Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
Bioresour Technol. 2011 May;102(10):6186-91. doi: 10.1016/j.biortech.2011.02.026. Epub 2011 Mar 3.
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 (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (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.
N-乙酰-D-氨基葡萄糖醛酸醛缩酶是生物技术生产 N-乙酰-D-氨基葡萄糖（唾液酸）的关键酶，可由 N-乙酰-D-甘露糖胺和丙酮酸通过沉淀 90%硫酸铵和交联 1%戊二醛固定为交联酶聚集体（CLEAs）。由于在往复式分散器（FastPrep）中仅能回收 40%的活性，因此通过将酶与 10mg/mL 牛血清白蛋白共聚集，然后用硼氢化钠处理，最后用 FastPrep 进行分散（FastPrep-CLEAs），制备了改良的 CLEAs。这使得活性提高了两倍，达到 86%，比交联包涵体（CLIBs）中报道的该醛缩酶活性提高了 30%。此外，这些 FastPrep-CLEAs 在 Neu5Ac 合成方面表现出显著的生物技术特性，包括在碱性 pH 值下具有良好的活性和稳定性、对 ManNAc 的高 K（M）值（对丙酮酸较低）和良好的操作稳定性。这些结果证实了在生物催化中使用 FastPrep-CLEAs 的实用性，从而降低了生产成本并有利于重复使用。
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