Site-Directed Immobilization

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

A variety of approaches exist for stabilizing proteins, for example, chemical modification, immobilization, and site-directed mutagenesis [95,96], but these techniques are not within the scope of this chapter. The focus here will be on stabilization of proteins via formulation development. The principal formulation strategy is to stabilize the protein using clinically acceptable additives (excipients) or through the use of suitable pharmaceutical-processing technologies. 

D.J.O. Shannessy and W.L. Hoffman, Site-directed immobilization of glycoproteins on hydrazide-containing solid supports. Biotechnol. Appl. Biochem. 9, 488—496 (1987). 

Kurzawa et al. [74] used SECM in the characterization of enzyme microstructures that were obtained by a new non-manual immobilization technique based on the site-directed deposition of polyelectrolytes induced by a local pH-shift. This method has already been used to modify microscopy sensor regions [75]. 

The following new trends in enzymatic synthesis can be delineated the development of new enzymatic reactions enzyme immobilization and stabilization the use of organic solvents and two phase systems site-directed mutagenesis chemical modification of enzymes antibody catalysis catalysis by RNA and DNA de novo design ofbiocatalists employment of recombinant DNA for production of enzymes and use computational and combinatorial methods... 

A different approach of site-specific polymerization and thus immobilization of a biorecognition element was shown by several research groups by utilizing electropolymerization [74-76]. In combination with microelectrodes in microchannel systems, a site directed simple immobilization of the biorecognition element could be achieved. Patterns can also be created by... 

Favourable orientation of antibodies also appears possible on immobilized metal ions supports. Hale and Beidler [80] observed that an innate histidine-rich sequence located in the C-terminal portion of the Fc region, well-conserved in every antibody classes investigated from several species, binds strongly to the Co2+-IDA resin. Thus, antibodies bound on the supports are oriented with their combining site directed away from the resin and facilitate maximum antigen binding [81]. 

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. 

In this section, we describe how a dextran-immobilized 27-MHz quartz crystal microbalance (QCM) is appUed to detect whether the site-directed... 

Hydrazine activation can also be used as the basis for site-directed immobilization or directional immobilization [16]. Directional immobilization is the immobilization of the sensor detector molecules in a highly ordered and reproducible manner. For example, in antibody-based biosensors requiring a monolayer coating of antibody on the transducer, it is critical to sensor functionality and sensitivity that as many of the antibodies as possible are immobilized such that their antigen binding sites are directed outward and easily accessible by antigen (analyte). 

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