A novel convenient procedure for extractive work‐up of whole‐cell biotransformations using de‐emulsifying hydrolases

Abstract

Extractive work-up of whole-cell biotransformations generally suffers from the formation of stable gels and slimes upon addition of the organic solvent to the cell suspension and the cell-free solution, respectively. This problem has been overcome by enzymatic lysis of emulsifying agents present in the medium through addition of hydrolases. Of these agents, proteases have exhibited the most powerful de-emulsifying activity. Enzyme treatment of cell-free culture media of Saccharomyces cerevisiae with pronase E drastically reduced phase separation time (t(p)) from 1 week to 30 min without significantly affecting product integrity. Yeast cell suspensions were de-emulsified best with protease N-01, where phase separation was complete after 1 h. As was exemplified with cell-free culture media of Lactobacillus kefir, wherein addition of pronase E or protease N-01 reduced t(p) from 1 week to 2 h each, this practical, ready-to-use method is appropriate for both fungal and bacterial biocatalysts.

Microbial De-emulsification: A Highly Efficient Procedure for the Extractive Workup of Whole-Cell Biotransformations

“…In case microbial methods are not applicable, enzymatic de-emulsification with hydrolases offer a powerful alternative, yet with potentially prolonged t p . , The use of high solvent/water ratios during extraction involves large amounts of solvents which need to be handled, recovered, and disposed of while the risk of emulsion formation is still there.…”

“…It was therefore a major breakthrough when bioemulsifiers responsible for gel formation during extractive work-ups were identified . Finally substantial progress had been made with the invention of hydrolases as de-emulsifying agents. , They act by cleaving emulsifying biomolecules, whereupon these lose their amphiphilic properties.…”

“…The gel and slime formation effect is caused by bioemulsifiers, surface-active substances, secreted by the microorganisms into the medium. , They are classified into (i) glycolipids, (ii) lipopeptides and lipoproteins, (iii) glycoproteins, and (iv) fatty acids, neutral lipids, and phospholipids. − The strategy behind enzymatic treatment of bioemulsifiers is to cleave the amphiphilic backbone into the hydrophobic and the hydrophilic structural unit. For these purposes protease treatment has furnished the best results thus far, but also glycosylases gave good phase separation times ( t p ), and fatty acids precipitated after lipase treatment …”

Effects of caffeine on stereoselectivities of high cell density biotransformations of cyclic β-keto esters with Saccharomyces cerevisiae

“…After incubation of the combined aqueous phases with 75 U Bacillus subtilis protease for 18 h (allowing phase separation according to the method described in ref. 36), the product was obtained through extraction (3×) with tertiary-butyl-methyl-ether (TBME). The combined organic phases were dried over MgSO 4 , filtered, and concentrated in vacuo.…”