Continuous lipase-catalyzed production of pseudo-ceramides in a packed-bed bioreactor

Abstract

Ceramides are spingolipid compounds that are very attractive as active components in both the pharmaceutical and the cosmetic industries. In this study, the synthesis of ceramide analogs, the so-called pseudo-ceramides, was carried out using for the first time a two-step continuous enzymatic process with immobilized Candida antarctica lipase B (Novozym ® 435) in a packed-bed bioreactor. The first step involved the selective N-acylation of 3-amino-1,2-propanediol using stearic acid as the first acyl donor (i). This was followed by the selective O-acylation of the N-stearyl 3-amino-1,2-propanediol synthesized in the first step, with myristic acid as the second acyl donor, to produce a N,O-diacyl 3-amino-1,2propanediol-type pseudo-ceramide, namely 1-O-myristyl,3-N-stearyl 3-amino-1,2propanediol (ii). The process was first optimized by evaluating the influences of three factors: feed flow rate, quantity of biocatalyst and substrate concentration. Under optimal conditions an amide synthesis yield of 92% and a satisfying production rate of almost 3.15 mmol h-1

Semi-pilot scale-up of a continuous packed-bed bioreactor system developed for the lipase-catalyzed production of pseudo-ceramides

“…The continuous enzymatic synthesis of 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides catalyzed by immobilized C. antarctica lipase B (Novozym ® 435) was conducted in a semi-pilot scaled-up packed-bed bioreactor system (Scheme 1) in two steps, according to the process previously developed at the laboratory scale (Le Joubioux et al, 2014). The operating conditions applied were very similar from those optimized at this scale, except that a stainless steel 1.5 cm long column with a 5 cm inner diameter, packed with Novozym ® 435, was used, giving a biocatalyst amount of 10 g, more than 10-fold higher (Tab.…”

“…The packed-bed bioreactor system used for the continuous two-step enzymatic synthesis of 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides catalyzed by immobilized C. antarctica lipase B (Novozym ® 435) was adapted from the one that we previously developed at the laboratory scale (Le Joubioux et al, 2014). For each step, the reaction mixture (substrates and solvent) was first homogenized for 15 min at 55°C while stirring at 250 rpm.…”

“…500 ml of a methanol/chloroform (50:50 v/v) were added to each sample in order to homogenize the reaction medium at room temperature. Structural and quantitative analyses of the reaction products (amides and pseudo-ceramides) were then conducted on these samples using a LC/MS-ES system from Agilent (1100 LC/MSD Trap mass spectrometer VL), according to the methodology from Le Joubioux et al (2014). The amide products were also characterized by infrared (IR) spectroscopy after liquid-liquid extraction and drying.…”

“…In order to overcome these disadvantages, teams, ours included, focused on developing enzymatic syntheses of pseudo-ceramides through immobilized lipase-catalyzed acylation or transacylation reactions carried out in organic solvents (Bakke et al, 1998;Le Joubioux et al, 2014;. Indeed, using lipases (E.C.…”

“…Starting from this overview, we previously developed an efficient process for the continuous enzymatic production of 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides using a packed-bed bioreactor containing immobilized Candida antarctica lipase B (Novozym ® 435) (Le Joubioux et al, 2014). In order to control the chemoselectivity of the reaction, the process was divided into two steps (Scheme 1), for the optimization of the selective diacylation of 3-amino-1,2-propanediol 1 conducted in a tert-amyl alcohol/n-hexane mixture (50:50 v/v), starting from various fatty acids as acyl donors: lauric acid 2a, myristic acid 2b, stearic acid 2d and linoleic acid 2e.…”