Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides

Abstract

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from alpha-cyclodextrin (alpha-CD) and n-dodecyl-(1,4)-beta-maltopyranoside (C(12)G(2)beta). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132 min at 60 degrees C and 18 min at 70 degrees C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4 degrees C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-beta-maltooctaoside (C(12)G(8)beta) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) immobilised on Eupergit C had good operational stability at 60 and 70 degrees C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C(12)G(8)beta due to extensive disproportionation reactions, giving a broad product range. (C) 2011 Elsevier B.V. All rights reserved. (Less)