Table 1. Mutated Cal-A libraries generated.
| Library name | Library type | Mutagenesis method | Theoretical library size | Variants screened per substrate | |
|---|---|---|---|---|---|
| 1 | Tyr93 | part 1 in mother vector | circular mutagenesis (NDT) | NS | NS |
| 2 | Tyr183 | part 1 in mother vector | circular mutagenesis (NDT) | NS | NS |
| 3 | Phe431 | part 3 in mother vector | circular mutagenesis (NDT) | NS | NS |
| 4 | Random | part 2 in mother vector | random mutagenesis | NS | NS |
| 5 a | Tyr93 | assembled gene in daughter vector | recombination: part 1* + WT parts 2,3 | 12 | 63 |
| 6 | Tyr183 | assembled gene in daughter vector | recombination: part 1* + WT parts 2,3 | 12 | 48 |
| 7 | Phe431 | assembled gene in daughter vector | recombination: part 3* + WT parts 1,2 | 144 | 48 |
| 8 | Tyr93/ Phe431 | assembled gene in daughter vector | recombination: parts 1*, 3* + WT 2 | 144 | 192 |
| 9 | Tyr183/ Phe431 | assembled gene in daughter vector | recombination: parts 1*, 3* + WT 2 | 144 | - b |
| 10 | Random | assembled gene in daughter vector | recombination: part 2* + WT parts 1,3 | >10180 | 384 |
*: mutated parts.
NS: non-screenable constructs.
a Library 5 was generated both in vector pET22b and in mother and daughter vectors. The library size and number of variants screened here refer to the library in pET22b.
b Upon screening library 6, it was recognized that the only substitutions at position 183 that maintain activity are the wild-type Tyr or Phe, hence library 9 was not screened.