Synthesis of Aspartame by Thermolysin: An X-ray Structural Study

Abstract

Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the artificial sweetener aspartame, a multiton peptide synthesis catalyzed by the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a crystal, rationalize the reaction's substrate preferences and reveal an unexpected form of substrate inhibition that explains its sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages.

Protein–ligand complex structure from serial femtosecond crystallography using soaked thermolysin microcrystals and comparison with structures from synchrotron radiation

“…S1). The present structures and four previously reported structures (PDB entries 3qgo, 3qh1, 3qh5 and 4ow3; Birrane et al, 2014;Hattne et al, 2014) share the same crystal packing; all of the crystals belong to the same space group, P6 1 22, with similar unit-cell parameters and contain a thermolysin monomer in the asymmetric unit. The final models of the present thermolysin structures with well defined electron densities contained entire amino-acid residues 1-316, a functional zinc ion at the active site and four structural calcium ions (Fig.…”

“…After the batch crystallization, the microcrystals were collected by centrifugation at 3000g, suspended in 500 ml harvest solution comprising 20% PEG 2000 MME, 0.1 M MES-NaOH pH 6.5, 5 mM CaCl 2 and filtered through a mesh with a 50 mm pore size. To remove a copurified ligand (Birrane et al, 2014), the microcrystal suspension was incubated at room temperature for 24 h (backsoaking). The back-soaked microcrystals were collected by centrifugation and resuspended in the harvest solution for the unliganded oil-SFX form, whereas they were resuspended in a soaking solution comprising 20% PEG 2000 MME, 60 mM N-carbobenzoxy-l-aspartic acid (ZA), 0.1 M MES-NaOH pH 6.5, 5 mM CaCl 2 for the liganded oil/water-SFX forms.…”

“…The details of cellulose as a water-based crystal carrier for SFX will be published elsewhere. Conventional macrocrystals soaked with ligand were prepared as reported by Birrane et al (2014) with slight modifications. Crystallization was performed by the hanging-drop vapour-diffusion method at 293 K using protein solution at a concentration of 25 mg ml À1 and a reservoir solution comprising 10% PEG 2000 MME, 0.1 M MES-NaOH pH 6.5, 5 mM CaCl 2 .…”

Highly efficient enzymatic synthesis of Z-aspartame in aqueous medium via in situ product removal

“…All of these three compounds were dissolved in buffer with different pH values (4)(5)(6)(7)(8) at 50 • C to form oversaturated solutions. Z-APM was dissolved in 0-50% DMSO solutions at pH 6.0.…”

“…More and more attention has been focused on the enzymatic synthesis of Z-APM from N-carbobenzoxy-l-Asp (Z-l-Asp) and l-Phenylalanine methyl ester (l-PheOMe) due to the enantiomer selectivity of protease. Thermolysin, an efficient biocatalyst, was utilized to catalyze the synthesis of Z-APM from Z-l-Asp and lPheOMe [7]. Nevertheless, thermolysin was relatively unstable and less active in the presence of organic solvents or in a low pH environment [8].…”