Stable monomeric mutases

A thermostable dimer was therefore considered as an alternative starting point for the design of a stable monomeric mutase. A large number of EcCM sequence homo-logues, some from thermophilic organisms, are known. For example, the hyperther-mophilic archaeon Methanococcus jannaschii produces a chorismate mutase (MjCM) that is 25 °C more stable than EcCM [37]. Despite only 21 % sequence identity, six prominent residues that line the active site are strictly conserved and the two enzymes have comparable activities. Since the hydrophobic core of MjCM is very similar to that of EcCM, interactions distant from the dimer interface must be responsible for its additional stability. These same interactions were expected to stabilize the desired monomer. 

In contrast to the selection experiments described above which were carried out on solid media, the library was grown in liquid culture lacking tyrosine and phenylalanine. This allows a direct competition for resources and amplification of the small 

The combination of selection ( 100-fold enrichment) and screening (1 in 26) shows that fewer than 0.05 % of the possible turn sequences are capable of yielding well-behaved, monomeric proteins. This result again contradicts the simple expectation 

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