Submitted to: Applied Microbiology and Biotechnology
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Supporting Information
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Efficient two-step chemo-enzymatic synthesis of all-trans-retinyl palmitate with
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high substrate concentration and product yield
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Authors and Their affiliation:
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Zhi-Qiang Liu1, Ling-Mei Zhou1, Peng Liu1, Peter James Baker1, Shan-Shan Liu1, Ya-Ping Xue1,
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Ming Xu2, and Yu-Guo Zheng1*
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Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China
Zhejiang Laiyi Biotechnology Co. Ltd., Shengzhou 312400, P.R. China
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*Corresponding author:
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Tel: +86-571-88320614, Fax: +86-571-88320630, E-mail: zhengyg@zjut.edu.cn
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Contents:
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Material and method
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Upscale production of recombinant lipase
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Purification of lipase
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Results
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High-cell-density fermentation of P. pastoris harboring lipase genes
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Lipase purification
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Material and method
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Upscale production of recombinant lipase
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High-density fermentation of recombinant P. pastoris harboring lipase gene was performed
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in a 700-L fermentor. Based on the optimal parameters of shaking flasks, 257 L basal salt
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medium (glycerol 40 g/L, calcium sulphate 0.93 g/L, potassium sulfate 18.2 g/L,
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magnesium sulfate•7H 2O 14.9 g/L, potassium hydroxide 4.13 g/L, and 85% phosphoric
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acid 26.7 mL/L)
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4.7 L/h for 8 h in yeast growth stage. When the glycerol was consumed, the fed -batch
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medium containing methanol and PTM1 Trace Salts (40:1, v/v) was fed, stepwise. PTM1
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Trace Salts was composed of cupric sulfate•5H 2O (6.0 g/L), sodium iodide (0.08 g/L),
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manganese sulfate•H2O (3.0 g/L), sodium molybdate•2H 2O (0.2 g/L), boric acid (0.02 g/L),
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cobalt chloride (0.5 g/L), zinc chloride (20.0 g/L), ferrous sulfate•7H 2O (65 g/L), biotin
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(0.2 g/L), and sulfuric acid (5.0 mL/L). Methanol was added to induce the lipase
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production. Meanwhile, tryptone (0.5%, w/v) and yeast extract (0.25%, w/v) were added to
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extend the induction time at an interval of 24 h. Ammonium hydroxide was batch-fed to
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keep the pH value at 5.5 in the fermentation process. In order to maintain over 20% of air
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saturation, dissolved oxygen (DO) was controlled by changing agitation speed, airflow
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stepwise and the rate of methanol. The airflow rate was set at 10 L/min. The stirring speed
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and temperature were controlled at 800 rpm/min and 28 °C, respectively.
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Purification of lipase
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After the fermentation, cells were separated from the fermentation broth by centrifugation
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at 12,000 rpm for 20 min. The supernatant was collected and stored at 4 °C for further
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treatment. Hollow fiber membrane with a diameter of below 0.1 µm was firstly used for the
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treatment of supernatant to remove the low suspended solids. Then an ultrafiltration unit
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(Millipore TFE system) with a 10 kDa membrane was used for concentrating the
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supernatant. The concentrate was then washed twice with distilled water. To obtain a
was recruited. 50% glycerol was added into the fermentor at the rate of
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substantially pure enzyme, the purification process was further performed using
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Nickel-affinity chromatography system with a 16 mmD×100 mmL POROS MC 20 mm
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column (Applied Biosystems Co., Foster City CA 94404, USA). 7 mL of enzyme sample
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was loaded on the POROS MC 20 mm column. A starting buffer (50 mM NaH 2PO4 buffer
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with 0.5 mM imidazole, pH 8.0) was applied to remove unbound proteins. The column was
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eluted with 50 mM NaH 2PO4 buffer, pH 8.0, containing 300 mM imidazole. The eluted
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fraction was collected and dialyzed to remove salt in distilled water. All eluted fractions
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were assayed both for lipase activity as well as total protein. The specific activity and
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purification fold were calculated.
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Results
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High-cell-density fermentation of recombinant P. pastoris harboring lipase genes
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The higher amount of recombinant lipase by P. pastoris harboring lipase gene was
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achieved through fed-batch fermentation in a 700-L fermentor. The lipase activity and
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growth curves were shown in Fig. S1. The maximum growth rate was typically observed
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during the early stages of cultivation. The growth rate slowed gradually when methanol as
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inducer was added into the fermentor after 45 h of cultivation. This was because methanol
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was toxic to the cells and reduced the cells growth. The activity of lipase increased rapidly
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after induction and reached a peak at 151 h. At the end of fermentation, the wet cell
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concentration reached 311 g/L and the lipase activity reached 8.0 U/mL.
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The enzyme production and cell biomass have certain correlation in recombinant P.
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pastoris cultivating process. The cell biomass was improved, which further contributed to
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the enzyme production. The glycerol could increase the wet cell concentration in fermented
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liquid. 1.5 folds of the original amount of glycerol were selected to add and the result was
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shown in Fig. S2. In the adding process of glycerol wet cell concentration increased rapidly,
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achieving at 230 g/L after cultivating 50 h. At the end of the fermentation, lipase activity
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reached 10 U/ml. Thus the excess adding of glycerol can improve the wet cell
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concentration at the beginning stage of fermentation, but has no impact to the final wet cell
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concentration and lipase activity.
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The growth of recombinant P. pastoris is aerobiotic, and it secreted proteases which
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degraded the secreted proteins. It was reported that the adding of yeast extract and peptone
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can effectively reduce the degradation of secreted proteins. 0.5% (w/v) of yeast extract and
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1% (w/v) of peptone were combined to add, one time every 48 h (3 times the whole
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fermentation process). The results showed (Fig. S3) that wet cell concentration reached 311
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g/L and lipase activity reached 18 U/ml after adding tryptone and yeast extract. Thus in the
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process of high density cultivation of recombinant P. pastoris, the excess amount of
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tryptone and yeast extract can inhibit the degradation of lipase, improving the lipase
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activity.
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Lipase purification
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The hydrolysis activity of recombinant enzyme and concentration of protein were determined
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respectively for each purification step. The result of enzyme purification was listed in Table S1.
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The purity of protein was ascertained by using SDS-PAGE (Fig. S4). The molecular weight of
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lipase was about 37 kDa. The activity assay showed that specific activity was enhanced up to 1.1
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folds, and lipase yield was 84.3% after ultrafiltration. Affinity chromatography could further
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improve the specific activity to 1.8 folds, and the final yield of lipase was 81.6%.
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Table S1. The efficiency of purification of recombinant lipase.
Steps
Total
Total activity
Specific activity
Purification
Yield
protein
(U)
(U/mg)
(fold)
(%)
(mg)
Crude enzyme
1072.2
4758.2
4.4
1
100
Ultrafiltration
861.3
4013.2
4.7
1.1
84.3
Affinity
480.6
3880.6
8.1
1.8
81.6
chromatography
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Table S2. The influence of organic solvent for the immobilized lipase.
organic solvent
Log P
Specific activity
Residual activity
(U/g)
(%)
Control
/
1020.5
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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
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Figure captions:
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Fig. S1. The lipase activity and cell growth curve of recombinant strain through fed-batch fermentation
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in a 700-L fermentor.
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Fig. S2. Effects of adding the feeding amount of glycerol on the fermentation process.
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Fig. S3. Effects of adding yeast extracts and peptone on the fermentation process.
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Fig. S4. The SDS-PAGE of the recombinant lipase. M: molecular mass marker proteins; Lane 1:
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purified enzyme; Lane 2: samples after concentration; Lane 3: crude enzyme; Lane 4: non-target
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protein.
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Fig. S5. HPLC Chromatograms. A) Retinyl acetate, B) Standard of retinol, C) Incomplete hydrolysis of
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retinyl acetate and D) Complete hydrolysis of retinyl acetate to retinol.
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Fig. S6. HPLC Chromatograms. A) Standard of retinyl palmitate, B) Purified retinyl palmitate, C)
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Standard of all-trans-retinyl palmitate and D) Purified all-trans-retinyl Palmitate. The retention times
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for retinyl palmitate and all-trans-retinyl palmitate are about 8.0 and 4.4 min, respectively.
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Fig. S7. FTIR infrared spectra. (A) Purified all-trans-retinyl palmitate and (B) Standard of
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all-trans-retinyl palmitate.
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Fig. S8. MS spectra. (A) Purified all-trans-retinyl palmitate, (B) Standard of all-trans-retinyl palmitate.
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Fig. S9. NMR spectra. (A) 1H NMR spectra of purified all-trans-retinyl palmitate, (B) 1H NMR spectra
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of standard of all-trans-retinyl palmitate, (C)
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and (D) 13C NMR spectra of standard of all-trans-retinyl palmitate.
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C NMR spectra of purified all-trans-retinyl palmitate
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Wet cell concentration
Lipase activity
280
210
6
140
4
70
2
0
0
137
138
8
20
40
60
0
80 100 120 140 160 180
Time (min)
Fig. S1
Lipase activity (U/mL)
Wet cell concentration (g/L)
350
5
141
9
100
Concentration
OD600
120
6
50
Lipase activity
pH
60
3
20
40
60
0
80 100 120 140 160 180
Time (min)
Fig. S2
0
4
3
pH
180
150
0
140
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Lipase activity (U/ml)
240
200
0
139
15
250
OD600
Wet cell concentration (g/L)
300
2
1
0
240
16
12
160
160
80
0
0
Concentration
OD600
80
Lipase activity
pH
0
20 40 60 80 100 120 140 160 180
Time (min)
142
143
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Fig. S3
8
4
0
4
3
pH
240
5
Lipase activity (U/ml)
320
OD600
Wet cell concentration (g/L)
20
320
2
1
0
145
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Fig. S4
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(A)
(B)
Retinol
Retinyl acetate
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(C)
(D)
Retinyl
acetate
Retinol
Retinol
150
151
Fig. S5
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(A)
Retinyl palmitate
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(B)
Retinyl palmitate
Retinol
155
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(C)
(D)
All-trans-retinyl
palmitate
All-transretinyl palmitate
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158
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Fig. S6
160
(A)
100
90
516.03
80
70
%T
60
721.32
50
40
30
1114.05
20
1358.02
1453.22
1738.84
1161.65 968.26
2848.59
10
0
-10
4000
161
162
3000
2000
Wave numbers (cm-1)
1000
(B)
100
90
516.03
80
70
721.32
%T
60
50
40
30
1114.05
20
2848.59
10
1738.84
0
163
164
-10
4000
3000
2000
Wave numbers (cm-1)
Fig. S7
1358.02
1453.22
1161.65 968.26
1000
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(A)
1#
HQ
Shimadzu Biotech Axima Performance 2.8.4.20081127: Mode Reflectron, Power: 116, Blanked, P.Ext. @ 500 (bin 50)
%Int.
9.4 mV[sum= 311 mV]
Profiles 1-33 Smooth Av 5 -Baseline 80
524.3
100
90
540.3
80
480.3
70
60
50
439.0
541.3
40
30
481.3
20
437.9
10
410.9
486.6
444.2
557.3
556.3
526.3
709.0
629.1
0
360
380
400
420
440
460
480
500
520
540
560
166
167
580
m/z
600
620
640
660
680
700
720
740
760
780
800
740
760
780
800
(B)
2#
HQ
Shimadzu Biotech Axima Performance 2.8.4.20081127: Mode Reflectron, Power: 116, Blanked, P.Ext. @ 500 (bin 50)
%Int.
9.4 mV[sum= 311 mV]
Profiles 1-33 Smooth Av 5 -Baseline 80
524.3
100
90
540.3
80
480.3
70
60
50
439.0
541.3
40
30
481.3
20
437.9
10
410.9
486.6
444.2
557.3
556.3
526.3
709.0
629.1
0
360
380
400
420
440
460
480
500
520
540
560
168
169
170
Fig. S8
580
m/z
600
620
640
660
680
700
720
171
(A)
172
173
174
(B)
(C)
(D)
Fig. S9
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