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小麦种子过氧化物酶WP1属于含血红素的植物Ⅲ型过氧化物酶,该酶不仅具有抗真菌活性,而且影响面粉加工品质。为提高WP1在大肠杆菌中的功能性表达,构建了用于提高大肠杆菌内源血红素合成,包含hemA和hemL基因的重组质粒pACYC-A-L,将其分别与包含WP1基因的分泌型和非分泌型表达载体pMAL-p4x-WP1与pET21a-MBP-WP1共同转化大肠杆菌T7 Express菌株;利用直链淀粉(Amylose)亲和层析柱纯化获得MBP-WP1融合蛋白,并以2,2’-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐(ABTS)为底物检测重组WP1的催化能力。结果表明,含pACYC-A-L的宿主菌28℃诱导12 h后,培养液中5-氨基乙酰丙酸(5-ALA)含量可达146.73 mg/L,卟啉类物质含量也显著上升。共转化pACYC-A-L和pMAL-p4x-WP1比单独转化pET21a-MBP-WP1获得的重组WP1的比活力提高14.6倍。该研究不仅成功地增强了小麦WP1在大肠杆菌中的功能性表达,同时为其他具有重要生物学功能并含血红素辅基蛋白的功能性表达提供了有益参考。
Wheat seed peroxidase WP1 is a heme-containing plant type III peroxidase that not only has anti-fungal activity but also affects flour processing quality. In order to improve the functional expression of WP1 in E. coli, a recombinant plasmid pACYC-AL was constructed for increasing the endogenous heme synthesis in E. coli, containing the hemA and hemL genes, which were respectively fused with the secreted and non-secreted The expression vector pMAL-p4x-WP1 was co-transformed into E. coli T7 Express strain with pET21a-MBP-WP1. The MBP-WP1 fusion protein was purified by Amylose affinity chromatography, Nitrogen - bis (3 - ethyl benzothiazole - 6 - sulfonic acid) diammonium salt (ABTS) as a substrate to detect the catalytic activity of recombinant WP1. The results showed that the concentration of 5-aminolevulinic acid (5-ALA) in the culture medium reached 146.73 mg / L after 12 h induction at 28 ℃, and the content of porphyrins increased significantly in the medium containing pACYC-A-L. The specific activities of pACYC-A-L and pMAL-p4x-WP1 were 14.6-fold higher than that of recombinant WP1 transformed with pET21a-MBP-WP1 alone. This study not only succeeded in enhancing the functional expression of wheat WP1 in E. coli, but also provided a useful reference for other important biological functions and functional expression of heme-containing proteins.