Development and Optimization of an In Vitro Multienzyme Synthetic

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Bioactive Constituents, Metabolites, and Functions

Development and Optimization of an in vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin Zhiping Zhang, Yanzhi He, Yue Huang, Li Ding, Lei Chen, Yaxian Liu, Yesen Nie, and Xinyue Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01299 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Development and Optimization of an in vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin Zhiping Zhang1, #, Yanzhi He1, #, Yue Huang1, Li Ding1, Lei Chen1, Yaxian Liu1, Yesen Nie1, Xinyue Zhang1, 2, 3, 4, * 1

College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu 225009, China.

2

Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for

Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University (26116120), Yangzhou, Jiangsu 225009, China. 3

Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University,

Yangzhou, Jiangsu 225009, China. 4

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases

and Zoonoses, Yangzhou, Jiangsu 225009, China.


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ABSTRACT

2

An in vitro multienzyme synthetic system was developed and optimized to efficiently

3

produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the

4

biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and

5

overexpressed in Escherichia coli. The recombinant proteins were purified through affinity

6

chromatography and showed activities of flavanone 3-hydroxylase and flavonol synthase,

7

respectively, followed by development of an in vitro synthetic system for producing

8

kaempferol. The system contains 8.2 mM α-ketoglutaric acid, 0.01 mM ferrous ion, 25

9

µg/mL of each recombinant enzyme, and 10% glycerol in 100 mM Tris-HCl (pH 7.2).

10

When the reaction was carried out at 40 °C for 40-50 min, the yield of kaempferol was

11

37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%.

12

Overall, this system provides a promising and efficient approach to produce kaempferol

13

economically.

14

15

16

17

Key Words: kaempferol, in vitro synthesis, optimization, flavanone 3-hydroxylase,

18

flavonol synthase.

19

20



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21

22

Flavonoids are a large group of plant-derived natural products and can be further classified

23

into six major subgroups depending on the variations in the heterocyclic C-ring: the

24

catechins or flavanols, flavones, flavonols, flavanones, anthocyanidins, and isoflavones 1-3.

25

These compounds show a wide range of biological activities

26

of studies on these secondary metabolites 7-12.

27

Kaempferol (KMF), also known as robigenin or 3,4’,5,7-tetrahydroxyflavone, is a natural

28

flavonol possessing numerous pharmacological and biological properties including

29

antioxidant, anti-inflammatory, anticancer, antimicrobial, cardioprotective, neuroprotective,

30

antidiabetic, anti-osteoporotic, estrogenic/anti-estrogenic, anxiolytic, analgesic, and

31

anti-allergic activities

32

However, the cost of KMF production is high due to the very low content of KMF in plants

33

15, 16

34

producing KMF.

35

Many methods have been developed to synthesize flavonoids

36

synthesis of flavonoids often requires multiple steps, toxic reagents, and extreme reaction

37

conditions

38

another crucial challenge in chemical synthesis. Hence, chemical synthesis is not

39

economically feasible for large-scale production of flavonoids 1.

40

Since the biosynthetic pathway of flavonoids has been successfully elucidated in plants 2, it

41

is a promising alternative strategy to synthesize these natural compounds using a microbial

42

cell factory. Recently, some flavonoids have been successfully biosynthesized by

INTRODUCTION

3-6

, leading to quite a number

13, 14

. KMF is mainly produced from traditional plant extraction.

. Therefore, it is crucial to develop alternative strategies to reduce the cost for

17, 18

. However, chemical

1, 17

. In addition, the chiral synthesis to produce active flavonoid compounds is


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43

engineered microbes, mainly including Escherichia coli and Saccharomyces cerevisiae 19-23.

44

For example, Duan, et al have constructed a microbial cell factory for KMF production by

45

introducing a biosynthetic pathway of KMF into the budding yeast S. cerevisiae and

46

achieved the highest production of KMF at 66.29 mg/L

47

microbes produce desired products due to the well-known complexity of a cellular system,

48

incompatibility of artificially synthesized genetic elements and hosts, inhibition of host

49

cells by target products, and instability of an engineered biosystem 25.

50

Another promising alternative strategy is to produce flavonoids using an in vitro

51

multienzyme synthetic system. Recently, Cheng, et al have reported the successful

52

multienzyme synthesis of enterocin polyketides by assembling a complete type II

53

polyketide synthase enzymatic pathway to natural products in a single reaction vessel

54

This cell-free synthetic biotechnology overcomes the disadvantages of a microbial cell

55

factory and is economically feasible for commercial-scale production of flavonoids 25.

56

As we know, there are two key enzyme genes, f3h and fls1, involved in the biosynthetic

57

pathway of KMF 1, 2. These two genes encode flavanone 3-hydroxylase (F3H) and flavonol

58

synthase (FLS), respectively. In this article, we report a detailed study of the in vitro

59

multienzyme synthesis of KMF in a single reaction tube from cheap chemicals including

60

the substrate naringenin (NRN). Firstly, we cloned Atf3h and Atfls1 genes from Arabidopsis

61

thaliana and expressed them in E. coli BL21(DE3), followed by the purification of

62

recombinant proteins. Then, we determined the enzyme activities of the purified

63

recombinant proteins and their kinetic parameters. Finally, we developed a single reaction

64

system to produce KMF and optimized a series of reaction parameters including pH value,

24

. However, not all engineered


26

.


65

reaction temperature and time, and total amount and ratio of two recombinant enzymes to

66

find the most efficient and economic conditions for KMF production. To our best

67

knowledge, there is no report on the development of an in vitro multienzyme synthetic

68

system to produce KMF.

69

MATERIALS AND METHODS

70

Plants, Bacterial Strains, Plasmids, and Chemicals. Four-week-old seedlings of

71

Arabidopsis thaliana were a gift from Dr. Lin Wang of Yangzhou University. Escherichia

72

coli DH5α and BL21(DE3) were purchased from Beijing CoWin Biotech Co., Ltd. (Beijing,

73

China) and used for plasmid maintenance and gene expression, respectively. The bacteria

74

were cultured in Luria–Bertani (LB) broth or on agar supplemented with 100 µg/mL

75

ampicillin when necessary. Prokaryotic expression vector pET-32a(+) (Novagen) was used

76

for cloning purposes. Authentic naringenin (NRN), dihydrokaempferol (DHK), kaempferol

77

(KMF), and other chemicals are products of Sigma-Aldrich (St. Louis, MO). Restriction

78

enzymes were bought from New England Biolabs (Hertfordshire, UK). T4 DNA ligase was

79

a product of Vazyme Biotech Co., Ltd (Nanjing, China). TRIzol® Reagent and Isopropyl

80

β-D-1-thiogalactopyranoside (IPTG) were purchased from Thermo Fisher Scientific Inc.

81

(MA, USA).

82

Buffers used. The buffers used for enzyme assays in this study were prepared based on

83

the published recipes with slight modifications 27, 28. F3H buffer contains 100 mM Tris-HCl

84

(pH 7.5), 0.4% sodium ascorbate, 10% glycerol, 0.174 mM α-ketoglutaric acid (α-KG), and

85

0.1 mM FeSO4. FLS buffer contains 100 mM Tris-HCl (pH 7.2), 0.4% sodium ascorbate,

86

10% glycerol, 8.2 mM α-KG, and 0.01 mM FeSO4. Based on the F3H and FLS buffers, we


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designed a panel of KMF synthetic buffers consisting of 100 mM Tris-HCl (pH 7.2 or 7.5),

88

10% glycerol, 0.4% sodium ascorbate, 0.174 mM or 8.2 mM α-KG, and 0.01 mM or 0.1

89

mM FeSO4, as shown in Table 1.

90

Isolation and reverse transcription of total RNA and construction of plasmids. Total

91

RNA was isolated from seedlings of A. thaliana using TRIzol® Reagent and reversely

92

transcribed into complementary DNA (cDNA) using the SuperRT cDNA Synthesis Kit

93

(Beijing CoWin Biotech Co., Ltd., Beijing, China) according to the manufacturer’s

94

protocol. The cDNAs were used for cloning Atf3h (Genbank accession no.

95

NM_001203121.1) and Atfls1 (Genbank accession no. NM_120951.3) genes. All primers

96

used in a specific polymerase chain reaction (PCR) are listed in Table 2 and were

97

synthesized by GENEWIZ, Suzhou, China. The Atf3h and Atfls1 genes were

98

PCR-amplified and cloned into pET-32a(+) following a standard protocol as described

99

elsewhere

100

29

. The DNA sequences of cloned Atf3h and Atfls1 genes were determined by

GENEWIZ, Suzhou, China.

101

Expression and purification of recombinant proteins. The recombinant plasmids were

102

transformed into BL21(DE3) and colonies were inoculated into LB broth containing 100

103

µg/mL ampicillin. Three milliliters of overnight culture were reinoculated into 150 mL LB

104

broth containing 100 µg/mL ampicillin and cultured at 220 rpm and 37 °C until the optical

105

density at 600 nm was between 0.4 – 0.6. Then, 0.2 mM IPTG was added into the culture to

106

induce gene expression at 20 – 22 °C for 3 h. The bacteria were harvested and resuspended

107

in 1/20 volume of bacterial lysis buffer (50 mM Tris-Cl, pH8.0, 0.1% Triton X-100, 1 mM

108

EDTA, 10% Glycerol, 150 mM NaCl, 0.5 mM DTT, and 0.1 mM PMSF, 1 µg/mL aprotinin,



109

1 µg/mL leupeptin, and 1 µg/mL pepstatin). The suspension was sonicated and

110

centrifugated at 13000 g for 10 min at 4 °C. TrxA-His-tagged recombinant proteins were

111

purified from the supernatant using Ni-IDA Agarose Resins (Beijing CoWin Biotech Co.,

112

Ltd., Beijing, China). Purification was performed according to the manufacturer’s protocol.

113

The purity of the purified proteins was analyzed on a 10% sodium dodecyl sulfate

114

polyacrylamide gel electrophoresis (SDS-PAGE) gel and the bands were visualized by

115

coomassie blue staining.

116

Enzyme assays. The enzyme assays were analyzed according to the reported protocols 27, 28

117

with slight modifications

, and the final volumes for all assays were 100 µL. In brief,

118

the F3H assay contained 0.5 mM NRN and 3.3 µg TrxA-His-AtF3H in F3H Buffer, and the

119

FLS assay included 0.5 mM DHK and 1.9 µg TrxA-His-AtFLS1 in FLS Buffer. The assays

120

were incubated at 30 °C in open 2-mL tubes with continuous shaking for 45 min or 3 h,

121

respectively. The assays were terminated with 100 µL of ethyl acetate and 10 µL of acetic

122

acid. Two hours later, the organic phases were transferred to 1.5 mL tubes for vacuum

123

freeze drying in a FreeZone 1 Liter Benchtop Freeze Dry System (Labconco, MO, USA).

124

The isolated flavonoids were then redissolved in methanol and subjected to polyamide thin

125

layer chromatography (TLC), high performance liquid chromatography (HPLC), and

126

electrospray ionization mass spectrometry (ESI-MS) analyses. The dots on the TLC plates

127

were visualized under a Tanon-2500 Gel Imaging System (Tanon Science & Technology

128

Co. Ltd., Shanghai, China).

129

To determine the enzyme activity of the TrxA-His-AtF3H, the concentration of NRN

130

was changed into 0.1 mM and the assay was incubated at 30 °C for 45 min. To determine


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that of the TrxA-His-AtFLS1, the concentration of DHK was changed into 0.08 mM and

132

the assay was incubated at 30 °C for 30 min. The calibration curves of authentic DHK and

133

KMF were made based on the HPLC data and the product amount was thus calculated. The

134

enzyme activity was evaluated from three individual experiments and expressed as the

135

amount of the recombinant protein needed to convert 1 µmol substrate into the product at

136

30 °C within one minute. The values were expressed as mean ± standard deviation.

137

Determination of the Michaelis-Menten constant Km of the recombinant enzymes.

138

To determine the Michaelis-Menten constant of the TrxA-His-AtF3H, a series of NRN

139

concentrations were prepared in the F3H assay, including 0.04 mM, 0.06 mM, 0.08 mM,

140

0.10 mM, and 0.12 mM. To determine that of the TrxA-His-AtFLS1, the concentrations of

141

DHK included 0.01 mM, 0.02 mM, 0.04 mM, 0.08 mM, and 0.16 mM in the FLS assay,

142

respectively. Then, the Michaelis-Menten constants were calculated using the data analysis

143

software OriginPro (version 9.0.0). The measurements were repeated three times and the

144

Km value of each recombinant enzyme was expressed as mean ± standard deviation.

145

Development and optimization of an in vitro multienzyme synthetic system for KMF

146

production. Based on the components in F3H and FLS buffers, a KMF multienzyme

147

synthetic system was developed to produce KMF from NRN in a single reaction tube. The

148

system contained 0.5 mM NRN, 2.0 µg TrxA-His-AtF3H, and 2.0 µg TrxA-His-AtFLS1 in

149

100 µL of various KMF synthetic buffers as shown in Table 1. The reactions were

150

incubated at 30 °C with continuous shaking for 3 h and terminated by addition of 100 µL of

151

ethyl acetate and 10 µL of acetic acid. Two hours later, the organic phase was subjected to

152

vacuum freeze drying. The powder was dissolved in methanol and analyzed by polyamide



153

TLC and HPLC-MS.

154

After initial setup of the synthetic system, the reaction parameters were further

155

investigated to maximize the efficiency of KMF production. Briefly, the pH value and the

156

concentrations of α-KG and FeSO4 in the reaction system were optimized according to

157

Table 1 by incubating the reaction tubes at 30 °C for 3 h. After the pH value and the

158

concentrations of α-KG and FeSO4 were fixed at an optimum value, the reaction time was

159

optimized by incubating the reaction mixture at 30 °C with stirring for 0, 10, 20, 30, 40, 50,

160

and 60 min. Next, the optimum reaction temperature was explored by incubating the

161

reaction mixture at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C for an optimum reaction time.

162

Then, the optimum ratio of the recombinant enzymes was analyzed by setting the ratio of

163

TrxA-His-AtF3H and TrxA-His-AtFLS1 at 1:3, 1:2, 1:1, 2:1, and 3:1. Finally, the total

164

amount of TrxA-His-AtF3H and TrxA-His-AtFLS1 was optimized by adding 1 µg, 2 µg, 3

165

µg, 4 µg, 5 µg, and 6 µg of the enzymes at an optimum ratio into a 100 µL of reaction

166

mixture. All measurements were repeated three times and the results were expressed as

167

mean ± standard deviation. TLC analysis. Analysis of flavonoid compounds by TLC was widely reported elsewhere

168 169

30

170

(Sinopharm Chemica Reagent Co., Ltd, Shanghai, China) in comparison to authentic

171

samples. The sample-loaded TLC plates were run in a solvent system consisting of

172

chloroform/methanol/ethyl acetate/formic acid at a ratio of 5.0:1.5:1.0:0.5. Then, the plates

173

were sprayed with 1% ethanolic solution of aluminum chloride (AlCl3) and subjected to

174

UV illumination at 254 nm to visualize spots. The gray value of each spot was analyzed by

. In brief, the reaction products were primarily analyzed by TLC in polyamide 6 plates


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175


the image processing software ImageJ 1.51j8.

176

HPLC-MS analysis. The HPLC-MS was performed to analyze the synthesized

177

flavonoid compounds according to the published work 31 with slight modifications. In brief,

178

the samples were processed sequentially through 0.45 µm and 0.22 µm filters and analyzed

179

using an Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole

180

LC/MS system. Data were collected and analyzed using Agilent MassHunter Workstation

181

(version B.02.00) and MassHunter Quantitative analysis software (version B.01.04),

182

respectively. The samples were separated at 30 °C using an Agilent C18 column (150×4.6

183

mm, 5 µm, Thermo Fisher Scientific. Inc., CA, USA). The separation was achieved using a

184

combination of two liquids, distilled water and acetonitrile. The column was eluted at 0.2

185

mL/min by a 10% – 85% (v/v) gradient of acetonitrile in water under the following

186

conditions: 0 – 10 min, a linear gradient of 10% – 25% (v/v) acetonitrile concomitant with

187

90-75% water; 10 – 35 min, a linear gradient of 25% – 50% acetonitrile; 35 – 45 min, a

188

linear gradient of 50% – 85% acetonitrile; 45 – 50 min, a linear gradient of 85% – 10%

189

acetonitrile; 50 – 60 min, 10% acetonitrile in 95% water. Absorbance of the eluate was

190

monitored from 200 nm to 800 nm. Single wavelength chromatograms at 280 nm, 320 nm,

191

and 370 nm were extracted using the software Xcalibur v2.0.7. The reaction products were

192

quantified by comparing the peak area of a compound in the reaction sample with that of a

193

known amount of an authentic compound.

194

Isolation of KMF. The isolation of KMF was a modification of the method developed

195

by Leite, et al. 32. In brief, the reaction was terminated by adding an equal volume of ethyl

196

acetate. The organic phase was transferred to 1.5 mL tubes for vacuum freeze drying as



197

described above. The dried powder was re-dissolved in methanol and processed through a

198

0.45 µm filter. Next, the solution was subjected to chromatography over a Sephadex LH-20

199

column eluted with methanol at 0.5 mL/min. The fractions were collected and combined

200

together according to the results of TLC analysis. The isolated KMF was further confirmed

201

by HPLC-MS analysis as described above and used for calculating the yield.

202

RESULTS AND DISCUSSION

203

Cloning, expression and purification of the key enzymes for KMF biosynthesis. As

204

shown in Figure 1, AtF3H and AtFLS1 are two key enzymes for the biosynthesis of KMF

205

in plants. To develop an in vitro system for multienzyme synthesis of KMF, we first cloned

206

Atf3h and Atfls1 genes from A. thaliana into prokaryotic expression vectors.

207

Generally speaking, soluble expression of recombinant proteins in bacteria maintains its

208

enzymatic activity. To increase the relative amount of soluble recombinant proteins

209

expressed in E. coli, we first compared the difference between pET-32a(+) and pGEX-5X-1,

210

two common prokaryotic expression vectors, in expressing recombinant proteins AtF3H

211

and AtFLS1. We found that pET-32a(+) provided a higher level of soluble AtF3H and

212

AtFLS1 proteins, which possibly resulted from the role of TrxA fragment in pET-32a(+) in

213

promoting soluble expression. Therefore, we chose pET-32a(+) as a vector for constructing

214

recombinant plasmids. Sequence analysis verified that there was no mutation occurring in

215

the PCR-generated DNA fragments.

216

Then, we transformed the recombinant plasmids into E. coli strain BL21(DE3) and

217

optimized the expression conditions mainly by changing two important induction

218

parameters – the induction temperature and the concentration of IPTG. When we set the


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temperature at 20 – 22 °C and the IPTG concentration at 0.1 – 0.2 mM, we found that the

220

bacteria expressed a high amount of soluble recombinant proteins. Then, we induced the

221

expression for 3 h and purified the recombinant proteins by affinity chromatography

222

through a nickel iminodiacetic acid (Ni-IDA) agarose resin. All the procedures for protein

223

purification were carried out at 4 °C to maintain the enzymatic activities of the recombinant

224

proteins to the highest extent. As shown in Figure 2, the purity of the purified recombinant

225

proteins, TrxA-His-AtF3H and TrxA-His-AtFLS1, was >95% on a 10% SDS-PAGE gel,

226

which were pure enough for subsequent enzyme assays.

227

Determination of the enzyme activities and the Michaelis-Menten constants of the

228

purified recombinant proteins. To determine the flavanone 3-hydroxylase activity of the

229

TrxA-His-AtF3H protein, we set up a reaction system containing 0.5 mM NRN and 3.3 µg

230

purified recombinant protein in F3H buffer. The reaction products were analyzed by

231

polyamide TLC and HPLC-MS. As shown in Figure 3A, there was a new spot on a

232

polyamide TLC plate with a migration distance similar to that of DHK. The HPLC-MS

233

analysis demonstrated that the new chemical showed a retention time of 12.50 min (Figure

234

3D) and yielded a quasi-molecular ion peak [M-H]- at m/z 287.1000 (Figure 4B), identical

235

to those of DHK. These data indicated that the new chemical was DHK and thus the

236

purified TrxA-His-AtF3H protein possessed a flavanone 3-hydroxylase activity. Enzyme

237

activity analysis indicated that 14.99 ± 0.34 µg of the TrxA-His-AtF3H was needed to

238

convert 1 µmol NRN into DHK at 30 °C in the F3H buffer within one minute. Further

239

dynamic

240

Michaelis-Menten constant of 0.081 ± 0.025 mM.

analysis

demonstrated

that

this

recombinant


enzyme

possessed

a


Page 14 of 32

241

To measure the flavonol synthase activity of the TrxA-His-AtFLS1 protein, we

242

established a 100-µL reaction system containing 0.75 mM DHK as a substrate and 1.9 µg

243

purified protein in FLS buffer. Polyamide TLC analysis indicated that the reaction

244

produced a new spot with a similar migration distance to that of KMF (Figure 3B). HPLC

245

analysis demonstrated that the new chemical possessed a retention time of 20.46 min

246

(Figure 3D) and further confirmation by ESI-MS generated a quasi-molecular ion peak

247

[M-H]- at m/z 285.1000 (Figure 4C), identical to those of KMF. These data demonstrated

248

that the new chemical molecule was KMF and the purified TrxA-His-AtFLS1 protein had a

249

flavonol synthase activity. However, due to an excessive amount of the substrate (0.5 mM)

250

and less amount of the TrxA-His-AtFLS1 in the FLS assay, the peak for KMF was

251

comparatively low in the HPLC chromatograms (Figure 3D), seemingly indicating a low

252

catalytic efficiency of the TrxA-His-AtFLS1. Therefore, we further analyzed the enzyme

253

activity of the TrxA-His-AtFLS1. The data demonstrated that 10.78 ± 0.61 µg of the

254

purified recombinant protein was needed to convert 1 µmol DHK into KMF at 30 °C in the

255

FLS buffer within one minute. Further dynamic analysis showed that the TrxA-His-AtFLS1

256

possessed a Michaelis-Menten constant of 0.072±0.052 mM. These data indicated that the

257

catalytic efficiency of the TrxA-His-AtFLS1 was relatively high enough for subsequent

258

experiments.

259

Development of the in vitro multienzyme synthetic system. To produce KMF in a 27, 28

260

single reaction tube, we analyzed the published buffer components for F3H and FLS

261

and found that both of them are very similar except for the pH value and the concentrations

262

of α-KG and FeSO4. In the beginning, we arbitrarily fixed the pH value at 7.2 and the


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263

concentrations of α-KG and FeSO4 at 8.2 mM and 0.01 mM, respectively, to explore the

264

possibility that KMF might be synthesized from NRN by AtF3H and AtFLS1 in a single

265

reaction tube. The reaction was incubated at 30 °C for 3 h. Polyamide TLC analysis

266

indicated that the reaction produced two new spots with a migration distance similar to that

267

of DHK and KMF, respectively (Figure 3C). HPLC analysis demonstrated that there were

268

two new peaks at 12.50 min and 20.46 min, identical to that of DHK and KMF,

269

respectively (Figure 3D). Further ESI-MS analysis confirmed that these two new molecules

270

yielded quasi-molecular ion peaks [M-H]- at m/z 287.1000 and 285.1000, respectively

271

(Figure 4B and 4C). These data indicated that we set up an in vitro multienzyme synthetic

272

system to produce KMF in a single reaction tube.

273

Optimization of the in vitro multienzyme synthetic system. To improve the yield of

274

KMF, we optimized the system by exploring a series of parameters. First, we prepared a

275

panel of KMF synthetic buffers as listed in Table 1 and compared their efficiency in

276

producing KMF to find the optimum pH value and concentrations of α-KG and FeSO4. The

277

reaction was incubated at 30 °C with stirring for 3 h. Polyamide TLC analysis showed that

278

the KMF synthetic buffer 7 demonstrated the highest yield when compared to others (P

Development and Optimization of an In Vitro Multienzyme Synthetic

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