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首页> 美国卫生研究院文献>Frontiers in Physiology >Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae)
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Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae)

机译：菜青虫中肠中消化α-淀粉酶的特征（鳞翅目：菜青虫）

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The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones.
机译：目前的研究通过特定的抑制剂阿卡波糖的纯化，酶促表征，基因表达和体内作用来处理菜青虫（Pieris brasicae L.）幼虫中的消化α-淀粉酶。尽管α-淀粉酶活性在幼虫的整个肠匀浆中最高，但淀粉分解活性的区室化显示后中肠（PM）和前中肠（AM）具有相同的活性。使用硫酸铵，Sepharyl G-100和DEAE-Cellulose Fast flow进行的三步纯化显示出一种酶，其比活性为5.18 U / mg，回收率为13.20，纯化倍数为19.25，分子量为88 kDa。纯化的α-淀粉酶在最适pH和8和35°C的温度下具有最高的活性。此外，以淀粉和糖原为底物，该酶的Vmax值分别为4.64和3.02 U / mg蛋白，Km值为1.37和1.74％。不同浓度的阿卡波糖，乙二胺四乙酸和乙二醇-双（β-氨基乙基醚）N，N，N'，N'-四乙酸显着降低了纯化的α-淀粉酶的活性。用0.22 mM的阿卡波糖将芸苔假单胞菌的四龄幼虫喂食处理过的萝卜（Raphanus sativus L.）叶片，以发现其对营养指标，α-淀粉酶活性和基因表达的体内影响。仅在消化食品的转化效率，相对生长速率以及对照和阿卡波糖喂食幼虫的代谢成本方面发现了显着差异。而且，通过生化和天然PAGE实验，在处理的幼虫中淀粉分解活性显着降低。 RT-PCR结果显示，一个长度为621 bp的基因负责α-淀粉酶的表达，该基因与Papilio xuthus和P. polytes有75％的同一性。最后，qRT-PCR揭示了与阿卡波糖喂养的相比，对照幼虫中α-淀粉酶的表达更高。

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