Submitted to: Applied Microbiology and Biotechnology
Supporting Information
Efficient two-step chemo-enzymatic synthesis of all-trans-retinyl palmitate with high substrate concentration and product yield
Authors and Their affiliation:
Zhi-Qiang Liu1, Ling-Mei Zhou1, Peng Liu1, Peter James Baker1, Shan-Shan Liu1, Ya-Ping Xue1, Ming Xu2, and Yu-Guo Zheng1*
1Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China
2Zhejiang Laiyi Biotechnology Co. Ltd., Shengzhou 312400, P.R. China
*Corresponding author:
Tel: +86-571-88320614, Fax: +86-571-88320630, E-mail:
Contents:
Material and method
Upscale production of recombinant lipase
Purification of lipase
Results
High-cell-density fermentation of P. pastoris harboring lipase genes
Lipase purification
Material and method
Upscale production of recombinant lipase
High-density fermentation of recombinant P. pastoris harboring lipase gene was performed in a 700-L fermentor. Based on the optimal parameters of shaking flasks, 257 L basal salt medium (glycerol 40 g/L, calcium sulphate 0.93 g/L, potassium sulfate 18.2 g/L, magnesium sulfate•7H2O 14.9 g/L, potassium hydroxide 4.13 g/L, and 85% phosphoric acid 26.7 mL/L) was recruited. 50% glycerol was added into the fermentor at the rate of 4.7 L/h for 8 h in yeast growth stage. When the glycerol was consumed, the fed-batch medium containing methanol and PTM1 Trace Salts (40:1, v/v) was fed, stepwise. PTM1 Trace Salts was composed of cupric sulfate•5H2O (6.0 g/L), sodium iodide (0.08 g/L), manganese sulfate•H2O (3.0 g/L), sodium molybdate•2H2O (0.2 g/L), boric acid (0.02 g/L), cobalt chloride (0.5 g/L), zinc chloride (20.0 g/L), ferrous sulfate•7H2O (65 g/L), biotin (0.2 g/L), and sulfuric acid (5.0 mL/L). Methanol was added to induce the lipase production. Meanwhile, tryptone (0.5%, w/v) and yeast extract (0.25%, w/v) were added to extend the induction time at an interval of 24 h. Ammonium hydroxide was batch-fed to keep the pH value at 5.5 in the fermentation process. In order to maintain over 20% of air saturation, dissolved oxygen (DO) was controlled by changing agitation speed, airflow stepwise and the rate of methanol. The airflow rate was set at 10 L/min. The stirring speed and temperature were controlled at 800 rpm/min and 28 °C, respectively.
Purification of lipase
After the fermentation, cells were separated from the fermentation broth by centrifugation at 12,000 rpm for 20 min. The supernatant was collected and stored at 4 °C for further treatment. Hollow fiber membrane with a diameter of below 0.1 µm was firstly used for the treatment of supernatant to remove the low suspended solids. Then an ultrafiltration unit (Millipore TFE system) with a 10 kDa membrane was used for concentrating the supernatant. The concentrate was then washed twice with distilled water. To obtain a substantially pure enzyme, the purification process was further performed using Nickel-affinity chromatography system with a 16 mmD×100 mmL POROS MC 20 mm column (Applied Biosystems Co., Foster City CA 94404, USA). 7 mL of enzyme sample was loaded on the POROS MC 20 mm column. A starting buffer (50 mM NaH2PO4 buffer with 0.5 mM imidazole, pH 8.0) was applied to remove unbound proteins. The column was eluted with 50 mM NaH2PO4 buffer, pH 8.0, containing 300 mM imidazole. The eluted fraction was collected and dialyzed to remove salt in distilled water. All eluted fractions were assayed both for lipase activity as well as total protein. The specific activity and purification fold were calculated.
Results
High-cell-density fermentation of recombinant P. pastoris harboring lipase genes
The higher amount of recombinant lipase by P. pastoris harboring lipase gene was achieved through fed-batch fermentation in a 700-L fermentor. The lipase activity and growth curves were shown in Fig. S1. The maximum growth rate was typically observed during the early stages of cultivation. The growth rate slowed gradually when methanol as inducer was added into the fermentor after 45 h of cultivation. This was because methanol was toxic to the cells and reduced the cells growth. The activity of lipase increased rapidly after induction and reached a peak at 151 h. At the end of fermentation, the wet cell concentration reached 311 g/L and the lipase activity reached 8.0 U/mL.
The enzyme production and cell biomass have certain correlation in recombinant P. pastoris cultivating process. The cell biomass was improved, which further contributed to the enzyme production. The glycerol could increase the wet cell concentration in fermented liquid. 1.5 folds of the original amount of glycerol were selected to add and the result was shown in Fig. S2. In the adding process of glycerol wet cell concentration increased rapidly, achieving at 230 g/L after cultivating 50 h. At the end of the fermentation, lipase activity reached 10 U/ml. Thus the excess adding of glycerol can improve the wet cell concentration at the beginning stage of fermentation, but has no impact to the final wet cell concentration and lipase activity.
The growth of recombinant P. pastoris is aerobiotic, and it secreted proteases which degraded the secreted proteins. It was reported that the adding of yeast extract and peptone can effectively reduce the degradation of secreted proteins. 0.5% (w/v) of yeast extract and 1% (w/v) of peptone were combined to add, one time every 48 h (3 times the whole fermentation process). The results showed (Fig. S3) that wet cell concentration reached 311 g/L and lipase activity reached 18 U/ml after adding tryptone and yeast extract. Thus in the process of high density cultivation of recombinant P. pastoris, the excess amount of tryptone and yeast extract can inhibit the degradation of lipase, improving the lipase activity.
Lipase purification
The hydrolysis activity of recombinant enzyme and concentration of protein were determined respectively for each purification step. The result of enzyme purification was listed in Table S1. The purity of protein was ascertained by using SDS-PAGE (Fig. S4). The molecular weight of lipase was about 37 kDa. The activity assay showed that specific activity was enhanced up to 1.1 folds, and lipase yield was 84.3% after ultrafiltration. Affinity chromatography could further improve the specific activity to 1.8 folds, and the final yield of lipase was 81.6%.
Table S1. The efficiency of purification of recombinant lipase.
Steps / Total protein (mg) / Total activity (U) / Specific activity (U/mg) / Purification (fold) / Yield (%)Crude enzyme / 1072.2 / 4758.2 / 4.4 / 1 / 100
Ultrafiltration / 861.3 / 4013.2 / 4.7 / 1.1 / 84.3
Affinity chromatography / 480.6 / 3880.6 / 8.1 / 1.8 / 81.6
Table S2. The influence of organic solvent for the immobilized lipase.
organic solvent / Log P / Specific activity (U/g) / Residual activity (%)Control / / / 1020.5 / 100
Anhydrous ethanol / -0.24 / 875.4 / 85.8
Butyl acetate / 1.7 / 956.4 / 93.7
Cyclohexane / 3.2 / 998.8 / 97.9
Petroleum ether / ≈3.5 / 987.1 / 96.7
n-Hexane / 3.5 / 979.1 / 96.0
n-Heptane / 4 / 989.2 / 96.9
Isooctane / 4.7 / 984.4 / 96.5
Dodecane / 6.6 / 981.7 / 96.2
Figure captions:
Fig. S1. The lipase activity and cell growth curve of recombinant strain through fed-batch fermentation in a 700-L fermentor.
Fig. S2. Effects of adding the feeding amount of glycerol on the fermentation process.
Fig. S3. Effects of adding yeast extracts and peptone on the fermentation process.
Fig. S4. The SDS-PAGE of the recombinant lipase. M: molecular mass marker proteins; Lane 1: purified enzyme; Lane 2: samples after concentration; Lane 3: crude enzyme; Lane 4: non-target protein.
Fig. S5. HPLC Chromatograms. A) Retinyl acetate, B) Standard of retinol, C) Incomplete hydrolysis of retinyl acetate and D) Complete hydrolysis of retinyl acetate to retinol.
Fig. S6. HPLC Chromatograms. A) Standard of retinyl palmitate, B) Purified retinyl palmitate, C) Standard of all-trans-retinyl palmitate and D) Purified all-trans-retinyl Palmitate. The retention times for retinyl palmitate and all-trans-retinyl palmitate are about 8.0 and 4.4 min, respectively.
Fig. S7. FTIR infrared spectra. (A) Purified all-trans-retinyl palmitate and (B) Standard of all-trans-retinyl palmitate.
Fig. S8. MS spectra. (A) Purified all-trans-retinyl palmitate, (B) Standard of all-trans-retinyl palmitate.
Fig. S9. NMR spectra. (A) 1H NMR spectra of purified all-trans-retinyl palmitate, (B) 1H NMR spectra of standard of all-trans-retinyl palmitate, (C) 13C NMR spectra of purified all-trans-retinyl palmitate and (D) 13C NMR spectra of standard of all-trans-retinyl palmitate.
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