重组小麦静息巯基氧化酶的表达、酶学特性及其对面包品质的影响

Expression and characterization of recombinant wheat quiescin sulfhydryl oxidase and its effect on bread quality

为发展新型面粉改良酶制剂,利用大肠杆菌Escherichia coli原核表达了小麦静息巯基氧化酶(Wheat quiescin sulfhydryl oxidase,wQSOX)。将合成的wqsox基因构建至pMAL-c5x载体,并在大肠杆菌中进行表达,优化蛋白表达条件后对重组蛋白进行分离纯化及融合标签切除,获得的重组wQSOX蛋白用于酶学性质探究以及面包品质改良。结果表明,合成的截短wqsox基因包含1359 bp,编码453个氨基酸,理论蛋白分子量51 kDa;构建的pMAL-c5x-wqsox重组质粒在E.coli Rosetta gamiB(DE3)中可溶表达了重组蛋白MBP-wQSOX,其最佳表达条件为:诱导温度25℃,诱导剂IPTG浓度0.3 mmol/L,诱导时间6 h;利用Xa因子蛋白酶切除了MBP融合标签,亲和层析纯化得到了wQSOX;wQSOX可催化DTT、GSH和Cys氧化,并伴随着H2O2的生成,其中对DTT表现出最高的底物特异性;酶学性质研究发现,wQSOX最适反应温度和pH分别为50℃和10.0,在高温和碱性环境条件下表现出较好的稳定性;每克面粉中添加1.1 U wQSOX能够显著(P<0.05)提高26.4%的面包比容,降低20.5%的面包芯硬度和24.8%的咀嚼性,表现出了较好的改良面包加工品质能力。研究结果对丰富新型面粉改良酶制剂种类以及推动wQSOX在焙烤行业的应用奠定了理论基础。

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 1359 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℃,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℃ 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.