Bacillus thermoproteolyticus, thermolysin

Bacillus subtilis, proteases from, 28 327, 328 Bacillus thermoproteolyticus, thermolysin from, 28 326... 

The metalloprotease thermolysin, obtained from Bacillus thermoproteolyticus, a strain of B. stearothermophilus, is used as a crude preparation in an aqueous medium. The enzyme is recovered from the reaction mixture by ultrafiltration with a yield of >95%. 

Figure 10-3. The environment of the four structural calcium ions in the enzyme thermolysin isolated from Bacillus thermoproteolyticus. All of the calcium ions are associated with oxygen donor ligands.

Figure 10-5. The environment of the metal in a series of zinc metalloproteins. The proteins are (a) human carbonic anhydrase II, (b) thermolysin from Bacillus thermoproteolyticus, and (c) bovine pancreas carboxypeptidase. Each of these enzymes is, essentially, hydrolytic.

Thermolysin, originally derived from Bacillus thermoproteolyticus, can be categorized as a neutral metalloprotease. The optimum conditions for this enzyme are pH 7.0 and 60-80 °C. Thermolysin also requires catalytically active zinc,and calcium for thermostability (Whitaker, 1994). When these conditions are met, heat-stable thermolysin specifically hydrolyzes peptide bonds involving isoleucine, leucine, valine, and phenylalanine (Adler-Nissen, 1986). It has been extensively utilized in the production of antihypertensive peptides which will be discussed later in this review. 

Thermolysin family Bacillus thermoproteolyticus ILND HiSa 3 HiSa 19 Glrw H2O ... 

Thermolysine,a protease from Bacillus thermoproteolyticus, effects selective peptide bond hydrolysis on the amino side of hydrophobic amino add residues (Leu, He, Val, Phe), thus enabling removal of a supporting peptide ester sequence from the protected target fragment. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

Another example of a protease-catalyzed commercial process, which in this case uses the enzyme in a synthetic mode, is the completely regio- and stereoselective production of the low-caloric sweetener aspartame developed by DSM-TOSOH [15] (Fig. 7.9). Aspartame is a dipeptide consisting of the amino acids phenylalanine and aspartic acid, which are coupled by the enzyme thermolysin from Bacillus thermoproteolyticus. For an efficient coupling, relatively high temperatures are required and the amount of water in the system must be kept low to drive the reaction in the desired direction. Thermolysin, which is a metallo-endoprotease, meets these two requirements. It is thermostable, and it works in an organic solvent, which is required to keep the water activity low. In practice, however, organic solvents were not necessary, since the product aspartame forms an insoluble complex with unreacted D-Phe-OMe, which crystallizes out of the aqueous medium. 

The thermal stability of thermolysin, a peptidase from the thermo-phillic bacterium Bacillus thermoproteolyticus, is apparently conferred by calcium bound to the enzyme (194), which has a molecular weight of 35,000 (195), and binds one mole of zinc (195) and three moles of calcium per mole of protein (196). Thermolysin contains no non-protein constituents (197) and consists of a single peptide chain containing neither cysteine nor cystine (197). Thermolysin has a high content of aspartic... 

Thermolysin. Bacillus thermoproteolyticus neu -tral proteinase, E.C. 3.4.24.4. Proteolytic enzyme of mol wt 37,500 that hydrolyzes protein bonds on the JV-terminal side of hydrophobic amino acid residues. Contains a zinc atom essentia] for activity and four Ca2+ ioos essentia] for thermal and conformational stability. Isolo from Bacillus thermopro-leolyticus S. Endo, J. Ferment. Technol. 40, 346 (1962). Properties and amino acid composition Y. Ohta ei of., J. Biol. Chem. 241, 5919 (1966). Site of enzymatic hydrolysis Y. Ohta, Y. Ogura, J. Biochem. 58, 607 (1965). Substrate specificity studies H. Matsubara ei al.. Biochem. Biophys. Res. Commun. 21, 242 (1965) 24, 427 (1966) K. Morihara, H. Tsuzuki, Biochim. Biophys. Acta 118, 215 (1966). Stability studies Y. Ohta, J. Biol. Chem. 242, 509 (1967). Inhibition studies H. Matsubara et al., Biochem. Biophys. Res. Commun. 34, 719 (1969) J. Murphy et al., Arch. Biochem. Biophys. 202, 405 (1980). Purification H. Matsubara,... 

Production of the artificial low-calorie sweetener aspartame from Z-L-aspartate and D/L-phenylalanine methylester by peptide bond formation with immobilized thermolysin from Bacillus thermoproteolyticus (Tosoh Corp., Ajinomoto, Toyo-Soda, DSM, annual world production approx. 10000 tons). Aspartame is about 200 times as sweet as sucrose, and is used in drinks such as Coca Cola and Pepsi Cola Light. In contrast to the older chemical process, the enzymatic process can - due to the L-selectivity of the enzyme - use the cheaper D/L-phenylalanine methylester instead of the pure L-form. The enzymatic process (Fig. 15) yields a-aspartame exclusively, whereas the chemical route yields a mixture of a-aspartame and bitter-tasting (5-aspartame, thus requiring an additional separation step. 

Thermolysin is a potent endopeptidase isolated from the organism Bacillus thermoproteolyticus. In contrast to the other zinc metalloenzymes discussed in this review, thermolysin has not yet been subjected to rigorous chemical and kinetic investigation. Therefore, although the complete amino acid sequence is known [184), and although Matthews and co-workers [43 6) have carried the X-ray structural analysis to 2.3 A resolution, it is neither possible to discuss the thermolysin catalytic mechanism nor to propose a role for zinc ion in thermolysin catalysis. Therefore, the following discussion is restricted to a brief review of the current status of the physical and chemical properties of thermolysin, and to a... 

Thermolysin (TEN EC 3.4.24.28), a thermostable bacterial protease isolated from Bacillus thermoproteolyticus, has been studied as the prototype of zinc-metallopeptidases at a time where no crystal structure was available for this class of proteases [122]. Crystallographic analysis of a number of TLN/inhibitor complexes has allowed an understanding of the binding mode of these inhibitors and allowed the mechanism of action of this protease to be determined [122]. These seminal studies have greatly inspired the development of NEP inhibitors, given the close stmctural relationship between TEN and NEP [123]. To examine further the structural relationships between these two peptidases, various phosphinic peptides were prepared. One of these compounds (58, Table 1) exhibits a Ki value of 26 nM toward thermolysin and 22 nM toward NEP [124]. 

Thermolysin (EC 3.4.24.4) a heat-stable, zinc- and calcium-containing neutral protease, M, 37,500, from Bacillus thermoproteolyticus, with a substrate specificity similar to that of Subtilisin (see). After one hour at 80 °C, T. still has 50% original activity. This high heat stability of T. is attributed to the large number of hydrophobic regions and the presence of four bound calcium ions, which serve in place of disulfide bridges (T. contains no disulfide bridges) to maintain the compact shape of the molecule. T. is neither a thiol nor a serine enzyme. 

In another approach, the C-2 position of taxol is the site of manipulation. Thus, a two-step enzymatic acylation in organic solvents has been employed to synthesize water-soluble taxol (paclitaxd) derivatives. In the first step, taxol 241 is reacted with the bifunctional acylating reagent 282, as catalyzed by thermolysin (from Bacillus thermoproteolyticus rokko), to give the activated acyl derivative 283. This is then used as a complex acyl donor in the second step [204]. 

In place of selective chemical degradation, enzymic digestion using a thermostable bacterial protease, thermolysin, obtained from Bacillus thermoproteolyticus Rokko (Endo, 1962 Matsubara et aL, 1965), was successfully applied to clupeine YI [Ando and Suzuki, 1967 Suzuki and Ando, 1972 (2)]. 

An increase in thermostability of a neutral protease from Bacillus stearothermophilus (NprT) was achieved from a rational approach by comparing its sequence with the thermostable thermolysin from B. thermoproteolyticus[33]. The enzymes were 85% identical, while the thermostability of NprT at 75 C was significantly lower than that... 

Formation of the peptide bond is catalyzed by thermolysin, a neutral zinc protease (Fig. 4-7). The enzyme employed was found in the bacterial strain Bacillus proteo-licus/thermoproteolyticus in the Rokko Hot Spring in Japan. The enzyme is stable up to temperatures of 60 °C. It is extracted and used in a form soluble in water. In this process the amino acids phenylalanine and asp>artic acid are coupled by the enz5Tne. The process, including the main steps, is shown schematically in Fig. 4-7. 

Several groups of workers have studied proteases from moderately thermophilie Bacillus strains, and thermolysin, from B. thermoproteolyticus, has been the subject of intensive research [279]. Heinen and Heinen [280] presented one of the earliest reports on the production of an extracellular protease from a Bacillus strain growing at 72 °C. 

---