Screening of a novel glucosyl condensing enzyme showing the activity of glycoside synthesis and the application of this enzyme to useful glycoside production

具有糖苷合成活性的新型葡萄糖基缩合酶的筛选及其在有用糖苷生产中的应用

• 批准号: 12650792
• 负责人: KINO Kuniki
• 金额: $ 2.05万
• 依托单位: Waseda University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12650792/
• 关键词: Glucosyl transfer enzyme; Glucosyl condensing enzyme; Glucosidase; Glycoside; Arbutin; Enzyme screening; Gene expression; Maltose phosphorylase

In this study, the novel method of the glucoside production using microbial enzymes was investigated. Xanthomonas campestris WU-9701 produces a novel enzyme catalyzing α-anomer-selective glucosylation. Using this enzyme, α-anomer-selective glucosylation of alcoholic and phenolic -OH groups was performed. Moreover, it was identified that the enzyme has also an activity of α-anomer-selective glucosylation of -SH group. This enzyme was purified and characterized, and the properties of this enzyme were clarified. The gene (xgtA) encoding the enzyme catalyzing α-anomer-selective glucosylation was cloned. The nucleotide sequence of xgtA shows homologies to those of several glucosidases. In addition, the 3D structure of XgtA was predicted. The gene xgtA was highly expressed in Escherichia coli, and the recombinant enzyme produced in E. coli was utilized to the production of α-arbutin, a useful glucoside. The synthesis of a glucoside by condensing was investigated. In the case of commercial enzymes, α-arbutin was synthesized by α-glucosidase of Aspergillus niger from hydroquinone and glucose. It was cleared that maltose was synthesized using glucose, and α-arbutin was synthesized by glucosetranslation from this maltose. Arbutin was similarly synthesized by β-glucosidase of A. niger from hydroquinone and glucose. Through the screening, strain (YS003) was obtained. A glucose condensing activity was not confirmed, but YS003 produced an enzyme catalyzing β-anomer-selective glucosylation of hydroquinone. On the other hand, it was identified that a commercial maltose phosphorylase (EC 2.4.1.8, MPase) has an activity of the production of α-glucoside using maltose as a glucosyl donor. It was confirmed that the glucosylation of an alcoholic -OH group by MPase.

研究了利用微生物酶法生产葡萄糖苷的新方法。油菜黄单胞菌WU-9701产生一种催化α-氨基选择性糖基化的新酶。使用该酶，α-异头选择性糖基化醇和酚-OH基团。此外，该酶还具有α-异头体选择性糖基化-SH基团的活性。对该酶进行了纯化和表征，并阐明了该酶的性质。克隆了α-异头选择性糖基化酶的编码基因xgtA。xgtA的核苷酸序列与几种葡萄糖苷酶具有同源性。此外，对XgtA的三维结构进行了预测。xgtA基因在大肠杆菌中高表达，重组酶被用于生产α-熊果苷，这是一种有用的葡萄糖苷。研究了缩合法合成一种葡萄糖苷的方法。以工业酶为原料，采用黑曲霉α-葡萄糖苷酶以对苯二酚和葡萄糖为原料合成α-熊果苷。结果表明，麦芽糖是由葡萄糖合成的，α-熊果苷是由麦芽糖经葡萄糖翻译合成的。同样地，黑曲霉β-葡萄糖苷酶以对苯二酚和葡萄糖为原料合成熊果苷。通过筛选得到菌株YS003。葡萄糖浓缩活性未被证实，但YS003产生一种酶催化对苯二酚的β-异丙基选择性糖基化。另一方面，确定了一种麦芽糖磷酸化酶（EC 2.4.1.8, MPase）以麦芽糖为糖基供体具有生产α-葡萄糖苷的活性。证实了MPase对乙醇-OH基的糖基化作用。

项目成果

JUN KUROSU et al.: "Enzymatic Synthesis of α-Arbutin by α-Anomer-Selective Glucosylation of Hydroquinone Using the Lyophilized Cells of Xanthomonas campestris WU-9701"Journal of Bioscience and Bioengineering. Vol. 93, No. 3. 328-330 (2002)

JUN KUROSU 等人：“利用野油菜黄单胞菌 WU-9701 的冻干细胞对氢醌进行 α-端基选择性糖基化酶促合成 α-熊果苷”，《生物科学与生物工程杂志》，第 93 卷，第 328-330 期。 (2002)

HIROYUKI NAKAGAWA: "α-Anomer-selective glucosylation of menthol with high yield through a crystal accumulation reaction using lyophilized cells of Xanthomonas campestris WU-9701"Journal of Bioscience and Bioengineering. Vol.89,No.2. 138-144 (2000)

HIROYUKI NAKAGAWA：“使用野油菜黄单胞菌 WU-9701 的冻干细胞进行高产率的α-端基选择性糖基化”，生物科学与生物工程杂志，第 89 卷，第 2 期（2000 年）。

HIROYUKI DO et al.: "Enzymatic Synthesis of l-Menthyl α-Maltoside and l-Menthyl α-Maltooligosides from l-Menthyl α-Glucoside by Cyclodextrin Glucanotransferase"Journal of Bioscience and Bioengineering. Vol. 94, No. 2. 119-123 (2002)

HIROYUKI DO 等人：“通过环糊精葡糖基转移酶从 1-薄荷基 α-葡萄糖苷酶促合成 1-薄荷基 α-麦芽糖苷和 1-薄荷基 α-麦芽糖苷”，《生物科学与生物工程杂志》，第 2 期，第 94 期，第 119 期。 123（2002）

JUN KUROSU: "Enzymatic Synthesis of α-Arbutin by α-Anomer-Selective Glucosylation of Hydroquinone Using Lyophilized Cells of Xanthomonas campestris WU-9701"Journal of Bioscience and Bioengineering. Vol.93, No.3. 328-330 (2002)

JUN KUROSU：“利用野油菜黄单胞菌 WU-9701 的冻干细胞通过对苯二酚的 α-端基选择性糖基化酶促合成 α-熊果苷”，生物科学与生物工程杂志，第 93 卷，第 328-330 期。

HIROYUKI DO: "Enzymatic synthesis of l-menthyl α-maltoside and l-menthyl α-maltooligosides from l-menthyl α-glucoside by cyclodextrin glucanotransferase"Journal of Bioscience and Bioengineering. Vol.94,No.2. 119-123 (2002)

HIROYUKI DO：“通过环糊精葡聚糖转移酶从 L-薄荷基 α-麦芽糖苷酶促合成 L-薄荷基 α-麦芽糖苷和 L-薄荷基 α-麦芽寡糖苷”《生物科学与生物工程杂志》第 94 卷，第 2 期（2002 年）。 ）