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  Section: Molecular Biology of Plant Pathways » Enzyme Engineering
 
 
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Identifying Appropriate Starting Enzyme(S)

 
     
 

The first step in any enzyme engineering project is to choose a source or arental enzyme(s). Because sequence space is vast and mostly devoid of function, selecting the most appropriate starting point for a desired activity is critical. The parental enzyme should be the closest activity available to the desired enzyme because this minimizes the sequence space that needs to be traversed in order to achieve the desired property (Fig. 2.3). For any particular biotransformation, an ideal starting point would be an enzyme that performs the desired activity as a side reaction. For example, a galactosidase can also perform a fructosidase reaction albeit very inefficiently (Zhang et al., 1997). Enzymes to be used for reengineering projects can be identified from the biochemical literature and genes can be isolated from the many publicly funded seed and culture collections. An alternate, and particularly appealing, strategy for identifying starting enzymes is to screen samples from multiple environments for the desired enzymatic activity (Gray et al., 2003). This can be achieved by isolating total DNA from a particular environment and creating an expression library that is then screened for the desired activity. This circumvents the classical microbiological route of identification of an organism capable of performing a specific biotransformation, followed by protein purification/gene isolation of the corresponding activity. The approach has advantages in that many organisms from a particular environment are screened simultaneously, even ones for which culture conditions have not been developed. A disadvantage of this approach is that it may fail because genetic control elements from one organism may not be functional in the expression host organism. Also, multicomponent activities may be difficult to isolate in this manner if one or more of the components is unsuited to heterologous expression.
 
     
 
 
     



     
 
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