Aspartase activity, stabilization

A new system for the enzymatic production of L-aspartate was proposed and started in the 1990s. In this system, resting intact cells of coryneform bacteria were used without immobilization and with an ultrafiltration membrane. This bacterial strain possesses high maleate isomerase and aspartase activities thorough transformation of their genes. The plasmids introduced were stabilized and the cells were reused many times without any loss of activity and lysis [17]. 

The aspartase activity and the operational stability of immobilized E. coZi cells with K-carrageenan were compared with that of immobilized one with polyacrylamide. As shown in Table 2,... 

HARDENING TREATMENT FOR STABILIZATION OF ASPARTASE ACTIVITY OF IMMOBILIZED EichiXLchlo. COtC... 

L-aspartic acid ammonia lyase, or aspartase (E.C. 4.3.1.1) is used on a commercial scale by Kyowa Hakko, Mitsubishi, Tanabe and DSM to produce L-aspartic acid, which is used as a building block for the sweetener Aspartame, as a general acidulant and as a chiral building block for synthesis of active ingrediants[1]. The reaction is performed with enzyme preparations from E. coli, Brevibacterium jlavum or other coryneform bacteria either as permeabilized whole cells or as isolated, immobilized enzymes. The process is carried out under an excess of ammonia to drive the reaction equilibrium from fumaric acid (1) in the direction of L-aspartic acid (l-2) (see Scheme 12.6-1) and results in a product of excellent quality (over 99.9% e.e.) at a yield of practically 100%. The process is carried out on a multi-thousand ton scale by the diverse producers of L-aspartic acid. Site directed mutagenesis of aspartase from E. coli by introduction of a Cys430Trp mutation has resulted in significant activation and stabilization of the enzyme P1. 

This reaction has been carried out by batch procedure, which has disadvantages for Industrial purpose. Thus, we studied the continuous production of L-aspartlc acid using a column packed with immobilized aspartase. As the aspartase is an Intracellular enzyme, it was necessary to extract the enzyme from microbial cells before Immobilization. Extracted Intracellular enzyme is generally unstable, and most of the Immobilization methods we tried resulted in low activity and poor yield. Although entrapment into polyacrylamide gel lattice gave relatively active immobilized aspartase, its operational stability was not sufficient,... 

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