Effect of water and enzyme concentration on thermolysin-catalyzed solid-to-solid peptide synthesis

Abstract

We have studied a thermolysin‐catalyzed solid‐to‐solid dipeptide synthesis using equimolar amounts of Z‐Gln‐OH and H‐Leu‐NH2 as model substrates. The high substrate concentrations make this an effective alternative to enzymatic peptide synthesis in organic solvents. Water content was varied in the range of 0 to 600 mL water per mol substrate and enzyme concentration in the range of 0.5 to 10 g/mol of substrates. High yields around 80% conversion and initial rates from 5 to 20 mmol s−1 kg−1 were achieved. The initial rate increases 10‐fold on reducing the water content, to reach a pronounced optimum at 40 mL water per mol substrate. Below this, the rate falls to much lower values in a system with no added water, and to zero in a rigorously dried system. This behavior is discussed in terms of two factors: At higher water contents the system is mass transfer limited (as shown by varying enzyme content), and the diffusion distances required vary. At low water levels, effects reflect the stimulation of the enzymatic activity by water. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:68–72, 1998.

Dynamic Combinatorial Chemistry

“…However, water is essential for proteases; mixed aqueous media usually reduce enzyme activity considerably, and insufficiently hydrated catalyst can even be completely inactive. [80][81][82][83] The major limitations of enzyme-mediated transamidation are the high substrate selectivity of the catalyst that may bias the reaction and the fact that the equilibrium typically lies on the side of the starting materials (acids and amines). This latter drawback can be eliminated by using an organic cosolvent in the presence of a hydrophilic solid phase.…”

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…After 15 min, the dry enzyme powder was added straight from the bottle. The complete reaction mixture was then mixed and separated into individual samples as previously described (Erbeldinger et al, 1998a). Each reaction mixture contained (unless otherwise stated) 2 mmol of both substrates, 200 L water, and 10 mg thermolysin or 300 mg Thermoase P160.…”

Chemo-enzymatic synthesis of arginine-based gemini surfactants

“…These results were notably lower than those obtained at 0.15 mmol (70% yield and 96% conversion). Erbeldinger et al (1998) reported that in solid-to-solid biotransformations there is an optimal buffer or solvent content for each reaction. On this basis, we considered the possibility that the optimal mL ethanol/mol substrate ratio could be different, depending on the amount of substrate.…”