of cells have not yet been developed. To develop versatile immobilization methods and techniques, this chapter discusses various immobilization methods. It describes immobilization by the induction of polymer gelation as the best approach, because in this case, an enzyme is not denatured during immobilization. As a result, several polymers that form gel lattice suitable for entrapping microbial cells have been found. Among the polymers tested, κ-carrageenan was the most suitable for the immobilization of microbial cells. The procedures for the immobilization of microbial cells with κ-carrageenan are also described. Advantages of the immobilization method using κ-carrageenan are that the immbolization can be performed under very mild conditions without the use of chemicals that destroy the enzyme activity of microbial cells. Another advantage of this method is that various shapes of immobilized cells can be easily tailor-made for particular application purposes.

[41] Production of l-malic acid by immobilized microbial cells

Publisher Summary This chapter describes the production of L-malic acid by immobilized microbial cells. As the immobilized cells have an activity that forms succinic acid as a by-product, succinic acid and unconverted fumaric acid accumulate in the reaction mixture. Although fumaric acid can be easily precipitated by acidifying the reaction mixture with hydrochloric acid, it is very difficult to separate succinic acid from L-malic acid. The key step to a successful process, therefore, is to prevent the formation of succinic acid. Recently, an industrially very advantageous method for carrying out the above fumarase reaction has been developed. That is, microbial cells having high fumarase activity are immobilized in polymer gel matrices, such as polyacrylamide and K-carrageenan. The immobilized cells are packed into a column, and a substrate solution (sodium fumarate) is continuously passed through the immobilized cell column. An effluent containing L-malate is obtained without contamination from impurities—such as microbial cells, components of cultural medium, and others.

Stabilization of fumarase activity ofBrevibacterium flavum cells by immobilization with k-carrageenan

Whole cells ofBrevibacterium flavum having fumarase activity were immobilized using K-carrageenan. The stabilities of fumarase activity in the immobilized cells against external factors, including heat, pH, organic solvents, and protein denaturing reagents, were compared with those of free cells and native enzyme. The stabilities of fumarase activity in immobillized cells against external factors were highest, and those of native enzyme were lowest. In the “gel-state,” K-carrageenan-immobilized cells showed a much higher stabilization effect for external factors than “sol-state” immobilized cells.