Table 1.
UniKP and EF-UniKP assisted enzyme mining and evolution
| Entry | Category | TALs | kcat (s−1) | Km (mM) | kcat / Km (s−1⋅mM−1) |
|---|---|---|---|---|---|
| 1 | UniKP: Enzyme mining | RgTAL | 117.8 | 0.36 | 327.2 |
| HiTAL | NA | NA | NA | ||
| PcTAL | 66.57 | 0.17 | 391.6 | ||
| SsTAL | 58.87 | 0.17 | 346.3 | ||
| AsTAL | 448.8 | 0.54 | 831.1 | ||
| IsTAL | 119.7 | 0.57 | 210.0 | ||
| 2 | UniKP: Enzyme evolution | RgTAL | 117.8 | 0.36 | 327.2 |
| MT-613P | NA | NA | NA | ||
| MT-603P | 52.57 | 1.15 | 45.71 | ||
| MT-366H | 40.31 | 0.48 | 83.98 | ||
| MT-366W | 31.02 | 0.85 | 36.49 | ||
| MT-587V | 162.9 | 0.33 | 493.6 | ||
| MT-10Y | 884.4 | 0.82 | 1079 | ||
| MT-337C | 96.81 | 6.48 | 14.94 | ||
| MT-668S | 53.60 | 3.85 | 13.92 | ||
| MT-489T | 816.8 | 0.71 | 1150 | ||
| MT-337D | 34.77 | 0.85 | 40.91 | ||
| 3 | EF-UniKP | TALclu | 16.54 | 0.05 | 330.80 |
| TrTAL | 34.54 | 0.04 | 863.50 | ||
| HiTAL | 76.00 | 0.21 | 361.90 | ||
| LeTAL | 33.85 | 0.07 | 483.57 | ||
| PpTAL | 28.09 | 0.04 | 702.25 | ||
| AaTAL | 25.24 | 0.03 | 841.33 |
Entry 1) The kinetic characteristics of wild-type Tyrosine ammonia lyase (RgTAL) from Rhodotorula glutinis and newly discovered TALs mined from non-redundant protein database by performing BLASTp. The top 5 sequences with the highest predicted kcat values by UniKP were selected for experimental validation, including HiTAL from Heterobasidion irregulare TC 32-1 (XP_009553370.1), PcTAL from Puccinia coronata f. sp. avenae (PLW06342.1), SsTAL from Sporidiobolus salmonicolor (CEQ38810.1), AsTAL from Armillaria solidipes (BK74450.1), IsTAL from Ilyonectria sp. MPI-CAGE-AT-0026 (KAH6995648.1). NA denotes the enzyme that was not soluble and showed no catalytic activity. Entry 2) The kinetic characteristics of RgTAL and mutants generated by UniKP. All variants of single-point mutations were generated for RgTAL, where each variant involved mutating an amino acid at a specific position to one of the other 19 canonical amino acids, which resulted in a total number of variants equal to the product of 19 and the length of the sequence (19*693 = 13,167). Through an in-silico screening of all 13,167 single-point mutations of RgTAL using UniKP, the top 5 mutants ranked by their predicted kcat or kcat / Km values were chosen from each screening (kcat or kcat / Km) for experimental validation. NA denotes the enzyme that was not soluble and showed no catalytic activity. And MT denotes the mutated form of RgTAL. Entry 3) The kinetic characteristics of wild-type Tyrosine ammonia lyase from Chryseobacterium luteum sp. nov (TALclu) and newly discovered TALs mined from non-redundant protein database by performing BLASTp. The top 5 sequences with the highest predicted kcat values by UniKP were selected for experimental validation, including TrTAL from Tephrocybe rancida (KAG6920185.1), HiTAL from Heterobasidion irregulare TC 32-1 (XP_009553370.1), LeTAL from Lentinula edodes (KAF8828722.1), PpTAL from Pleurotus pulmonarius (KAF4563271.1), AaTAL from Aspergillus arachidicola (KAE8337485.1). All the experiments were conducted under a pH of 9.5.