Mahmoudian M, Noble D, Drake C S, Middleton R F, Montgomery D S, Piercey J E, Ramlakhan D, Todd M, Dawson M J
Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Herts, United Kingdom.
Enzyme Microb Technol. 1997 Apr;20(5):393-400. doi: 10.1016/s0141-0229(96)00180-9.
N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation. Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac. The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction. Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach. This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin. In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc. ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate. Neu5Ac can be isolated from such reaction mixtures by a simple crystallization. This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.
N-乙酰-D-神经氨酸（Neu5Ac）醛缩酶（EC 4.1.3.3）已被报道可用于合成Neu5Ac，[1-5] 但尚无关于大规模操作时不存在重大缺陷的工艺报道。在此，来自大肠杆菌过表达重组菌株的Neu5Ac醛缩酶已被用于开发一种固定化酶工艺来生产Neu5Ac。该酶被固定在Eupergit-C上，并且可以在反应中多次重复使用。N-乙酰-D-葡萄糖胺（GlcNAc）的碱催化差向异构化产生了GlcNAc/N-乙酰-D-甘露糖胺（ManNAc）混合物（约4:1），其可直接用于醛缩酶反应；然而，GlcNAc对该酶的抑制限制了通过这种方法可用于反应的ManNAc浓度。这就需要加入大量摩尔过量的丙酮酸（五到七倍）来推动平衡向Neu5Ac方向移动；尽管如此，已开发出一种方法，通过与亚硫酸氢盐络合有效地去除过量的丙酮酸，从而使Neu5Ac能够通过吸附到阴离子交换树脂上进行回收。在第二种方法中，已开发出一种方法来富集GlcNAc/ManNAc混合物中的ManNAc。ManNAc可在反应中以高浓度使用，从而无需使用大量摩尔过量的丙酮酸。Neu5Ac可通过简单的结晶从这种反应混合物中分离出来。这项工作表明了综合工艺解决方案对于生物转化反应有效放大的重要性。
