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首页> 外文期刊>Applied Microbiology and Biotechnology >Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase
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Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase

机译：固定化N-乙酰基-d-氨基葡萄糖-2-表异构酶和N-乙酰基-d-神经氨酸裂解酶的偶联生物转化制备N-乙酰基-d-神经氨酸

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摘要

N-Acetyl-d-neuraminic acid (Neu5Ac) can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-d-manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-d-neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-d-glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was ～73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.
机译：N-乙酰基-d-神经氨酸（Neu5Ac）可以由N-乙酰基-d-葡萄糖胺（GlcNAc）和丙酮酸通过化学酶促过程制得，在化学过程中，碱催化的差向异构化将GlcNAc转化为N-乙酰基-d-甘露糖胺（ManNAc ）。然后在N-乙酰基-d-神经氨酸裂解酶（NAL）的存在下，通过丙酮酸将ManNAc进行生物催化缩合，或者通过两步完全酶促过程进行纯化，包括使用N-乙酰基-d-将GlcNAc转化为ManNAc，将ManNAc转化为Neu5Ac。氨基葡萄糖2-表异构酶（AGE）和NAL。该技术存在一些缺点，例如反应时间长，并且在两步酶促过程中使用可溶形式的酶时转化率低。在这项研究中，通过基于FP的亲和层析纯化上清中表达大肠杆菌的AGE和NAL，然后将其固定在Amberzyme环氧乙烷树脂上。将这两种固定的酶（对于AGE具有78.18 U / g的比活性和对于NAL具有69.30 U / g的比活性）偶联以在一个反应​​器中直接将GlcNAc转化为Neu5Ac。从GlcNAc到Neu5Ac的两步反应转化率在24 h内为〜73％。此外，固定化的AGE和NAL都可以使用多达五个反应周期，而不会损失活性或明显降低转化率。

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