Ification of recombinant BglPm The genomic DNA from P. mucilaginosus KCTC 3870T was extracted working with a genomic DNA extraction kit. The gene encoding b-glucosidase was amplified from the genomic DNA as a template by means of a polymerase chain reaction employing Pfu DNA polymerase. The sequence on the oligonucleotide primers used for the gene cloning was depending on the DNA sequence of b-glucosidase. Forward and reverse primers have been created as primers to introduce the BamHI and XhoI restriction sites, respectively, and Characterization of a Novel b-glucosidase Substrate preference was examined using 2.0 mM chromogenic o-nitrophenyl and p-nitrophenyl as substrates at 37uC for ten min, with 1 activity unit being defined as the release of 1 mmol o-nitrophenol or p-nitrophenol per min. The following substrates were tested: PNP-b-D-glucopyranoside, PNP-b-Dgalactopyranoside, PNP-b-D-fucopyranoside, PNP-N-acetyl-b-Dglucosaminide, PNP-b-L-arabinopyranoside, PNP-b-D-mannopyranoside, CASIN web PNP-b-D-xylopyranoside, PNP-a-D-glucopyranoside, PNP-a-L-arabinofuranoside, PNP-a-L-arabinopyranoside, PNPa-L-rhamnopyranoside, PNP-a-D-mannopyranoside, PNP-a-Dxylopyranoside, ONP-b-D-glucopyranoside, ONP-b-D-galactopyranoside, ONP-b-D-fucopyranoside and ONP-a-D-galactopyranoside. two.five. Determination of kinetic parameters Kinetic studies were performed with freshly purified enzymes utilizing pNPGlc at 0.110.0 mM, Rb1, Gyp XVII and Rd at concentrations from 0.two mM to five.0 mM. One unit of activity was defined as the volume of protein needed to produce 1 mmol of pnitrophenol or to convert 1 mmol of Rb1 or Gyp XVII or Rd per minute. All assays had been performed in triplicate. The parameters, Km and Vmax, were determined applying the enzyme kinetics program described by Cleland. 2.6. Biotransformation activity of PPD ginsenosides utilizing BglPm The initial biotransformation experiments employing the big ginsenosides Rb1 and Rd as substrates revealed that the GSTfused enzyme will not impact the activities of BglPm. Thus, the fusion protein was applied to determine the specificity and selectivity of the enzymes for the hydrolysis of the glucose moieties attached in the C3 and C20 web sites inside the seven PPD ginsenosides. The enzyme options at a concentration of 0.1 mg/ml in 50 mM of sodium phosphate buffer were reacted with an equal volume of Rb1, Rb2, Rb3, Rc, Rd, Gyp XVII and Rg3 option at a concentration of 0.1% in 50 mM of sodium phosphate buffer at 37uC. The samples were taken at typical intervals and analyzed by means of TLC or HPLC after pretreatment. thiogalactopyranoside with a final concentration of 0.1 mM with feeding 2% glucose. The bacterial cells were incubated for any further 24 h at 18uC and had been then harvested through centrifugation at 5,000 rpm for 20 min at 4uC. For the production in the recombinant BglPm, the LB medium supplemented with ampicillin was utilised to cultivate the E. coli harboring pGEX-bglPm in a ten L stirred-tank reactor with a five L functioning volume at 12926553 500 rpm. The pH value of your medium was adjusted to 7.0 using 100 mM of sodium phosphate. The culture was incubated at 37uC until the culture reached an OD of 3.0 at 600 nm. The protein expression was induced via the GW-0742 chemical information addition IPTG with a final concentration of 0.1 mM. The bacterial cells had been incubated to get a further 18 h at 30uC and had been then harvested by means of centrifugation at 5,000 rpm for 20 min at 4uC. The cells suspended in 100 mM of phosphate buffer had been disrupted through sonication, and then the intact cells and debris have been removed through.Ification of recombinant BglPm The genomic DNA from P. mucilaginosus KCTC 3870T was extracted employing a genomic DNA extraction kit. The gene encoding b-glucosidase was amplified in the genomic DNA as a template by means of a polymerase chain reaction making use of Pfu DNA polymerase. The sequence with the oligonucleotide primers employed for the gene cloning was determined by the DNA sequence of b-glucosidase. Forward and reverse primers were developed as primers to introduce the BamHI and XhoI restriction websites, respectively, and Characterization of a Novel b-glucosidase Substrate preference was examined utilizing 2.0 mM chromogenic o-nitrophenyl and p-nitrophenyl as substrates at 37uC for 10 min, with 1 activity unit getting defined as the release of 1 mmol o-nitrophenol or p-nitrophenol per min. The following substrates have been tested: PNP-b-D-glucopyranoside, PNP-b-Dgalactopyranoside, PNP-b-D-fucopyranoside, PNP-N-acetyl-b-Dglucosaminide, PNP-b-L-arabinopyranoside, PNP-b-D-mannopyranoside, PNP-b-D-xylopyranoside, PNP-a-D-glucopyranoside, PNP-a-L-arabinofuranoside, PNP-a-L-arabinopyranoside, PNPa-L-rhamnopyranoside, PNP-a-D-mannopyranoside, PNP-a-Dxylopyranoside, ONP-b-D-glucopyranoside, ONP-b-D-galactopyranoside, ONP-b-D-fucopyranoside and ONP-a-D-galactopyranoside. two.five. Determination of kinetic parameters Kinetic research were performed with freshly purified enzymes utilizing pNPGlc at 0.110.0 mM, Rb1, Gyp XVII and Rd at concentrations from 0.two mM to five.0 mM. One particular unit of activity was defined as the amount of protein necessary to create 1 mmol of pnitrophenol or to convert 1 mmol of Rb1 or Gyp XVII or Rd per minute. All assays had been performed in triplicate. The parameters, Km and Vmax, had been determined using the enzyme kinetics program described by Cleland. two.six. Biotransformation activity of PPD ginsenosides making use of BglPm The initial biotransformation experiments making use of the main ginsenosides Rb1 and Rd as substrates revealed that the GSTfused enzyme doesn’t impact the activities of BglPm. Therefore, the fusion protein was utilized to identify the specificity and selectivity in the enzymes for the hydrolysis of the glucose moieties attached at the C3 and C20 web sites within the seven PPD ginsenosides. The enzyme solutions at a concentration of 0.1 mg/ml in 50 mM of sodium phosphate buffer have been reacted with an equal volume of Rb1, Rb2, Rb3, Rc, Rd, Gyp XVII and Rg3 answer at a concentration of 0.1% in 50 mM of sodium phosphate buffer at 37uC. The samples have been taken at common intervals and analyzed through TLC or HPLC right after pretreatment. thiogalactopyranoside using a final concentration of 0.1 mM with feeding 2% glucose. The bacterial cells had been incubated to get a additional 24 h at 18uC and have been then harvested through centrifugation at five,000 rpm for 20 min at 4uC. For the production from the recombinant BglPm, the LB medium supplemented with ampicillin was used to cultivate the E. coli harboring pGEX-bglPm inside a ten L stirred-tank reactor with a five L working volume at 12926553 500 rpm. The pH value from the medium was adjusted to 7.0 utilizing one hundred mM of sodium phosphate. The culture was incubated at 37uC till the culture reached an OD of three.0 at 600 nm. The protein expression was induced via the addition IPTG using a final concentration of 0.1 mM. The bacterial cells have been incubated for a additional 18 h at 30uC and were then harvested via centrifugation at 5,000 rpm for 20 min at 4uC. The cells suspended in 100 mM of phosphate buffer had been disrupted through sonication, and then the intact cells and debris were removed via.
