Screening of various glycosidases for the synthesis of octyl glucoside.

Thirteen glycosidases of microbial origin and almond beta-glycosidase were assayed in octanol/DMF (80:20, v/v), using a combination of hydrolysis, transglycosylation, and condensation reactions, in order to assess their potential for the production of alkyl glucosides. The two mesophile enzymes were highly impaired by the organic media. Three of the 11 thermophile enzymes gave interesting results in the hydrolysis and transglycosylation reactions, but they were highly inhibited by glucose. This made their use in a condensation reaction less interesting than the use of almond beta-glucosidase, which has a lower activity but shows less inhibition by the glucose.

Enzymatic hydrolysis of nitriles by an engineered nitrile hydratase (papain Gln19Glu) in aqueous-organic media.

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.

Microbial resolution of baclofen by a new isolate of Streptomyces halstedii.

We attempted to isolate microorganisms able to metabolize (S)-baclofen while showing little or no activity with (R)-baclofen. A culture collection of 218 microbial isolates was established from several soil samples using various approaches. Subsequent chiral HPLC analysis revealed that only two cultures showed promise. Initial shake flask experiments demonstrated a correlation between culture growth and (S)-baclofen metabolism. (S)-baclofen was metabolized much more rapidly than (R)-baclofen. The best culture, identified as Streptomyces halstedii, was used to scale up the process in small-scale fed-batch fermentations using pulse addition of racemic baclofen to yield extracellular fractions showing enantiomeric excess (e.e.) values of greater than 90% in favour of the (R)-isomer. Resting cells also were able to carry out the wanted reaction but initial attempts using cell free extracts were negative. It is speculated that the initial attack on (S)-baclofen may be catalyzed by a mono- or dioxygenase.