Kozo Yamamoto

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 .

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

SummaryContinuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

SummaryFor the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate β-decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.

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.

Characteristics and applications of N-(ω-aminohexyl--aspartic acid sepharose as an affinity adsorbent☆

1. 1. The affinity adsorbent, N-(ω-aminohexyl)-l-aspartic acid-Sepharose 6B (AHA-Sepharose), was prepared by covalently linking AHA to Sepharose 6B previously activated with CNBr. 2. 2. The enzymes relating to the metabolism of l-aspartic acid showed a group-specific adsorption to AHA-Sepharose column. Asparaginase, aspartase, aspartate-β-decarboxylase and modified asparaginase with tetranitromethane were adsorbed to AHA Sepharose column and eluted by increasing the ionic strength with NaCl. Asparaginase and aspartase were eluted with the respective substrate or product of lower ionic strength, whereas holo- and apo-enzymes of aspartate β-decarboxylase and the modified asparaginase could not be eluted with the respective substrate or product of lower ionic strength. 3. 3. Fumarase was not adsorbed to this column. 4. 4. By the affinity chromatography employing the AHA-Sepharose column, asparaginase from Proteus vulgaris was purified easily and in a high yield.

[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.

Applications of Immobilized Enzymes and Immobilized Microbial Cells for L-Amino Acid Production

Recently, utilization of L-amino acids for medicines, food and animal feed has been rapidly developing, and the economical production of optically active amino acids and the related compounds has been needed.

[44] Immobilizedl-aspartic acid

Publisher Summary An affinity adsorbent prepared by immobilizing a ligand of high specificity may be expected to have high adsorption specificity. On the other hand, an affinity adsorbent prepared with a ligand of lower specificity may be useful for “group-specific” adsorption. This chapter describes a method of immobilization of L-aspartic acid to agarose with interposed hexamethylenediamine as a spacer and the application of the immobilized L-aspartic acid (L-Asp) to the purification of several enzymes for which this ligand has affinity. A group of enzymes related to the metabolism of L-aspartic acid is adsorbed onto the immobilized L-Asp column. These enzymes include both asparaginases from E. coli and Proteus vulgaris, aspartase and apo- and holoenzymes of aspartate β-decarboxylase and can be eluted by increasing the ionic strength of the eluent with NaCI.