Extracellular production of neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae
Extracellular production of neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae
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DOI：
10.1128/aem.72.5.3716-3723.2006
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发表时间：
2006-05-01
影响因子：
4.4
通讯作者：
Abe, Keiko
中科院分区：
文献类型：
--
作者：
Nakajima, Ken-ichiro;Asakura, Tomiko;Abe, Keiko
Neoclin(NCL)是一种甜度约为糖500倍的蛋白质，可用作非血糖甜味剂。它还具有将酸味转化为甜味的调味活性。NCL是由N-糖基化的酸性亚基(NAS)和碱性亚基(NBS)组成的杂二聚体，它们通过二硫键连接在一起。为了使米曲霉产生重组NCL(RNCL)，在NAS和NBS的上游融合了带有KEX2裂解位点的α-淀粉酶-K-R-，并同时表达了融合蛋白。为了准确和有效地切割KEX2样酶的融合构建物，在KEX2裂解位点之后插入了一个甘氨酸基序。由于NBS的生产效率低于NAS，所以在共转化过程中，NBS表达载体的数量多于NAS表达载体，从而成功地在培养基中表达了rNCL。此外，为了获得更高的rNCL产量，我们克隆了HACA基因的活性形式，该基因编码一种可诱导未折叠蛋白反应的转录因子，并进行了结构性表达。这导致rNCL的产量增加了1.5倍(2.0毫克/升)。通过层析纯化rNCL，发现其NAS如预期的那样是N-糖基化的。用表达人甜味受体的人胚胎肾细胞进行钙成像和感官测试证实，rNCL的原始甜度和味觉改变活性与天然NCL相当。
Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, alpha-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.