d-lactate-selective amperometric biosensor based on the cell debris of the recombinant yeast Hansenula polymorpha.
d-lactate-selective amperometric biosensor based on the cell debris of the recombinant yeast Hansenula polymorpha.
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DOI：
10.1016/j.talanta.2014.02.041
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发表时间：
2014-07
期刊：
影响因子：
6.1
通讯作者：
O. Smutok;K. Dmytruk;Maria Karkovska;W. Schuhmann;M. Gonchar;A. Sibirny
中科院分区：
文献类型：
--
作者：
O. Smutok;K. Dmytruk;Maria Karkovska;W. Schuhmann;M. Gonchar;A. Sibirny
利用重组耐热甲基营养型多形汉逊酵母（Hansenula polymorpha）的细胞碎片，过量产生d-乳酸：细胞色素氧化还原酶（EC 1.1.2.4，d-乳酸脱氢酶（细胞色素），DlDH），开发了Ad-乳酸选择性生物传感器。TheH.在两个步骤中构建多晶型DlDH生产者。首先，在H.多晶型体在葡萄糖阻遏中受损并且缺乏过氧化氢酶活性以避免特异性1-乳酸-细胞色素氧化还原酶活性。第二，编码DlDH的同源基因DLD 1在强H.将多形醇氧化酶启动子插入到质粒的框架中，用于在Δ cyb 2菌株中进行多拷贝整合。由于DlDH与线粒体膜有很强的结合，因此将细胞碎片用作生物传感器的生物识别元件。将重组细胞机械破碎后的细胞碎片固定在石墨工作电极上，用Os-络合物修饰的阴极电沉积聚合物电化学生成层。细胞色素作为额外的天然电子介体，以改善电子从还原DlDH到工作电极的转移。所构建的乳酸脱氢酶选择性生物传感器的特征在于高灵敏度（46.3-61.6 A M-1 m-2）、高选择性和足够的储存稳定性。
Ad-lactate-selective biosensor has been developed using cells׳ debris of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha, overproducingd-lactate: cytochromec-oxidoreductase (EC 1.1.2.4,d-lactate dehydrogenase (cytochrome), DlDH). TheH. polymorphaDlDH-producer was constructed in two steps. First, the geneCYB2was deleted on the background of the С-105 (gcr1 catX) strain ofH. polymorphaimpaired in glucose repression and devoid of catalase activity to avoid specificl-lactate-cytochromecoxidoreductase activity. Second, the homologous geneDLD1coding for DlDH was overexpressed under the control of the strongH. polymorphaalcohol oxidase promoter in the frame of a plasmid for multicopy integration in theΔcyb2strain. The selected recombinant strain possesses 6-fold increased DlDH activity as compared to the initial strain.The cells׳ debris was used as a biorecognition element of a biosensor, since DlDH is strongly bound to mitochondrial membranes. The cells׳ debris, prepared by mechanic disintegration of recombinant cells, was immobilized on a graphite working electrode in an electrochemically generated layer using an Os-complex modified cathodic electrodeposition polymer. Cytochromecwas used as additional native electron mediator to improve electron transfer from reduced DlDH to the working electrode. The constructedd-lactate-selective biosensors are characterized by a high sensitivity (46.3–61.6 A M−1m−2), high selectivity and sufficient storage stability.