Thermodynamically Controlled Synthesis of Amide Bonds Catalyzed by Highly Organic Solvent-Resistant Penicillin Acylase Derivatives

Abstract

A study of various direct condensations between different amines, having very high pK values, and unmodified acyl donors has been performed. This has been possible by the use of a very stable PGA derivative. First, it has been found that the higher the cosolvent concentration, the higher the pK of the acyl donor and thus the higher the yield. Therefore, these high concentrations of cosolvents seem to be a requisite for certain enzymatic condensations. Using ethanolamine and 2-hydroxy-2-phenylethyl-amine as nucleophiles and phenyl acetic acid as the acyl donor, the increase in the diglyme concentration from 50 to 90% (v/v) permitted improvement of not only the yield (reaching values higher than 99% in both cases) but also the reaction rates (by 360- or 3-fold, respectively). However, even when using PGA preparations stabilized by multipoint covalent attachment, it was not possible to obtain these results by inactivation of the enzyme derivative. Thus, in the protection of the octylamine with phenylacetic acid in 90% diglyme, the enzymatic activity was more than 20-fold higher using the hydrophilized derivative than the glyoxyl PGA, which allowed us to obtain a yield higher than 99%. Thus, the use of hydrophilized derivatives that are very stable even in the presence of high concentrations of organic solvents opens new opportunities in the use of PGA in organic chemistry.

Improved Stabilization of Genetically Modified Penicillin G Acylase in the Presence of Organic Cosolvents by Co‐ Immobilization of the Enzyme with Polyethyleneimine

“…These derivatives may be used in many reactions where stability in organic solvents is a limiting parameter. [14,22,23,25,26].…”

Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System

“…The stabilization effect was subsequently enhanced by adding successive layers of the polymers and including sulfate dextran to increase the "saline" effect of the modification [253,254]. These biocatalysts, displaying this "hyperstabilization" effect versus the deleterious influence of organic solvents, could be efficiently used in several applications, such as the hydrolysis of penicillin G in the presence of methyl isobutyl ketone (used to extract penicillin G from the fermentation medium) without a significant decrease in enzyme activity/stability [255], the production of amides using a thermodynamically-controlled synthesis in media containing a high percentage of cosolvent [256], or the resolution of racemic phenylacetamides in the above-mentioned media [257].…”

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

“…Second the immobilized-stabilized PGA was coated with a layer of very hydrophilic polymers (simulating a polymeric salt around the enzyme) that greatly reduced the presence of organic solvent in the enzyme environment by a partition phenomenon. Thus, by reducing the cause of the enzyme inactivation and by reducing the effects of the organic solvents, different biocatalysts of PGA specially designed to be used in the presence of organic media have been developed using different supports [67][68][69][70] or even coaggregation of PGA and polymers to give crosslinked enzyme aggregates (CLEAs) [71][72][73]. These biocatalysts may be used in the presence of high concentrations of organic solvents.…”

“…4). The glyoxyl-PGA coated with hydrophilic polymers can be used in the presence of 90-95% of diglyme or tetraglyme, with good activity retention after some weeks of use [66][67][68][69][70]. Other alternative is the co-aggregation of PGA and polymers to yield CLEAs with high stability in organ in the presence of organic solvents [71][72][73].…”