New stabilized FastPrep-CLEAs for sialic acid synthesis

作者：Inmaculada Garcia Garcia Maria; Sola Carvajal Agustin; Sanchez Carron Guiomar; Garcia Carmona Francisco; Sanchez Ferrer Alvaro^*

来源：Bioresource Technology, 2011, 102(10): 6186-6191.

DOI：10.1016/j.biortech.2011.02.026

• CLEAs
• Sialic acid
• N-acetylneuraminate lyase
• Kinetic parameters
• Synthesis

N-acetyl-D-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-D-neuraminic acid (sialic acid) from N-acetyl-D-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (Fast Prep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10 mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by Fast Prep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K-m for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.

• 出版日期2011-5