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

doi:10.7503/cjcu20180149

Abstract

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:

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

Figures/Tables 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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