Thermolysin, stability

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

Tetrhedral intermediate, 172 Thermodynamic cycles, 186 Thermolysin, zinc as cofactor for, 204 Thrombin, 170 Torsional potential, 111 Transition states, 41-42,44, 45,46, 88, 90-92 in amide hydrolysis, 219-221 oxyanion hole and, 181 stabilization of, 181,181 carbonium ion, 154,155,156-161, 167-169 for gas-phase reactions, 43... 

Resistance to peptidases was also reported for the octapeptoid 6.106 when incubated with papain, chymotrypsin, or thermolysin [229], However, resistance to peptidases may not be synonymous with a long half-life in vivo, since many factors beside peptidases can be expected to contribute to the elimination of peptoids. An indirect indication of this effect can be found for antimicrobial peptoids and particularly compound CHIR29498 (6.107) [232], In mice infected with Staphyllococus aureus, this peptoid was less active when injected 2 h post-infection compared to 0 h or 0.5 h. The conclusion drawn by the authors was that the compound requires optimization for improved absorption or stability within the body. 

Thermolysin is a single-chain protease composed of 316 amino acids its amino acid sequence and three-dimensional structure (Colman et at, 1972) have been determined. The native protein has no disulfide bonds, but does contain both Ca2+ and Zn2+, which strongly stabilize the structure. 

Several proteins from different sources have been shown to maintain stability at high temperatures and NMR studies have been carried out in order to reveal their structures and/or to understand their activity. The most relevant references of a miscellany of thermostable proteins are reported in Table 3. Some of them such as bovine pancreatic trypsin inhibitor (BPTI), thermolysin and lysozyme have been widely studied as model systems in protein science. 

Thermolysin is a zinc-dependent proteinase (Mr = 34.6K) that requires Ca for optimal thermal stability. The thermolysin (3TLN) crystal structure contains four bound Ca ions. Two of these lie only 3.8 A... 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

Both the IA and IIA cations have important roles in the neutralization of negative charge on various organic polymers, and in the stabilization of certain structures. Thus K+ and Mg2+ stabilize ribosomes, while the binding of Ca2+ may stabilize enzymes against denaturation, such as thermolysin which functions at high temperatures. 

The ability of Ca2+ to crosslink groups and so stabilize proteins against thermal denaturation or hydrolytic attack is well illustrated by thermolysin, which involves four Ca2+ and a catalytic Zn2+. The function of the calcium is to stabilize the protein structure.404 Calcium also protects surface protein of the neural retina from tryptic cleavage.405... 

First and foremost, cross-linked enzyme crystals are crystals. Within the crystal lattice the concentration of protein approaches the theoretical limit. This is important to the process development chemist, who would much rather use a small quantity of a very active catalyst in a reactor than fill it with an immobilized enzyme. Typically an immobilized enzyme contains only 1-10% by weight enzyme, with the remaining carrier material simply occupying valuable reactor space. The crystallinity is absolutely required to achieve the stability exhibited by CLCs [8], Cross-linked soluble thermolysin and cross-linked precipitate of thermolysin are no more stable than the soluble enzyme. Crystals of proteins... 

Figure 3 Stability of thermolysin CLEC at elevated temperatures. In comparison, the half-life of soluble thermolysin is only 10 hr at 50°C.

Thermolysin is a zinc metallo enzyme which binds Ca2+ ions. It consists of a single polypeptide chain of M.W. 34600 containing 316 amino acid residues. Four Ca2+ ions per molecule give thermal stability to the enzyme. The crystal structure of both Ln3+ substituted and unsubstituted enzyme has been determined [54],... 

---