Significant enhancement of (R)-mandelic acid production by relieving substrate inhibition of recombinant nitrilase in toluene-water biphasic system
Significant enhancement of (R)-mandelic acid production by relieving substrate inhibition of recombinant nitrilase in toluene-water biphasic system
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DOI：
10.1016/j.jbiotec.2011.01.013
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发表时间：
2011-03-10
影响因子：
4.1
通讯作者：
Liu, You-Yan
中科院分区：
文献类型：
--
作者：
Zhang, Zhi-Jun;Pan, Jiang;Liu, You-Yan
表达腈水解酶活性的重组大肠杆菌全细胞对扁桃腈的对映选择性水解受到高浓度（> 300 mM）底物的严重抑制，导致目标产物（R）-（-）-扁桃酸的产率较低。为了减轻底物抑制并提高 (R)-(-)-扁桃酸生产率，本工作尝试了八种水-有机溶剂双相系统。在测试的溶剂中，发现甲苯是最适合作为有机相的溶剂。在摇瓶中系统地检查和优化了各种参数。相体积比、缓冲液 pH 值和反应温度是影响双相系统中产物的产率和对映体纯度的敏感参数。在优化条件下，底物耐受性从200 mM显着提高到500 mM，平均生产率从179.6 g l(-1) d(-1)显着提高到352.6 g l(-1) d(-1)。随后，通过重复使用海藻酸钙包埋的细胞5批，在搅拌反应器（2升规模）中成功进行扁桃腈的生物催化水解，得到110.7g（R）-（-）-扁桃酸，98.0％ee（对映体过量），比产量为13.8g（扁桃酸）g（-1）（细胞）， 分别。 (C) 2011 Elsevier B.V. 保留所有权利。
The enantioselective hydrolysis of mandelonitrile with whole cells of a recombinant Escherichia coli expressing nitrilase activity was severely inhibited by the substrate at high concentrations (> 300 mM), which resulted in a low yield of the target product (R)-(-)-mandelic acid. To relieve the substrate inhibition and to enhance the (R)-(-)-mandelic acid productivity, eight water-organic solvent biphasic systems were attempted in this work. Toluene was found to be the most suitable solvent as the organic phase among the solvents tested. Various parameters were systematically examined and optimized in shake flasks. The phase volume ratio, buffer pH and reaction temperature were shown to be sensitive parameters affecting both the yield and the enantiopurity of product in the biphasic system. Under the optimized conditions, significant enhancement of substrate tolerance from 200 mM to 500 mM and average productivity from 179.6 g l(-1) d(-1) to 352.6 g l(-1) d(-1) were achieved. Subsequently, the biocatalytic hydrolysis of mandelonitrile was successfully carried out in a stirred reactor (2-l scale) by repeated use of the calcium alginate entrapped cells for 5 batches, affording 110.7 g (R)-(-)-mandelic acid in 98.0% ee (enantiomeric excess) and a specific production of 13.8 g (mandelic acid) g(-1) (cell), respectively. (C) 2011 Elsevier B.V. All rights reserved.