Du Nian, Deng Yuanyuan, Wei Zhencheng, Zhang Yan, Tang Xiaojun, Li Ping, Zhou Pengfei, Liu Guang, Zhang Mingwei
College of Life Science, Yangtze University, Jingzhou 434020, Hubei, China.
Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Affairs/Guangdong Key Laboratory of Agricultural Products Processing, Sericultural and Agri-Food Research Institute Guangdong Academy of Agricultural Sciences, Guangzhou 510610, Guangdong, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Feb 25;37(2):593-603. doi: 10.13345/j.cjb.200365.
Wheat quiescin sulfhydryl oxidase was expressed in Escherichia coli for developing a new biological flour improver. The synthesized wqsox gene was constructed into the vector pMAL-c5x and expressed in E. coli, then the expression conditions of recombinant protein was optimized. The MBP fusion label in recombinant protein was removed by protease digestion after affinity purification. Moreover, enzymatic properties of the purified wQSOX and its effect on bread quality were investigated. The synthesized wqsox gene contained 1 359 bp and encoded 453 amino acids with a deduced molecular weight of 51 kDa. The constructed recombinant vector pMAL-c5x-wqsox could successfully express soluble recombinant protein MBP-wQSOX in E. coli Rosetta gamiB(DE3), and the optimal induced expression conditions for recombinant protein were 25 °C, 0.3 mmol/L IPTG and 6 h. MBP fusion tag was cut out by factor Xa protease and wQSOX was prepared after affinity purification. wQSOX could catalyze the oxidation of DTT, GSH and Cys, accompanying the production of H2O2, and exhibited the highest substrate specificity for DTT. Furthermore, enzymatic properties results demonstrated that the optimal temperature and pH for wQSOX catalyzing oxidation of DTT was 50 °C and 10.0, respectively, and wQSOX presented a good stability under high temperature and alkaline environment. The addition of wQSOX with 1.1 U/g flour significantly (P<0.05) increased 26.4% specific volume of the bread, and reduced 20.5% hardness and 24.8% chewiness of bread crumb compared to the control, indicating a remarkable ability to improve the quality of bread.
为开发一种新型生物面粉改良剂，在大肠杆菌中表达了小麦静息素巯基氧化酶。将合成的wqsox基因构建到载体pMAL-c5x中并在大肠杆菌中表达，随后对重组蛋白的表达条件进行优化。亲和纯化后通过蛋白酶消化去除重组蛋白中的MBP融合标签。此外，还研究了纯化的wQSOX的酶学性质及其对面包品质的影响。合成的wqsox基因包含1359 bp，编码453个氨基酸，推导分子量为51 kDa。构建的重组载体pMAL-c5x-wqsox能够在大肠杆菌Rosetta gamiB(DE3)中成功表达可溶性重组蛋白MBP-wQSOX，重组蛋白的最佳诱导表达条件为25℃、0.3 mmol/L IPTG和6 h。用因子Xa蛋白酶切除MBP融合标签，亲和纯化后制备得到wQSOX。wQSOX能够催化DTT、GSH和Cys的氧化，伴随H2O2的产生，并且对DTT表现出最高的底物特异性。此外，酶学性质结果表明，wQSOX催化DTT氧化的最佳温度和pH分别为50℃和10.0，并且wQSOX在高温和碱性环境下表现出良好的稳定性。与对照相比，添加1.1 U/g面粉的wQSOX显著（P<0.05）提高了面包26.4%的比容，降低了面包心20.5%的硬度和24.8%的咀嚼性，表明其具有显著的改善面包品质的能力。
