毕赤酵母中新型耐热α-淀粉酶的高效表达及其在麦芽糖浆生产中的应用。
High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production.
机构信息
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China.
出版信息
Int J Biol Macromol. 2019 Apr 15;127:683-692. doi: 10.1016/j.ijbiomac.2019.01.162. Epub 2019 Jan 29.
A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.
从嗜热真菌Thermomyces dupontii L18 中克隆到一个新型的α-淀粉酶基因（TdAmyA），其开放阅读框为 1431bp，推断编码 476 个氨基酸。该重组α-淀粉酶在毕赤酵母中成功过表达。在 168 小时的高密度发酵后，获得了最高的 38314U/mL 的α-淀粉酶活性，蛋白含量为 28.7mg/mL。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化的 TdAmyA 的分子质量为 61.2kDa，凝胶过滤测定为 59.2kDa。TdAmyA 是一种糖蛋白，含有 5.3%（w/w）的碳水化合物。TdAmyA 的最适活性温度为 60°C，最适 pH 值为 6.5，在 pH 值 4.5-10.0 范围内可稳定至 55°C。它对直链淀粉底物的活性高于支链淀粉底物。水解产物主要由麦芽糖和麦芽三糖组成。经过 8 小时水解，TdAmyA 产生的麦芽糖含量最高，为 51.8%。因此，TdAmyA 可能是生产麦芽糖糖浆的候选α-淀粉酶。
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