Peptide Synthesis of Aspartame Precursor Using Organic-Solvent-Stable PST-01 Protease in Monophasic Aqueous-Organic Solvent Systems

Abstract

The PST-01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water-soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Cbz-Asp-Phe-OMe) synthesis from N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and L-phenylalanine methyl ester (Phe-OMe) in the presence of water-soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz-Asp-Phe-OMe were attained by the PST-01 protease when 30 mM Cbz-Asp and 500 mM Phe-OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 degrees C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz-Asp-Phe-OMe using the PST-01 protease attained 83% in the presence of 50% (v/v) DMSO.

Nature Versus Nurture: Developing Enzymes That Function Under Extreme Conditions

“…The protease PST-01, from Pseudomonas aeruginosa, was one of the first reported solvent-tolerant proteases and can be overproduced from the native host to ∼80 mg/liter of culture (103). The wild-type protease is stable to many polar organic solvents, but its peptide synthesis rate is low (104). However, the activity (k cat ) was improved tenfold by a single amino acid mutation of Y114S, made by comparison with a homologous thermophilic metalloprotease, Thermolysin (105).…”

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

“…The highest yield 82.2% in aqueous medium was obtained from 100 mM Z-l-Asp and 500 mM l-PheOMe at pH 6.0. The efficient synthesis of Z-APM from 30 mM Z-l-Asp and 500 mM l-PheOMe in 50% DMSO at pH 7.0 with the highest yield of 83.0% was reported by Ogino group using protease PST-01, which has the same amino acid sequence with protease PT121 [12]. The distinct effect of DMSO on Z-APM synthesis was probably Table 1 shows that the molar solubility of Z-APM was 8.2 × 10 −2 M at pH 7.0.…”

“…However, production of Z-APM is difficult to scale up due to the complicated purification of the product, as well as the cost of the reaction medium [11]. Z-APM was also synthesized by Pseudomonas aeruginosa protease PST-01, with an 83.0% yield, in the presence of 50% (v/v) dimethyl sulfoxide (DMSO) at pH 7.0 using 30 mM Z-l-Asp and 500 mM l-PheOMe [12].…”

Proteases as Powerful Catalysts for Organic Synthesis

“…The use of organic solvents, usually required for performing enzymatic peptide synthesis [301], imposes additional restrictions on process design and engineering, and contradicts the clean technology concept [245,302], so major efforts are being directed toward their replacement by greener systems, among which ionic liquids [82,198] and solid or semisolid reaction media are prominent [196,[303][304][305]. Recently, Meng et al [291] reported the enzymatic synthesis of the CCK pentapeptide (CCK-5) where the coupling was conducted in aqueous buffer and no hazardous chemicals were necessary.…”