Recombinant GST-BglPm contains 42.560.3% of total soluble protein in E. coli lysate. This high expression level in soluble form makes it more possible to industrial application. 3.3. Characterization of recombinant BglPm BglPm had optimal pH activity at 7.5 and stability at pH 6.0 to 7.5 in a sodium phosphate buffer at 37uC; from pH 8.0, the enzyme activity decreased swiftly, while at pH 5.0 the enzyme activity decreased to 17.4%. The optimal temperature activity was 45uC; at 30uC and 37uC, the enzyme has 63.7% and 73.5% of MedChemExpress SPDB relative activity, respectively, while thermostability was decreased from 37uC and at 45uC the enzyme has 53.1% of relative activity, and not detected at 55uC. Though BglPm has optimum temperature at 45uC for pNPGlc, ginsenoside-conversion reaction was occurred at 37uC for extension of stable transformation activity. The effects of metal ions, EDTA, b-mercaptoethanol, and SDS on BglQM activity were also investigated. BglPm activity was not affected by b-mercaptoethanol, which is well known thiol group inhibitors. These results suggested that sulfhydryl groups may not be involved in the catalytic center of the enzyme. The enzyme was not affected by 1 mM and 10 mM Na+, K+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+. The chelating agent EDTA did not inhibit BglPm activity, which indicated that divalent cations are not required for enzymatic activity. The enzyme activity appeared to be 100% inhibited in the presence of sodium dodecyl-sulfate and strongly inhibited in the presence of 10 mM Hg2+ and also inhibited in the presence of 10 mM Mg2+, which caused a 43% activity drop. However, no dramatic positive effects on the activity of the BglPm were found for the tested ion. The substrate specificity of BglPm was tested using 2.0 mM of p-nitrophenyl and o-nitrophenyl -glycosides with a and b configurations. The results showed that BglPm was only active against glucose moiety of pNP-b-D-glucopyranoside and oNP-b-D-glucopyranoside. It showed maximum activity towards pNP-b-D-glucopyranoside and 15% relative activity towards oNPb-D-glucopyranoside. 3.4. Determination of kinetic parameters The kinetic parameters of Vmax and Km of BglPm were determined by plotting the substrate concentration vs the initial velocity of each reaction and subjecting the data to no linear regression analysis. The Km, Vmax, kcat and kcat/Km PHCCC values for 4 substrates were presented in 6 Characterization of a Novel b-glucosidase 3.5. Ginsenoside-transformation characteristics of BglPm For the verification of the bioconversion pathways of the seven PPD type ginsenosides by BglPm, the TLC analyses were performed at regular intervals. As shown Fig. 4, it is clear that the BglPm could transform seven ginsenosides based on the Rf values. The transformed ginsenosides were also determined using their retention times in the HPLC. The proposed biotransformation pathways by BglPm for the PPD ginsenosides are as follows: Rb1 R Gyp XVII R 12926553 F2; Rd R F2; Rg3 R Rh2; Rb2 R C-O; Rb3 R C-Mx1; Rc R C-Mc1 via the stepwise hydrolysis of the outer glucose Glycoside hydrolase name BglPm b-glycosidase BglSp BglF3 AbfA Microorganism Paenibacillus mucilaginosus KCTC3870T Sulfolobus solfataricus Sphingomonas sp. 2F2 Flavobacterium johnsoniae Rhodanobacter ginsenosidimutans Gsoil 3054 Ginsenoside conversion pathway Rb1RGyp XVIIRF2, RdRF2 Rb1RRdRF2RC-K, Rb2RRdRF2RC-K, RcRC-McRC-K Rb1R Gyp XVIIRF2, Rb2RC-ORF2, RcRC-McRF2, RdRF2 Rb1RRd, Gyp XVIIRF2 C-Mc1RF2 Glycoside hydrolase family 1 1 1 3 51.Recombinant GST-BglPm contains 42.560.3% of total soluble protein in E. coli lysate. This high expression level in soluble form makes it more possible to industrial application. 3.3. Characterization of recombinant BglPm BglPm had optimal pH activity at 7.5 and stability at pH 6.0 to 7.5 in a sodium phosphate buffer at 37uC; from pH 8.0, the enzyme activity decreased swiftly, while at pH 5.0 the enzyme activity decreased to 17.4%. The optimal temperature activity was 45uC; at 30uC and 37uC, the enzyme has 63.7% and 73.5% of relative activity, respectively, while thermostability was decreased from 37uC and at 45uC the enzyme has 53.1% of relative activity, and not detected at 55uC. Though BglPm has optimum temperature at 45uC for pNPGlc, ginsenoside-conversion reaction was occurred at 37uC for extension of stable transformation activity. The effects of metal ions, EDTA, b-mercaptoethanol, and SDS on BglQM activity were also investigated. BglPm activity was not affected by b-mercaptoethanol, which is well known thiol group inhibitors. These results suggested that sulfhydryl groups may not be involved in the catalytic center of the enzyme. The enzyme was not affected by 1 mM and 10 mM Na+, K+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+. The chelating agent EDTA did not inhibit BglPm activity, which indicated that divalent cations are not required for enzymatic activity. The enzyme activity appeared to be 100% inhibited in the presence of sodium dodecyl-sulfate and strongly inhibited in the presence of 10 mM Hg2+ and also inhibited in the presence of 10 mM Mg2+, which caused a 43% activity drop. However, no dramatic positive effects on the activity of the BglPm were found for the tested ion. The substrate specificity of BglPm was tested using 2.0 mM of p-nitrophenyl and o-nitrophenyl -glycosides with a and b configurations. The results showed that BglPm was only active against glucose moiety of pNP-b-D-glucopyranoside and oNP-b-D-glucopyranoside. It showed maximum activity towards pNP-b-D-glucopyranoside and 15% relative activity towards oNPb-D-glucopyranoside. 3.4. Determination of kinetic parameters The kinetic parameters of Vmax and Km of BglPm were determined by plotting the substrate concentration vs the initial velocity of each reaction and subjecting the data to no linear regression analysis. The Km, Vmax, kcat and kcat/Km values for 4 substrates were presented in 6 Characterization of a Novel b-glucosidase 3.5. Ginsenoside-transformation characteristics of BglPm For the verification of the bioconversion pathways of the seven PPD type ginsenosides by BglPm, the TLC analyses were performed at regular intervals. As shown Fig. 4, it is clear that the BglPm could transform seven ginsenosides based on the Rf values. The transformed ginsenosides were also determined using their retention times in the HPLC. The proposed biotransformation pathways by BglPm for the PPD ginsenosides are as follows: Rb1 R Gyp XVII R 12926553 F2; Rd R F2; Rg3 R Rh2; Rb2 R C-O; Rb3 R C-Mx1; Rc R C-Mc1 via the stepwise hydrolysis of the outer glucose Glycoside hydrolase name BglPm b-glycosidase BglSp BglF3 AbfA Microorganism Paenibacillus mucilaginosus KCTC3870T Sulfolobus solfataricus Sphingomonas sp. 2F2 Flavobacterium johnsoniae Rhodanobacter ginsenosidimutans Gsoil 3054 Ginsenoside conversion pathway Rb1RGyp XVIIRF2, RdRF2 Rb1RRdRF2RC-K, Rb2RRdRF2RC-K, RcRC-McRC-K Rb1R Gyp XVIIRF2, Rb2RC-ORF2, RcRC-McRF2, RdRF2 Rb1RRd, Gyp XVIIRF2 C-Mc1RF2 Glycoside hydrolase family 1 1 1 3 51.

Recombinant GST-BglPm contains 42.560.3% of total soluble protein in E. coli lysate.

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