Immobilisation and kinetics of monoamine oxidase (MAO-N-D5) enzyme in polyvinyl alcohol gels
Immobilisation and kinetics of monoamine oxidase (MAO-N-D5) enzyme in polyvinyl alcohol gels
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DOI：
10.1016/j.molcatb.2016.04.009
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发表时间：
2016-07-01
影响因子：
--
通讯作者：
Rebros, Martin
中科院分区：
文献类型：
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作者：
Markosova, Kristina;Dolejs, Igor;Rebros, Martin
该研究集中于使用LentiKats技术在PVA凝胶中产生和固定重组单胺氧化酶（EC 1.4.3.4）的粗酶提取物，所述重组单胺氧化酶来源于尼日尔曲霉（MAO-N-D5）并在大肠杆菌中表达。MAO-Ns由于其立体选择性而在化学工业中是重要的酶，并且它们通常用于非光学纯的胺混合物的去外消旋化。本文详细描述了生物质生产、酶制备、酶固定化、固定化酶的工艺参数和酶的表征。在实验室生物反应器中制备生物质，并比较了两种不同的破碎技术。以仲胺3-氮杂双环[3，3，0]辛烷为底物，通过生物转化法测定酶的活性。粗酶提取物显示61.5%的全细胞的活性和固定化的酶比游离酶表现出更广泛的最适pH和温度范围。固定化单胺氧化酶粗酶提取物的初始比活性在12次重复生物转化后保持在80%。首次根据乒乓式双-双反应机理获得了固定化单胺氧化酶-N-D5的全动力学参数。比活性为0.29 U g（（Lentikats））（-1），Km为7.31 mM，与全细胞MAO-N-D5相比相似。与游离和全细胞MAO-N-D5相比，固定化的MAO-N-D5的表征在活性和稳定性方面显示出特别的益处;因此，该酶的固定化非常适合于工业应用。(C)© 2016 Elsevier B. V.版权所有。
This study focused on the production and immobilisation of the crude enzyme extract of recombinant monoamine oxidase (EC 1.4.3.4), originating from Aspergillus niger (MAO-N-D5) and expressed in Escherichia coli, in PVA gel using the LentiKats (R) technique. MAO-Ns are important enzymes in the chemical industry due to their stereoselectivity and they are often used for the deracemisation of non optically pure mixtures of amines. Biomass production, enzyme preparation, enzyme immobilisation, process parameters for the immobilised enzyme and characterisation of the enzyme are described in detail here. The biomass was prepared in laboratory bioreactors, and two different disruption techniques were compared. The activity of the enzyme was determined by biotransformation with secondary amine 3-azabicyclo [3,3,0] octane as a substrate. The crude enzyme extract showed 61.5% of the whole cell activity and the immobilised enzyme showed a wider optimum pH and temperature ranges than the free enzyme. The initial specific activity of the immobilised monoamine oxidase crude enzyme extract remained at 80% after 12 repeated biotransformations. For the first time, the full kinetic parameters of an immobilised MAO-N-D5 were obtained based on a ping-pong bi-bi reaction mechanism. The specific activity was 0.29 U g((Lentikats))(-1) and the K-m was 7.31 mM, which were similar in comparison to whole cell MAO-N-D5. Characterisation of immobilised MAO-N-D5 showed particular benefits in terms of activity and stability in comparison with free and whole cell MAO-N-D5; therefore, the immobilisation of this enzyme is very suitable for industrial applications. (C) 2016 Elsevier B.V. All rights reserved.