人参皂苷β-葡萄糖苷酶基因的毕赤酵母载体构建及生物转化

doi:10.7503/cjcu20180149

高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (11): 2451.doi: 10.7503/cjcu20180149

人参皂苷β-葡萄糖苷酶基因的毕赤酵母载体构建及生物转化

刘欣茹¹, 刘春莹², 徐龙权¹, 宋建国¹, 鱼红闪¹()

1. 大连工业大学生物工程学院, 大连116034
2. 大连大学生命科学与技术学院, 大连116622

收稿日期:2018-02-28 出版日期:2018-11-10 发布日期:2018-10-08
作者简介:联系人简介: 鱼红闪, 男, 博士, 教授, 博士生导师, 主要从事天然产物生物转化方面的研究. E-mail: hongshan@dlpu.edu.cn
基金资助:
国家高端外国专家项目(批准号: GDT20152100019)资助.

Construction of Ginsenoside-β-glucosidase Gene Vector and Biotransformation in Pichia Pastoris^†

LIU Xinru¹, LIU Chunying², XU Longquan¹, SONG Jianguo¹, YU Hongshan^1,^*()

1. School of Biology Engineering, Dalian Polytechnic University, Dalian 116034, China
2. School of Life Science and Technology, Dalian University, Dalian 116622, China

Received:2018-02-28 Online:2018-11-10 Published:2018-10-08
Contact: YU Hongshan E-mail:hongshan@dlpu.edu.cn
Supported by:
† Supported by the National High?end Foreign Expert Project, China(No.GDT20152100019).

摘要/Abstract

摘要：

将人参皂苷β-葡萄糖苷酶(GluGF)基因与表达载体pPIC9K连接, 构建重组质粒, 转化至毕赤酵母中表达, 并用于人参皂苷Rb₁的催化转化. 研究结果表明, 成功构建了重组表达质粒pPIC9K-GluGF, 转化后筛选到阳性重组毕赤酵母菌. 重组菌产酶的最佳甲醇诱导体积为0.5%, 诱导时间为168 h. 重组菌诱导培养制备的粗酶液具有GluGF的活性, 可以水解人参皂苷Rb₁, 产物中皂苷C-K含量达到0.09 mg/mL, 粗酶液中GluGF的比活力为0.67 U/mg.

关键词: 人参皂苷β-葡萄糖苷酶;, 人参皂苷Rb₁, 载体构建, 毕赤酵母, 生物转化

Abstract:

The ginsenoside-β-glucosidase(GluGF) gene was ligated with the expression vector pPIC9K to construct a recombinant plasmid. The recombinant plasmid was transformed into pichia pastoris to expresse and accomplish the biotransformate of ginsenoside Rb₁. The results showed that the recombinant plasmid pPIC9K-GluGF was successfully constructed and transformed into pichia pastoris GS115 competent cells. The optimal volume of methanol for recombinant strain was 0.5%, and the induction time was 168 h. The crude enzyme solution prepared by induction culture could hydrolyze ginsenoside Rb₁. The content of C-K in the product reached 0.09 mg/mL. The enzyme activity of GluGF in the crude enzyme solution was 0.67 U/mg.

Key words: Ginsenosides-β-glucosidase;, Ginsenoside Rb₁, Vector construction, Pichia, Biotransformation

TrendMD:

刘欣茹, 刘春莹, 徐龙权, 宋建国, 鱼红闪. 人参皂苷β-葡萄糖苷酶基因的毕赤酵母载体构建及生物转化. 高等学校化学学报, 2018, 39(11): 2451.

LIU Xinru, LIU Chunying, XU Longquan, SONG Jianguo, YU Hongshan. Construction of Ginsenoside-β-glucosidase Gene Vector and Biotransformation in Pichia Pastoris^†. Chem. J. Chinese Universities, 2018, 39(11): 2451.

图/表 8

Scheme 1 Mechanism of ginsenoside glycosidase and Rb1 reaction

Fig.1 Identification of recombinant plasmidLane M1: DL2000 DNA marker; lane M2: λ-Hind Ⅲ digest marker; lane 1: restriction enzyme digestion of recombinant plasmids.

Fig.2 Identification of recombinant by PCR analysisLane M1: λ-Hind Ⅲ digest; lanes 1—8: recombinant plasmid sieve product; lane M2: DL2000 DNA marker.

Fig.3 TLC detection of enzyme reaction products with different reaction time(A) Standard ginsenosides; (B) line 1: blank(substrate react with water); bacterial culture time of lines 2—9 were 24, 48, 72, 96, 120, 144, 168, 192 h, respectively.

Fig.4 TLC detection of enzyme reaction products with different concentrations of methanol(A) Standard ginsenosides; (B) line 1: blank(substrate react with water); methanol concentration of lines 2—5 were 0, 0.1%, 0.5%, 1%, respectively.

Fig.5 SDS-PAGE analysis of the expression productLane M: protein maker; lane 1: methanol-induced positive strain; lane 2: methanol-free positive strain; lane 3: methanol-induced negative strain; lane 4: purified enzyme solution.

Fig.6 TLC of inducement effect on expression TLCRb1, Rd, F2, C-K: Standard ginsenosides; lane 1: methanol-induced positive strain; lane 2: methanol-free strain; lane 3: methanol-induced negative strain; lane 4: enzyme reaction after purification.

Table 1 Chromatography data of each substance analysis

Ginsenoside	t/min	Area/(μV·s)	Mass fraction(%)
Rb₁	38.27	567971	76.4
Rd	40.09	70188	9.4
F₂	45.58	22857	3.1
C-K	54.15	82385	11.1

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人参皂苷β-葡萄糖苷酶基因的毕赤酵母载体构建及生物转化

Construction of Ginsenoside-β-glucosidase Gene Vector and Biotransformation in Pichia Pastoris^†

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人参皂苷β-葡萄糖苷酶基因的毕赤酵母载体构建及生物转化

Construction of Ginsenoside-β-glucosidase Gene Vector and Biotransformation in Pichia Pastoris†

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Construction of Ginsenoside-β-glucosidase Gene Vector and Biotransformation in Pichia Pastoris^†