2.1 Strains, plasmids, media, and chemical reagents
All plasmids and strains used in this work are listed in Table 1. LB medium and TB medium with appropriate antibiotics were used to culture the strains. Kaempferol and astragalin were purchased from MUST Bio-Technology (Chengdu, China). Microcrystalline cellulose (Avicel PH101) was purchased from Beijing Oka Biological Technology (Beijing, China). Cellulase was purchased from Sigma-Aldrich (St Louis, USA). Peptone,tryptone, yeast extract, polyethylene glycol 2000 (PEG 2000), Tween 20, and bovine serum albumin (BSA) were purchased from Sangon Biotech (Shanghai, China).
Table 1
Plasmids and Strains Used in This Study
Plasmids/strains | Description | References |
Plasmids pGEX-78D2 | pGEX-2T carrying AtUGT78D2 from A. thaliana | [16] |
pACYCDuet-cscB-Cep-UgpA Strains BL21 (DE3) BL21-78D2-cscB-cep-UgpA | pACYCDuet carrying sucrose permease gene (cscB) from E. coli W, cep from Saccharophagus degradans and UTP-glucose-1-phosphate uridylyltransferase gene (ugpA) from Bifdobacterium bifdum, T7 promoter Escherichia coli BL21 (DE3) BL21 (DE3) harboring pGEX-78D2 and pACYCDuet-cscB-Cep-UgpA | [40] Novagen This study |
2.2 Biotransformation of kaempferol using recombinant strains
The plasmids pGEX-78D2 and pACYCDuet-cscB-Cep-UgpA were co-transformed into E. coli BL21 (DE3) cells to obtain the recombinant strain BL21-78D2-cscB-cep-UgpA. Recombinant strains were inoculated into 5 mL of fresh LB medium containing 50 µg/mL ampicillin and 35 µg/mL chloramphenicol, and incubated overnight at 37℃ with shaking at 200 rpm. The strain solution was then inoculated in 5 ml of TB medium with 50 µg/mL ampicillin and 35 µg/mL chloramphenicol and were grown at 37°C, 200 rpm until the OD600 reached 1.2 ~ 1.4. Kaempferol was dissolved in dimethyl sulfoxide (DMSO) with a concentration of 200 g/L as a stock solution. A total of 1 g/L kaempferol, 10 g/L cellobiose, 0.02 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) were added to the culture, and the fermentation broth was incubated at 30℃ with shaking at 200 rpm for 48 h. Five volumes of methanol were added to the fermentation broth. Then the samples were centrifuged at 12000 rpm for 5 min to remove the precipitate. The supernatant was analyzed using high-performance liquid chromatography (HPLC).
2.3 Optimizing biotransformation conditions for the production of astragalin
The recombinant strains were inoculated in 5 mL of TB medium containing 50 µg/mL ampicillin and 35 µg/mL chloramphenicol and grown at 37°C until the OD600 reached about 1.2 ~ 1.4. The effects of IPTG concentration (0, 0.02, 0.05, 0.1 and 0.2 mM), induction temperature (20, 25, 30, 37, 45 ℃), DMSO concentration (1, 2, 3, 5 and 10% v/v), kaempferol concentration (0.5, 1, 2, 3, and 5 g/L), and timing of kaempferol addition (0, 2, 4, 6, 8, and 10 h) on astragalin production was determined. The fermentation broth was incubated at 30℃ with shaking at 200 rpm for 48 h. The samples were analyzed using HPLC.
2.4 Effects of the ratio of cellobiose to glucose on astragalin production
The recombinant strains were inoculated in 50 mL of TB medium containing 50 µg/mL ampicillin and 35 µg/mL chloramphenicol and grown at 37°C until the OD600 reached about 1.2 ~ 1.4. The broth was incubated at 30℃ for 6 h after adding 0.02 mM IPTG. Then, 3 g/L kaempferol and different ratios of cellobiose to glucose (cellobiose : glucose = 10:0, 9:1, 8:2 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 g/L) were added to the broth for 48 h of fermentation. The samples were analyzed using HPLC.
2.5 Optimizing the conditions of microcrystalline cellulose hydrolysis
The enzymatic solution, which contained 50 mM citrate buffer (pH 5.0), 5% (w/v) microcrystalline cellulose and 10 FPU/g cellulose in 100 mL, was incubated for 48 h at 50oC and 170 rpm. The effects of temperature (25, 30, 40, 50, and 60oC), pH (3.0, 4.0, 5.0, 6.0, and 7.0), cellulase dosage (5, 10, 15, 20, and 30 FPU/g cellulose), and additives (peptone, tryptone, yeast extract, BSA, PEG 2000, and Tween 20, 0.05 g/g cellulose) on the production of cellobiose and glucose was determined.
2.6 Effects of cellulase adsorption on the production of cellobiose and glucose
The adsorption mixture contained 50 mM citrate buffer (pH 5.0), 5% (w/v) microcrystalline cellulose and 5, 10, 15, or 20 FPU/g cellulose in 100 mL, was incubated for 30 min at 0oC. The microcrystalline cellulose with cellulase was recovered by 10,000 g for 30 min. The microcrystalline cellulose with cellulase was suspended in 100 mL 50 mM citrate buffer (pH 5.0) and incubated for 48 h at 50oC and 170 rpm. Cellobiose and glucose in the enzymatic solution were determined by HPLC.
Conversion yield (%) = 0.9 * 100 * (glucose (g) + 1.053 * cellobiose (g)) / initial cellulose (g)[22]
2.7 The production of astragalin with the cellulose enzymatic solution as the carbon source
The recombinant strains were inoculated in 50 mL of TB medium containing 50 µg/mL ampicillin and 35 µg/mL chloramphenicol and grown at 37°C until the OD600 reached about 1.2 ~ 1.4. The broth was incubated at 30℃ for 6 h after adding 0.02 mM IPTG. Then, 3 g/L kaempferol and different cellulose enzymatic solutions were added to the broth for 48 h of fermentation. The samples were analyzed using HPLC.
2.8 Determination of enzyme activities
The reaction mixture for β-glucosidase contained 50 mM citrate buffer (pH 5.0), 1 mM p-nitrophenyl-β-D-glucopyranoside (pNP-G), and a certain amount of cellulase in 100 µL[23, 24]. The reaction mixture was incubated for 10 min at 50oC. The reaction was stopped by the addition of 200 µL of 1 M Na2CO3 and detected at 405 nm. The reaction mixture for filter paper unit activity (FPU) contained 50 mM citrate buffer (pH 5.0), 50 mg Whatman filter paper, and a certain amount of cellulase in 1 mL. The reaction mixture was incubated for 60 min at 50oC and estimated by dinitrosalicylic acid method (DNS) method. One unit of enzyme activity was defined as the amount of enzyme necessary to liberate 1 µmol of pNP or glucose per min under the assay conditions.
2.9 HPLC analysis
HPLC analysis was conducted on kaempferol and astragalin using an Agilent HPLC 1200 system (Santa Clara, CA) equipped with a C18 column (250 by 4.6 mm; i.d., 5 µm). The mobile phase consisted of methanol (A) and distilled water (B) with a ratio of 60:40 (A/B) for a duration of 20 minutes. The flow rate was set at 0.8 mL/min, and absorbance at 368 nm was monitored for detection.
HPLC analysis was conducted on cellobiose and glucose using an Agilent HPLC 1200 system (Santa Clara, CA) equipped with a Prevail Carbohydrate ES column (250 mm × 4.6 mm; i.d., 5µm). The mobile phase consisted of acetonitrile (A) and distilled water (B) with varying A/B ratios of 75:25, 55:45, 75:25, and 75:25, respectively, for run times of 0, 13, 13.5, and 16 minutes. The flow rate was maintained at 0.8 mL/min, and the products were detected using an evaporative light-scattering detector, operated with an airflow of 2.0 L/min at 100°C.