Lets say I want to make an enzyme that catalyzes the conversion of A into B, where neither A nor B affect the cell's viability in any significant way. I know that there is an enzyme "X" which catalyzes a very similar reaction, but will not work on substrate A. I decide to create a library of mutants where 5 amino acids in the active site are randomized to the twenty amino acids. I now have 3.2x10^6 different enzymes, and I want to identify a specific one which catalyzes the reaction A -> most efficiently.
What screening methods are currently used to perform a search like this one? Are there any "in vivo" directed evolution approaches?
That is very difficult to answer, because screening methods depend largely on the properties of the substrate / product. For instance, if product B (but not substrate A) can be further metabolized by the cell, you can grow cells on substrate A as only carbon source. Only those cells able to transform the substrate will grow.
If your substrate has an analog that forms a colored product, you can monitor enzyme activity that way.
Some techniques such as Infrared or Raman spectroscopy allow detection of specific compounds in situ, and they can be adapted for high-throughput screening:
http://www.ncbi.nlm.nih.gov/pubmed/11391018
http://www.ncbi.nlm.nih.gov/pubmed/15300259
Or, if the product affects the growth of other organisms, an appropriate screening method can be applied:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397223/
To help you we should know the organism you want to express your proteins. As Gustavo Acevedo-Hernandez suggested also the biochemical/toxicological features of your substrate/product should be indicated.
Screening the substrate with all enzymes with bioinformatics methods may prove useful here. If you can nab out the enzymes with best binding affinity for your substrate it will be easy to proceed further.
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