Cloning and characterization of thermophilic endoglucanase and its application in the transformation of ginsenosides

Abstract

A novel endoglucanase (BcelFp) was identified from Fervidobaterium pennivorans DSM9078 which had biotransformation activity for protopanaxadiol (PPD)-type ginsenosides. Sequence analysis of BcelFp revealed that it could be classified into glycoside hydrolase family 5 (GH5). The gene encoding a 323-amino acid protein was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximate 37 kDa. The recombinant BcelFp exhibited an optimal activity at 95 ^oC and pH 5.5 and showed high thermostability. The endoglucanase had high selectivity for cleaving the outer glucose moiety at the C3 carbon of ginsenoside Rb1, Rb2, Rc and Rd, which produced stronger pharmacologically active gypenoside XVII (GypXVII), Compound O (CO), Compound Mc1 (CMc1) and F2, respectively. The K_m values for Rb1, Rb2, Rc and Rd were 3.66 ± 0.04 µM, 4.02 ± 0.12 µM, 5.95 ± 0.03 µM, 0.67 ± 0.006 µM, respectively. The k_cat/K_m value of BcelFp for ginsenoside Rd was 27.91 mM^-1s^-1, which was much higher than that of the previously enzymes. This study was the first report of the highly efficient and selective transformation of GypXVII, CO, CMc1 and F2 from Rb1, Rb2, Rc and Rd by a GH5-family thermophilic endoglucanase.

Keywords: Biotransformation; Endoglucanase; Fervidobaterium pennivorans DSM9078; Ginsenoside; Glycoside hydrolase family.

Fig. 1

Chemical structures of PPD-type ginsenosides. Glu β-D-glucopyranosyl, Arap α-L-arabinopyranosyl, Araf α-L-arabinofuranosyl

Fig. 2

Multiple amino acid sequence alignment of BcelFp with several characterized glycoside hydrolases from GH5. The accession numbers of the aligned sequences are for the following organisms: ADD73709, endoglucanase FnCel5A from Fervidobacterium nodosum Rt17-B1; AAD36816, endoglucanase from Thermotoga maritima MSB8; AXU72614. endoglucanase from Clostridioides difficile. The accession numbers were indicated to the left of the amino acid sequences. Identical residues are indicated by a red background. Symbols: ↑ amino acids forming a catalytic residues

Fig. 3

Phylogenetic analysis of BcelFp, and other characterized glycoside hydrolases from GH5. The units at the bottom of the tree indicate numbers of substitution events

Fig. 4

(A) Effect of pH on enzyme activity. (B) Effect of pH on enzyme stability. The activities were determined by assays with CMC as substrate following incubation of the enzyme at pH 4 (■), pH5(●), pH 6 (▶) and pH 7 (◀) for the indicated times. (C) Effect of temperature on enzyme activity. (D) Effect of temperature on enzyme stability. The activities were determined by assays with CMC as substrate following incubation of the enzyme at 85 °C (▲), 90 °C (●), and 95 °C (■) for the indicated times. (E) Differential Scanning Calorimetry (DSC) analysis of BcelFp. The enzyme was concentrated to 1.5 mg/ml in 50 mM PBS buffer (pH 6.0). The equilibrated enzyme was scanned from 35 to 120 ^oC at a rate of 2.0 K/min. The enzyme scan was corrected using a buffer–buffer baseline

Fig. 5

HPLC analysis of ginsenoside Rb1, Rb2, Rc and Rd during biotransformation process by using BcelFp. Ginsenoside standards were indicated on the peaks. Numbers were used to indicate the product peaks

Fig. 6

Biotransformation pathways of ginsenoside Rb1, Rb2, Rc and Rd by using BcelFp.