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首页> 外文期刊>Applied Microbiology >Heterologous Expression of a Bioactive β-Hexosyltransferase, an Enzyme Producer of Prebiotics, from Sporobolomyces singularis
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Heterologous Expression of a Bioactive β-Hexosyltransferase, an Enzyme Producer of Prebiotics, from Sporobolomyces singularis

机译：奇异孢子菌生物活性β-己糖基转移酶（益生元的酶生产者）的异源表达

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Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene ( Bht ) from Sporobolomyces singularis . The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed.
机译：低聚半乳糖（GOS）是不易消化的膳食纤维，能够到达胃肠道下部，并由促进健康的细菌进行选择性发酵。在本报告中，我们描述了奇异孢子菌的β-己糖基转移酶基因（Bht）的优化合成生产版本的异源表达。 Bht基因编码一个糖基水解酶（EC 3.2.1.21），充当半乳糖基转移酶，能够催化乳糖向GOS的一步转化。该酶在大肠杆菌中的表达产生了非活性的不溶蛋白，而甲基营养酵母巴斯德毕赤酵母GS115产生了生物活性的β-己糖基转移酶（rBHT）。该酶在3.5至6的pH值和40至50℃的温度下显示出更快的动力学。在低于40°C的温度下，酶的稳定性得到改善，发现葡萄糖是酶活性的竞争性抑制剂。巴斯德毕赤酵母将一部分生物活性rBHT分泌到发酵液中，而大部分酶仍与外膜结合。分泌形式和膜相关形式都能够有效地将乳糖转化为GOS。另外，具有膜结合酶的静止细胞以68％的收率（g / g）（最大理论值为75％）将90％的初始乳糖转化为GOS，而没有次级残留（葡萄糖或半乳糖）产物。这是奇异链球菌具有生物活性的BHT的首次报道，该生物活性BHT已异源表达。

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