Efficient Biocatalytic Synthesis of (R)‐Pantolactone

Abstract

Screening for stereoselective cyanohydrin synthesis in 96-well plates was employed in the development of an efficient, pH-stable hydroxynitrile lyase for the conversion of sterically hindered aliphatic aldehydes. Site-saturation mutagenesis (SSM) resulted in a powerful catalyst for the stereoselective conversion of hydroxypivalaldehyde and pivalaldehyde to their corresponding (R)-cyanohydrins (ee > 97%) which are used as chiral building blocks (e.g., for pantothenic acid production). Furthermore, redesigning the PaHNL5 gene and improving its expression by Pichia pastoris with the help of a new P AOX1 promoter variant and the helper protein PDI (protein disulfide isomerase) led to elevated amounts of todays most efficient biocatalyst for vitamin B 5 synthesis.

Immobilization of Prunus amygdalus Hydroxynitrile Lyase on Celite

“…As reported earlier [14,15,17], in this case, a slow reaction catalyzed by the buffer washed Celite R-633 tightly packed in a teabag was observed, approximately 5% in 5 h. This implies that the desired enantiopurities of ee > 97% can only be obtained if the PaHNL catalyzed reaction clearly outperforms the background reaction. Careful examination of the literature reveals that similar background reactions have been reported for Celite before [14,15,17] and that the best enantiopurity (ee ~ 97, albeit at only 37% conversion) was achieved when a high enzyme loading of 0.86 U PaHNL per mg of Celite was utilized [13]. In the other studies, the lower enzyme loadings were used because higher loadings yielded no advantage [14].…”

“…It is a natural material and exists in countless variations [18]. Unfortunately, the exact Celite type employed for the previous PaHNL immobilization studies was not described [11][12][13][14]17]. In this study, Celite R-633 was utilized to ensure comparability with other HNL immobilization studies that described the nature of the carrier [15,16].…”

“…Indeed, Celite is broadly used for the immobilization of many enzymes and successful applications have been described [7][8][9][10]. Earlier studies describe many different HNLs on Celite, normally with good success both for stability as well as enantioselectivity [3,[11][12][13][14][15][16]. PaHNL on Celite, however, seems to be the exception.…”

“…PaHNL on Celite, however, seems to be the exception. This FAD-containing HNL was immobilized on Celite for the production of pantolactone and even acid resistant mutants did not yield the desirable enantioselectivity of ee > 97% [11][12][13]. This is surprising as a study from 1990 demonstrated excellent stability and also enantioselectivity for PaHNL on Celite in mandelonitrile synthesis [14].…”

The microscale flocculation test (MFT)—A high-throughput technique for optimizing separation performance

“…It was considered ideal because it can mix all the wells of the microplate to a sufficient degree such that the polymer flocculants can penetrate into high solids-content digestate suspensions. Previous studies have used the same tumble stirrer for microplate studies that screened yeast transformants for their biological activity [23], measured partition coefficients in organic-aqueous mixtures [24], and developed ternary phase diagrams for therapeutic drug candidates [25].…”