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利用N-乙酰葡糖胺糖苷酶在乳酸乳球菌表面展示超氧化物歧化酶
引用本文:黄新凤,李小华,李林.利用N-乙酰葡糖胺糖苷酶在乳酸乳球菌表面展示超氧化物歧化酶[J].微生物学通报,2010,37(7):0992-0998.
作者姓名:黄新凤  李小华  李林
作者单位:华中农业大学农业微生物学国家重点实验室,湖北,武汉,430070
基金项目:国家自然科学基金项目(No. 30670054, 30370026)
摘    要:分别将乳酸乳球菌(Lactococcus lactis)N-乙酰葡糖胺糖苷酶基因(acmA)的信号肽序列(ss)、C-末端结构域(cA)及全长基因,与来自大肠杆菌(Escherichiacoli)的超氧化物歧化酶(SOD)基因sod构建成融合基因ss-cA-sod和acmA-sod,并连接于表达载体pMG36K,然后导入乳酸乳球菌ATCC11454菌株,获得了能在细胞表面展示SOD的重组工程菌MB193和MB194。经SDS-PAGE验证,重组菌MB193和MB194可分别表达产生分子量约为46和64kD的融合酶蛋白cA-SOD和AcmA-SOD。通过黄嘌呤氧化酶法测定MB193和MB194菌株的全细胞Mn-SOD酶活力分别为(2.63±0.51)U/mL和(3.51±0.64)U/mL,明显高于仅在细胞内表达产生SOD的对照重组菌MB192的酶活性(1.53±0.38)U/mL,且表达融合酶AcmA-SOD的重组菌MB194具有最大的表面展示效率(56.4%)。

关 键 词:细胞表面展示系统    N-乙酰葡糖胺糖苷酶    超氧化物歧化酶    全细胞催化剂

Display of Superoxide Dismutase on the Surface of Lactococcus lactis by Use of the N-acetylglucosaminidase Anchor
HUANG Xin-Feng,LI Xiao-Hua and LI Lin.Display of Superoxide Dismutase on the Surface of Lactococcus lactis by Use of the N-acetylglucosaminidase Anchor[J].Microbiology,2010,37(7):0992-0998.
Authors:HUANG Xin-Feng  LI Xiao-Hua and LI Lin
Institution:State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China;State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China;State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China
Abstract:In the present study, we utilized a previously characterized N-acetylglucosaminidase (AcmA) to develop a whole-cell catalyst of bacterial superoxide dismutase (SOD) in Lactococcus lactis. The truncated C-terminal domain (cA) and the full-length AcmA from L. lactis wild-type strain MB191, were used as the anchoring motifs to immobilize an Escherichia coli-derived SOD onto the surfaces of L. lactis ATCC11454 cells. The PCR-amplified cA fragment, the signal and the full-length sequences of acmA, were fused with sod to generate the recombinant ss-cA-sod and acmA-sod, respectively, followed by ligating into the expression vector pMG36K, yielding the recombinant strain MB193 (harboring the ss-cA-sod fusion gene) and MB194 (harboring acmA-sod), respectively. SDS-PAGE analysis showed that the substantial expression profile of the fusion enzymes cA-SOD and AcmA-SOD in the recombinant L. lactis MB193 and MB194, with the predicted Mr of 46 kD and 64 kD, respectively. The Mn-SOD activities of MB193 and MB194 cells were determined by using the standard xanthinoxidase assay procedure. It showed that MB193 and MB194 exhibited the higher whole-cell Mn-SOD activities by (2.63 ± 0.51) U/mL and (3.51 ± 0.64) U/mL, respectively, compared to the control strain MB192 by (1.53 ± 0.38) U/mL] that expressing the intracellular SOD. In addition, the recombinant MB194 cells exhibited the higher cell-surface display efficiency (by 56.4%) compared to MB193 cells (by 41.9%).
Keywords:Cell surface display system  N-acetylglucosaminidase  Superoxide dismutase  Whole-cell catalyst
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