Continuous Synthesis of N-(Benzyloxycarbonyl)-L-Aspartyl-L-Phenylalanine Methyl Ester with Immobilized Thermolysin in an Organic Solvent

Nakanishi, Koji; Kamikubo, Tadashi; Matsuno, Ryuichi

doi:10.1038/nbt0585-459

“…In 1979, Isowa et al 60 showed that the chiral aspartame precursor Z-L-Asp-L-Phe-OMe could be produced from the thermolysin-promoted condensation of Z-L-Asp and L-Phe-OMe. The results were confirmed by Wayne and Fruton 61 and Nakanishi et al 62,63 The rate of reaction is, however, slow. St. Clair and Navia 64 and Persichetti et al 65 stable in mixtures of water with various organic solvents such as dimethylformamide, tetrahydrofuran, or acetone.…”

Section: U S E O F T H E R M O L Y S I N I N P E P T I D E C O U P L supporting

confidence: 57%

Thermolysin

Matthews

¹

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

0

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Enzyme: thermolysin (TLN) (E.C.3.4.24.4); a metalloprotease with a single zinc ion in the active site. The enzyme also binds four calcium ions that contribute to thermostability.Thermolysin is an endopeptidase of molecular weight 34 600 Da isolated from the thermophile Bacillus thermoproteolyticus. 1 It preferentially cleaves peptides with a bulky, hydrophobic residue (phenylalanine or leucine) in the R 1 position. 2-4 As noted below, thermolysin is a member of a very large family and the different family members display a broad range of specificities. O C C U R R E N C EThermolysin is the prototypical member of the socalled 'M4' or 'thermolysin' family of secreted eubacterial endopeptidases defined by Rawlings and Barrett. 5 All members of the family contain the HEXXH sequence motif in which the two histidines coordinate the zinc ion and the 3D Structure Ribbon drawing showing the overall structure of thermolysin. The view is toward the active site with the zinc ion shown in light blue. A bound inhibitor (ZF P LA) is shown in dark blue. Four calcium ions are shown as green spheres. The amino-terminal domain is located in the top half of the figure. It includes an extended β-sheet that terminates across the 'top' of the substrate binding site and contributes to substrate binding. The carboxy-terminal domain, located in the bottom half of the figure, is predominantly α-helical. [Figure, based on PDB file 4TMH, courtesy of Doug Juers was prepared with MOLSCRIPT 17 and RASTER3D. 18 ]

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“…In 1979, Isowa et al 60 showed that the chiral aspartame precursor Z-L-Asp-L-Phe-OMe could be produced from the thermolysin-promoted condensation of Z-L-Asp and L-Phe-OMe. The results were confirmed by Wayne and Fruton 61 and Nakanishi et al 62,63 The rate of reaction is, however, slow. St. Clair and Navia 64 and Persichetti et al 65 have shown that cross-linked crystals of thermolysin are …”

supporting

confidence: 57%

Thermolysin

Matthews

¹

2004

Handbook of Metalloproteins

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Enzyme: thermolysin (TLN) (E.C.3.4.24.4); a metalloprotease with a single zinc ion in the active site. The enzyme also binds four calcium ions that contribute to thermostability.Thermolysin is an endopeptidase of molecular weight 34 600 Da isolated from the thermophile Bacillus thermoproteolyticus.1 It preferentially cleaves peptides with a bulky, hydrophobic residue (phenylalanine or leucine) in the R 1 position. [2][3][4] As noted below, thermolysin is a member of a very large family and the different family members display a broad range of specificities. O C C U R R E N C EThermolysin is the prototypical member of the socalled 'M4' or 'thermolysin' family of secreted eubacterial endopeptidases defined by Rawlings and Barrett. 5 All members of the family contain the HEXXH sequence motif in which the two histidines coordinate the zinc ion and the 3D Structure Ribbon drawing showing the overall structure of thermolysin. The view is toward the active site with the zinc ion shown in light blue. A bound inhibitor (ZF P LA) is shown in dark blue. Four calcium ions are shown as green spheres. The amino-terminal domain is located in the top half of the figure. It includes an extended β-sheet that terminates across the 'top' of the substrate binding site and contributes to substrate binding. The carboxy-terminal domain, located in the bottom half of the figure, is predominantly α-helical.[ Figure, based Thermolysin glutamic acid is critical for catalysis (see below). Family members include enzymes from the pathogens Legionella, Listeria, Pseudomonas, and Vibrio. 5 Proteins that are structurally and functionally related to thermolysin, and presumably evolved from a common precursor, extend well beyond the M4 family. Rawlings and Barrett 5 define the 'HEXXH + E Metallopeptidase Clan (MA).' This includes families M4 (thermolysin), M5 (mycolysin), M13 (neprilysin), M1 (membrane alanyl aminopeptidase), and M2 (peptidyl-dipeptidase A). All members of these families are clearly related to thermolysin. Rawlings and Barrett 5 also define the 'HEXXH + H Metallopeptidase Clan (MB).' This includes families M12 (astacin and reprolysin), M10 (interstitial collagenase, serralysin, and matrixins), M11 (autolysin), and M7 (Streptomyces small neutral protease). Members of the latter clan have also been defined as the metzincins 6 and have been shown to have significant structural relationship to thermolysin. B I O L O G I C A L F U N C T I O NThermolysin and its immediate family members are secreted eubacterial peptidases. As such, the presumed biological function of the enzyme is to degrade ambient peptides that can be utilized by the host. More distantly related relatives are involved in a broad range of digestive activities. They also are involved in the processing of biologically active peptides. Perhaps the best-known example is that of the angiotensin-converting enzyme for which the structure has recently been determined. 7 A M I N O A C I D S E Q U E N C E I N F O R M A T I O NThe amino acid sequence of thermolysin was deter...

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“…As a model system, we chose the commercially interesting enzymatic condensation (cf. [24]) of benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) with L-phenylalanine methyl ester (L-Phe-OMe) to the sweetener aspartame [25]. As seen in Figure 3, the route to aspartame formation using thermolysin is a straightforward process, starting from Z-L-aspartic acid and L-phenylalanine methyl ester, leading in a single condensation step to Z-~-aspartame.…”

Section: Equilibrium Shiftingmentioning

confidence: 99%