Immobilized horse liver alcohol dehydrogenase as an on-line high-performance liquid chromatographic enzyme reactor for stereoselective synthesis

Abstract

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.

Silica-immobilized enzyme reactors; application to cholinesterase-inhibition studies

“…Immobilized enzyme reactors (IMERs) have found application in catalysis and have also been used with a wide variety of receptor proteins for substrate interaction and inhibition studies [1][2][3][4][5][6][7]. The main advantages of immobilized enzyme systems are stability and reusability.…”

Immobilized enzyme reactors based upon the flavoenzymes monoamine oxidase A and B

“…IMERs have been created using non-cofactor dependent hydrolases such as ␣-chymotrypsin [12], trypsin [12] and lipase [13], and have been developed using co-factor dependent enzymes such as the NAD/NADH dependent horse liver alcohol dehydrogenase [14], d-glyceraldehyde-3-phosphate dehydrogenase [15] and cytochrome P450s [16], the ATP dependent glutamine synthetase [17], and transferases such as phenylethanolamine N-methyltransferase [18] and UDP-glucuronyltransferase [19]. These IMERs have been placed in standard high performance liquid chromatographic systems and used to carryout online synthesis [13,14,16,20,21] as well as standard Michaelis-Menten enzyme kinetic studies for the quantitative determination of enzyme kinetic constants such a K m and V max [12,15,[17][18][19][20][21][22][23]. These systems can also be used to identify specific inhibitors, to provide information regarding the mode of inhibition and to calculate the K i of the inhibitor using both zonal chromatography [12,15] and frontal chromatography techniques [22].…”

Imobilização de enzimas em suportes cromatográficos: uma ferramenta na busca por substâncias bioativas

“…75 A integração da pré-reação enzimática e etapas de separação do produto são de interesse na análise de metabólitos em matrizes complexas, como urina 73,76 e tecidos biológicos, 77 no clean up de interferentes presentes em matrizes complexas, 78 na síntese enantiosseletiva on line e na purificação de moléculas quirais. 25,26 Todas, áreas de interesse da indústria farmacêutica.…”