Increased Production of Food-Grade d-Tagatose from d-Galactose by

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Increased Production of Food-Grade D-Tagatose from DGalactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing DGalactose Isomerase from Geobacillus thermodenitrificans Kyung-Chul Shin, Dong-Hyun Sim, Min-Ju Seo, and Deok-Kun Oh J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03588 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 21, 2016

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Journal of Agricultural and Food Chemistry

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Increased Production of Food-Grade D-Tagatose from D-

2

Galactose by Permeabilized and Immobilized Cells of

3

Corynebacterium glutamicum, a GRAS Host, Expressing D-

4

Galactose Isomerase from Geobacillus thermodenitrificans

5

6

Kyung-Chul Shin, Dong-Hyun Sim, Min-Ju Seo, Deok-Kun Oh*

7 8

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, South Korea

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ACS Paragon Plus Environment


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ABSTRACT: The generally recognized as safe microorganism Corynebacterium glutamicum

11

expressing Geobacillus thermodenitrificans D-galactose isomerase (D-GaI) was an efficient

12

host for the production of D-tagatose, a functional sweetener. The D-tagatose production at

13

500 g/L D-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing

14

D-GaI,

15

(PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated

16

cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate

17

showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total D-tagatose concentration

18

in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L D-tagatose

19

after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed

20

significantly higher

21

tagatose production by PICG cells may be an economical process to produce food-grade D-

22

tagatose.

respectively. The

D-tagatose-producing

D-tagatose

activity of permeabilized C. glutamicum

productivity than that reported for other cells. Thus,

D-

23 24 25 26 27 28

KEYWORDS: Corynebacterium glutamicum, Geobacillus thermodenitrificans, D-galactose

29

isomerase, D-tagatose, permeabilized cells, immobilized cells, GRAS microorganism

30

2


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INTRODUCTION

32 33

D-Tagatose,

34

concentrations in dairy products.1,2

35

sucrose. The two sugars are both involved in browning reactions and lack laxative and

36

cooling effects. However, D-tagatose is a tooth-friendly and low-calorie sweetener, unlike

37

sucrose.3 Moreover, D-tagatose has many health benefits, including the prevention of weight

38

gain, increase in live births, and treatment of type 2 diabetes.4-6 Therefore, this sugar has

39

attracted significant attention in the field of functional foods.7

40

a hexoketose monosaccharide and an isomer of D-galactose, is present at low

L-Arabinose

D-Tagatose

has a taste and some properties similar to

isomerase (L-AI), which catalyzes the conversion of L-arabinose to L-ribulose,

41

can convert

D-galactose

42

configured substrates.8 Thus,

43

bacteria,

44

Anoxybacillus

45

stearothermophilus,13 Geobacillus thermodenitrificans,14 Lactobacillus fermentum,15,16

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Pediococcus pentosaceus,17 Shewanella sp.18 Thermoanaerobacterium saccharolyticum,19

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Thermotoga neapolitana,20,21 and Thermotoga maritima.22

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galactose isomerase (D-GaI) by increasing the isomerization activity of the enzyme for D-

49

galactose, based on a substrate-docking homology model.23 A triple-site (F280N-C450S-

50

N475K) variant of G. thermodenitrificans L-AI exhibited the highest turnover number for D-

51

galactose ever reported for L-AIs and D-GaI. The enzyme was identified as D-GaI because of

52

its preference for D-galactose over L-arabinose.23

including

to

D-tagatose,

owing to its broad substrate specificity for similar

D-tagatose

Alicyclobacillus

flavithermus,11

has been produced by many L-AIs from various acidocaldarius,9

Bacillus

Acidothermus

cellulolyticus,10

stearothermophilus,12

Geobacillus

L-AI

can be changed to

D-

53

Enzymatic reactions can obtain higher productivity and concentration for product than cell

54

reactions by using the high concentrations of enzymes.13 However, the stability and resistance

55

to environmental perturbations of cells are greater than those of enzymes. In addition, whole 3



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cell reactions are more commercially feasible than enzyme reactions because cells do not

57

require purification steps such as cell lysis, protein precipitation, and dialysis.24 Several

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whole cells have been reported for the industrial production of D-tagatose. Escherichia coli

59

expressing L-AI, originating from other bacterial strains, has been used as a host for

D-

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tagatose production.9-15,17-22 However, E. coli is not suitable for producing food-grade

D-

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tagatose because E. coli is not a generally recognized as safe (GRAS) host. Although several

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GRAS

63

thermophiles,28 and Saccharomyces cerevisiae,29 have been used for D-tagatose production,

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the superiority of these hosts to E. coli host for D-tagatose production has not been exhibited.

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Therefore, the value of these hosts is not clear. The GRAS host Corynebacterium glutamicum

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has numerous biotechnological advantages such as easy gene manipulation, high expression

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levels, high growth rate, high cell concentration, utilization of inexpensive media, and stable

68

lipid-rich outer cell wall.30-32 Moreover, C. glutamicum expression system was non-

69

pathogenic and not producing endotoxins, had minimal protease activities, and could secrete

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protein to the culture.32 Due to these advantages, whole cells using C. glutamicum can be

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suggested for the production of food-grade D-tagatose. To increase cell stability and product

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productivity, immobilized cells have been used for

73

productivity of D-tagatose is still low for industrial production. Thus, a more effective cell

74

conversion is needed.

hosts,

including

Lactococcus

lactis,25

Bacillus

subtilis,26,27

D-tagatose

Streptococcus

production. However, the

75

In this study, D-GaI from G. thermodenitrificans was cloned and expressed in the GRAS

76

microorganism C. glutamicum, which has not yet been applied to D-tagatose production. To

77

evaluate C. glutamicum as a

78

recombinant C. glutamicum expressing G. thermodenitrificans D-GaI were compared with

79

those of recombinant E. coli expressing D-GaI. To increase the productivity of D-tagatose

80

from D-galactose, the culture medium for recombinant C. glutamicum was optimized, and

D-GaI

host, the growth and

4


D-tagatose

production of

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whole cells were permeabilized and immobilized. D-Tagatose production by permeabilized

82

and immobilized C. glutamicum (PICG) cells was compared with those by permeabilized C.

83

glutamicum (PCG) and non-permeabilized C. glutamicum cells.

84 85

MATERIALS AND METHODS

86 87

Materials. D-Galactose and D-tagatose were purchased from Sigma (St. Louis, MO). Bio-

88

LC grade sodium hydroxide solution was purchased from Fisher Scientific (Hanover Park,

89

IL). Detergents were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

90 91

Microorganisms and Plasmids. The D-GaI gene of G. thermodenitrificans was cloned

92

and expressed in E. coli using the pET-15b plasmid as described previously.14,23 C.

93

glutamicum ATCC 13032 (ATCC, Manassas, USA) and the pEKEx2 plasmid (Juelich

94

Research Center, Juelich, Germany) were used as the host cells and E. coli−C. glutamicum

95

shuttle vector, respectively, for expression of G. thermodenitrificans

96

encoding G. thermodenitrificans D-GaI was inserted into the pEKEx2 plasmid. The primer

97

sequences used for gene amplification were based on the DNA sequence of G.

98

thermodenitrificans

99

CTGCAGAAAGGAGAATATAGATGCTGTCATTACGTCCTTATGAACTTTG-3′)

D-GaI.

The

forward

D-GaI.

The gene

primer

(5′-

100

included PstI (underlined) and ribosomal-binding site (bold) with extra nucleotide, and the

101

reverse primer (5′-GTCGACTTACCGCCCCCGCCAAAAC-3′) included a SalI restriction

102

site (underlined). These primers were synthesized by Macrogen (Seoul, Republic of Korea).

103

DNA fragments were amplified by PCR, purified, and ligated into the pEKEx2 vector

104

digested by the same restriction enzymes. The plasmid containing the D-GaI gene (800 ng)

105

was transformed into 50 μL of C. glutamicum ATCC 13032 as competent cells (500 cells/mL), 5



106

which were cultivated in AB minimal salts medium (25 mM Tris-HC1, pH 7.5, 0.5 mM

107

phosphate) supplemented with glucose (ABG) with 0.5% Tween 80 and 2.5% glycine, and

108

washed with 15% glycerol, using a MicroPulser Electroporator (Bio-Rad, California, USA)

109

with 2.5 kV of voltage and 4.6 msec of time constant in cuvette with 0.2 cm gap at 4 °C.

110

After the pulse, 1 mL of SMMC buffer (0.5 M sorbitol-20 mM maleate-20 mM MgCl2-20

111

mM CaC12, pH 7.0) was added to the cuvette, and then cells were incubated at 37 °C for 1 h.

112

The strain was plated on brain-heart infusion (BHI) agar containing 15 μg/mL kanamycin. A

113

kanamycin-resistant clone was selected, and the plasmid DNA was extracted from the

114

transformants using a plasmid purification kit (Intron, Deajeon, Republic of Korea). DNA

115

sequencing was conducted by Macrogen.

116 117

Culture Conditions. Recombinant E. coli containing the pET-15b/D-GaI gene of G.

118

thermodenitrificans was prepared, and D-GaI enzyme was purified for comparison with the

119

activities of cells as described previously.23 Recombinant C. glutamicum containing the

120

pEKEx2/D-GaI gene of G. thermodenitrificans was cultivated in a 2-L flask containing 500

121

mL of BHI, MB, CGXII, A, or Riesenberg medium supplemented with 15 μg/mL kanamycin

122

at 30 °C with shaking at 200 rpm. To induce D-GaI, when the optical density of the bacterial

123

culture reached 0.6 at 600 nm, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a

124

final concentration of 1.0 mM to the culture, and the cells were further incubated with

125

shaking at 200 rpm at 30 °C for 19 h. The cultured cells were collected from the culture broth

126

by centrifugation at 13,000 × g at 4 °C for 20 min and washed further twice with 0.85% NaCl

127

solution. The expression of D-GaI was investigated by substituting glucose with sugars such

128

as ribose, maltose, fructose, sucrose, and lactose in Riesenberg medium, consisting of 13.5

129

g/L KH2PO4, 4.0 g/L (NH4)2HPO4, 1.7 g/L citric acid, 1.4 g/L MgSO4·7H2O, 20 g/L glucose,

130

0.2 mg/L biotin, 4 g/L urea, and a 10 mL/L trace metal solution containing 10 g/L 6


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FeSO3·7H2O, 2.25 g/L ZnSO4·7H2O, 1.0 g/L CuSO4·5H2O, 0.5 g/L MnSO4·5H2O, 0.23 g/L

132

Na2B4O7·10H2O, 2.0 g/L CaCl2·2H2O, and 0.1 g/L (NH4)6Mo7O24. The cells grown on

133

different carbon sources were used to determine specific activity. After culture, the reactions

134

were performed in 50 mM EPPS buffer (pH 8.5) containing 15.6 g/L dry weight cells and 18

135

g/L D-galactose at 60 °C for 20 min. The specific activity was defined as the amount of

136

tagatose as a product per the amount of cells per unit reaction time (g/g/h) and the total

137

activity was defined as the amount of tagatose as a product per unit reaction time (g/L/h).

138 139

Permeabilization and Immobilization of C. glutamicum Cells. Harvested cells

140

were frozen and stored at −80 °C. To prepare PCG cells, cells were thawed and resuspended

141

in solutions containing detergents such as 2% and 5% (v/v) Tween 20, Tween 40, Tween 80,

142

Span 20, and Span 80; and 0.5% and 1% (v/v) Triton X-100. To investigate the effect of the

143

Triton X-100 concentration, its concentration was varied from 0 to 5% (v/v). Cell

144

suspensions were incubated at 4 °C for 15 min,33-36 and then washed twice with 50 mM 4-(2-

145

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 8.0). The treated cells

146

were used as PCG cells for the production of D-tagatose from D-galactose. For immobilizing

147

PCG cells, the mixed solution containing 160 g/L PCG cells and 3.0% (w/w) sodium alginate

148

was dropped into 0.6 M CaCl2 solution stirred on ice using a syringe with applying vacuum.

149

To increase hardness of beads, the obtained alginate beads confined into 0.6 M CaCl2

150

solution and then stirred slowly overnight. The influence of factors related to cell

151

immobilization were optimized by varying (1) the cell concentration from 80 g/L to 180 g/L

152

at fixed concentrations of 0.2 M Ca2+ and 2% alginate, (2) the alginate concentration from

153

1% to 4% at fixed concentrations of 0.2 M Ca2+ and 160 g/L cells, and (3) the Ca2+

154

concentration from 0.2 M to 0.8 M at fixed concentrations of 3% alginate and 160 g/L cells.

155 7



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156

Effects of Temperature and pH on the Activities of PCG and PICG Cells.

157

The effects of temperature and pH on the activities of PCG and PICG cells expressing D-

158

GaI from G. thermodenitrificans for the isomerization of

159

investigated by varying the temperature from 45 to 70 °C at a constant pH of 8.0 and varying

160

the pH from 7.0 to 9.0 at a constant temperature of 60 or 65 °C, respectively. The reactions

161

were carried out for 20 min in 50 mM HEPES buffer (pH 7.0−8.0), 50 mM 3-[4-(2-

162

hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS) buffer (pH 8.0−8.5), and 50 mM

163

N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer (pH 8.6−9.0) containing 15.6 g/L

164

cells and 18 g/L D-galactose for PCG cells and 500 g/L alginate beads with cells and 300 g/L

165

D-galactose

166

cells was examined by holding the cells at four temperatures (50 °C, 55 °C, 60 °C, and 65 °C)

167

for various periods of time. The activities of PCG and PICG cells were measured at specific

168

time intervals after the reactions, which were carried out in 50 mM HEPPS buffer (pH 8.5)

169

containing 15.6 g/L cells and 18 g/L D-galactose at 60 °C, and 50 mM HEPES buffer (pH

170

8.0) containing 500 g/L alginate beads with cells and 300 g/L D-galactose at 65 °C for 20 min,

171

respectively. The half-lives of PCG and PICG cells were calculated using Sigma Plot 9.0

172

software (Systat Software, San Jose, CA).

D-galactose

to

D-tagatose

were

for PICG cells. The influence of temperature on the stability of PCG and PICG

173 174

Reactions of PCG and PICG Cells. PCG and PICG cells were collected and washed

175

three times with 50 mM HEPPS buffer (pH 8.5) and 50 mM HEPES buffer (pH 8.0),

176

respectively, containing 300 g/L D-galactose. The batch reactions were performed at 55 °C for

177

3 h in 50 mM HEPPS buffer (pH 8.5) without boric acid or in 41.2 g/L boric acid buffer (pH

178

8.5) containing 160 g/L cells and 300 g/L D-galactose for PCG cells, and 50 mM HEPES

179

buffer (pH 8.0) containing 500 g/L alginate beads with cells and 300 g/L D-galactose for

180

PICG cells. In repeated batch reactions the substrate was replaced for cell reuse after batch 8


Page 9 of 39

181


culture for 3 h.

182 183

Analytical Methods. Cell concentrations were determined using a calibration curve

184

derived from the correlation between the dry cell weight and the optical density at 600 nm.

185

The concentrations of

186

system(Dionex ICS-3000, Sunnyvale, CA) and an electrochemical detector, and eluting 200

187

mM sodium hydroxide into a CarboPac PA10 column at 30 °C at a flow rate of 1 mL/min.

D-tagatose

and

D-galactose

were measured using a Bio-LC

188 189

RESULTS AND DISCUSSION

190 191

Suitability of C. glutamicum as a Host for the Production of D-Tagatose from

192

D-Galactose.

193

thermodenitrificans used in the present study for

194

mM/min, respectively, whereas those for L-arabinose were 769 1/min and 2.3 mM/min,

195

respectively, indicating that the enzyme is D-GaI.23 C. glutamicum and E. coli expressing G.

196

thermodenitrificans D-GaI were cultivated in Riesenberg medium because the medium has

197

been used to cultivate E. coli to high cell densities.37 The maximal specific growth rate and

198

cell concentration of recombinant C. glutamicum in Riesenberg medium were 1.05 h−1 and

199

7.1 g/L, respectively, which were 1.8- and 3.0-fold higher, respectively, than those of

200

recombinant E. coli (Fig. 1A). The effect of the concentration of D-galactose as a substrate on

201

D-tagatose

202

to 500 g/L with 25 g/L cells and 0.53 mg/mL purified enzyme, which was obtained from 25

203

g/L of E. coli expressing G. thermodenitrificans

204

recombinant enzyme, and E. coli and C. glutamicum increased with increasing the

205

galactose concentration, and the increasing rate for D-tagatose production by recombinant C.

The kcat and kcat/Km of the tagatose-producing enzyme from G. D-galactose

were 6245 1/min and 4.7

production was investigated by varying the concentration of D-galactose from 100

D-GaI. D-Tagatose

9


production by D-


Page 10 of 39

206

glutamicum was higher than that by recombinant E. coli (Fig. 1B). As a result, recombinant C.

207

glutamicum produced more D-tagatose than recombinant E. coli above 300 g/L D-galactose as

208

a substrate. These results may be due to the difference of membrane structures in E. coli and

209

C. glutamicum because the expression level of D-GaI in C. glutamicum was slightly lower

210

than that in E. coli (Fig. S1) and the two crude enzymes extracted from E. coli and C.

211

glutamicum, respectively, which were equivalent to 25 g/L cell concentration, showed similar

212

activities (Fig. S2). The productivity of recombinant C. glutamicum using 500 g/L

213

galactose was 1.4- and 1.9-fold higher than that of recombinant E. coli and purified enzyme,

214

respectively, indicating that C. glutamicum is a more suitable host than E. coli for the

215

production of D-tagatose from D-galactose.

D-

216 217

Optimization of Culture Medium for the Production of

D-Tagatose

from

D-

218

Galactose by Recombinant C. glutamicum Expressing G. thermodenitrificans

219

D-GaI.

220

in BHI,38 MB,39 CGXII,40 A,41 or Riesenberg medium,42 which has been used to cultivate C.

221

glutamicum. The cell concentration of recombinant C. glutamicum was the highest in CGXII

222

medium among the media tested. However, recombinant C. glutamicum grown on Riesenberg

223

medium showed the highest specific and total activities, followed by the cells grown on MB,

224

BHI, CGXII, and A media for specific activity, and by the cells grown on MB, CGXII, BHI,

225

and A media for total activity (Fig. 2A). Thus, Riesenberg medium was selected as the culture

226

medium for recombinant C. glutamicum to produce D-tagatose.

227

Recombinant C. glutamicum expressing G. thermodenitrificans D-GaI was cultivated

The specific D-tagatose-producing activity of recombinant C. glutamicum expressing G.

228

thermodenitrificans

229

maltose, fructose, sucrose, and lactose. The specific activity using lactose was very low. It

230

may due to that C. glutamicum does not utilize galactose and the host does not have its own

D-GaI

in Riesenberg medium was examined using ribose, glucose,

10


Page 11 of 39


and D-galactose isomerase.43 The specific activity was the highest when the original

231

L-AI

232

carbon source glucose was used (Fig. 2B). Therefore, the cells were cultivated in Riesenberg

233

medium containing glucose as a single carbon source.

234 235

Permeabilization

and

Immobilization

of Recombinant C.

glutamicum

236

Expressing G. thermodenitrificans D-GaI. Recombinant C. glutamicum expressing G.

237

thermodenitrificans D-GaI were permeabilized by treatment with detergents, including 2%

238

and 5% (w/v) Tween 20, Tween 40, Tween 80, Span 20, and Span 80; and 0.5% and 1% (w/v)

239

Triton X-100. Triton X-100 was used at lower concentrations than other detergents because

240

its concentration used for cell permeabilization was lower.35,36 When cells were treated with

241

0.5% and 1% Tween 20, Tween 40, Tween 80, Span 20, and Span 80, the

242

producing activity was almost the same as that of untreated control cells (data not shown).

243

PCG cells treated with 1% (w/v) Triton X-100 showed the highest activity with 2.1-fold

244

higher activity than that of untreated cells (Fig. 3A). The effect of Triton X-100 concentration

245

on the D-tagatose-producing activity of cells for D-tagatose production was investigated. The

246

activity increased with increasing Triton X-100 concentrations up to 1% (w/v) (Fig. 3B).

247

However, above 1% (w/v), the activity did not increase, indicating that Triton X-100

248

concentration was optimal at 1% (w/v). Thus, PCG cells treated with 1% Triton X-100 were

249

used for immobilization. To remove Triton X-100, which is not food-grade, PCG cells were

250

washed twice with HEPES buffer.

D-tagatose-

251

Cells have been immobilized in calcium alginate beads for D-tagatose production.13,16,21

252

Recombinant C. glutamicum expressing G. thermodenitrificans D-GaI were also immobilized

253

in calcium alginate beads. To optimize the immobilization conditions, the

254

producing activity of PICG cells was determined at various concentrations of cells, Ca2+, and

255

alginate. When 160 g/L PCG cells were immobilized with 3% alginate and hardened with 0.6 11


D-tagatose-


Page 12 of 39

256

M Ca2+, PICG cells exhibited the highest activity (Table 1) and were used for D-tagatose

257

production.

258 259

Effects of Temperature and pH on the Production of

D-Tagatose

from

D-

260

Galactose by PCG and PICG Cells Expressing G. thermodenitrificans

261

The maximum activities of PCG and PICG cells expressing G. thermodenitrificans D-GaI

262

were observed at pH 8.5 and 60 °C and at pH 8.0 and 65 °C, respectively (Fig. 4). The

263

activity of the purified

264

55 °C.23 The thermal stabilities of PCG and PICG cells were examined by determining the

265

activities of these cells after incubation at temperatures ranging from 50 °C to 65 °C for 20

266

min. The activities of PCG and PICG cells displayed first-order kinetics for thermal

267

inactivation (Fig. 5). The half-lives of PCG cells were 18.6 h, 8.9 h, 3.9 h, and 1.5 h at 50 °C,

268

55 °C, 60 °C, and 65 °C, respectively, and those of PICG cells were 57.5 h, 22.2 h, 5.5 h, and

269

1.6 h at the same temperatures, respectively, which were 3.1-, 2.5-, 1.4-, and 1.1-fold longer,

270

respectively, than those of PCG cells, indicating that cell immobilization increased the

271

thermal stability of cells. Although the maximum activity of PICG cells was observed at

272

65 °C, the reactions by PCG and PICG cells were performed at 55 °C to maintain the stability

273

of D-tagatose production for a longer period. The half-lives of the purified D-GaI were 30.8 h,

274

6.4 h, and 1.6 h at 55 °C, 60 °C, and 65 °C, respectively,23 which were 3.5-, 1.6- and 1.1-fold

275

longer than the half-lives of PCG cells, respectively. Although cells were more thermostable

276

than enzyme, enzyme showed higher thermostablity than permeabilized cells. Therefore, the

277

enzyme in permeabilized cells may be more easily deactivated by heat treatment for a long

278

time.

D-GaI

D-GaI.

from G. thermodenitrificans was maximal at pH 8.0 and

279

12


Page 13 of 39

280


Production of

D-Tagatose

from

D-Galactose

281

Expressing G. thermodenitrificans

282

production were performed using PCG and PICG cells containing 300g/L D-galactose for 3 h.

283

PICG cells produced 165 g/L D-tagatose after 3 h, with a conversion of 55% (w/w) and a

284

productivity of 55 g/L/h (Fig. 6). T. neapolitana L-AI showed the previously highest kcat.20

285

The kcat of G. thermodenitrificans D-GaI (6245 1/min) was 7.7-fold higher than that of T.

286

neapolitana L-AI (504 1/min).23 Due to its significant higher kcat, the conversion time reduced

287

to 3 h. PCG cells in the first batch of the repeated batch reactions produced 144 g/L D-

288

tagatose after 3 h, with a conversion of 48% (w/w) and a productivity of 48 g/L/h (Fig. 7).

289

Thus, the conversion and productivity of PICG cells in the batch reaction were 1.15-fold

290

higher than those of PCG cells, respectively. Several rounds of conversion, reusing PCG and

291

PICG cells, were performed (Fig. 7). With cell recycling, PICG cells produced 82 g/L D-

292

tagatose after nine recycling rounds, whereas PCG cells produced no D-tagatose after four

293

recycling rounds. These results may reflect the higher stability of PICG cells than PCG cells.

294

The total concentration of D-tagatose produced in nine recycling rounds by PICG cells was

295

1116 g/L, which was 3.1-times higher than that by PCG cells.

296

D-GaI.

by PCG and PICG Cells

The processes described in this work for

Time-course reactions for

D-tagatose

D-tagatose

production are compared to those

297

presented in the literature in Table 2. The D-tagatose productivity of PICG cells achieved in

298

the present study was 5.5-fold higher than that of immobilized L. fermentum, which

299

previously showed the highest productivity. Thus, the productivity of PICG cells is the

300

highest reported to date. L. lactis expressing Bifidobacterium longum L-AI in borate buffer

301

displayed the highest product concentration (186 g/L).25 PCG cells produced 180 g/L

302

tagatose in borate buffer with a conversion of 60.6% (data not shown). However, boric acid is

303

not suitable for the production of food-grade D-tagatose because it can be toxic in humans.

304

Previously, the highest D-tagatose concentration and conversion achieved without boric acid 13


D-


Page 14 of 39

305

was 75 g/L and 75% in Bacillus subtilis expressing L. fermentum L-AI, respectively.27 The D-

306

tagatose concentration (165 g/L) and conversion (55%) produced by PICG cells without boric

307

acid was 2.2-fold higher and 1.4-fold lower than that by recombinant B. subtilis cells,

308

respectively. In conclusion, the GRAS C. glutamicum host had advantages over E. coli in the production

309 310

of

D-tagatose,

including higher growth rate, cell concentration, and sugar tolerance. Cell

311

permeabilization increased the productivity of

312

substrate and product across the cell membrane. Cell immobilization also increased the

313

productivity because of the increased thermal stability of cells. C. glutamicum cells that were

314

both permeabilized and immobilized in the present study exhibited significantly higher D-

315

tagatose productivity than that of other cells reported. Thus,

316

permeabilized and immobilized cells using an efficient GRAS host may be an economically

317

feasible production process, and this process can contribute to the industrial production of

318

food-grade D-tagatose.

D-tagatose

by enhancing the transfer of

D-tagatose

production by

319 320 321

ASSOCIATED CONTENT

322 323

Supporting Information

324

Figure S1. SDS-PAGE analysis of G. thermodenitrificans D-GaI expressed in E. coli and C.

325

glutamicum

326

Figure S2. Effect of

327

galactose by crude enzymes extracted from C. glutamicum and E. coli expressing G.

328

thermodenitrificans D-GaI

D-galactose

concentration on the production of

329 14


D-tagatose

from

D-

Page 15 of 39

330


AUTHOR INFORMATION

331 332

Corresponding Author

333

* Phone: (822) 454-3118. Fax: (822) 444-5518. E-mail: [email protected]

334 335

Funding sources

336 337

This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea

338

Institute for Advancement of Technology (KIAT) through the Encouragement Program for

339

The Industries of Economic Cooperation Region.

340

15



341

Page 16 of 39

References

342 343 344 345 346 347 348 349 350 351 352 353 354

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presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-

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arabinose isomerase in Bacillus subtilis, a GRAS host, for the production of edible tagatose.

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tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase.

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octadecadienoic acid from alpha-linolenic acid by permeabilized cells of recombinant

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Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia.

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Biotechnol. Lett. 2013, 35, 1487-1493.

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(35) An, J. U.; Joo, Y. C.; Oh, D. K., New biotransformation process for production of the

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fragrant compound gamma-dodecalactone from 10-hydroxystearate by permeabilized

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Waltomyces lipofer cells. Appl. Environ. Microbiol. 2013, 79, 2636-2641.

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(36) Zhang, L.; Jiang, B.; Mu, W.; Zhang, T., Bioproduction of D-psicose using

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permeablilized cells of newly isolated Rhodobacter sphaeroides SK011. Font. Chem. Eng.

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(37) Riesenberg, D., High-cell-density cultivation of Escherichia coli. Curr. Opin. Biotechnol. 1991, 2, 380-384.

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(38) Yim, S. S.; An, S. J.; Kang, M.; Lee, J.; Jeong, K. J., Isolation of fully synthetic

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(39) Kim, T. H.; Kim, H. J.; Park, J. S.; Kim, Y.; Kim, P.; Lee, H. S., Functional analysis of

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sigH expression in Corynebacterium glutamicum. Biochem. Biophys. Res. Commun. 2005,

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458

(40) van Ooyen, J.; Emer, D.; Bussmann, M.; Bott, M.; Eikmanns, B. J.; Eggeling, L.,

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Citrate synthase in Corynebacterium glutamicum is encoded by two gltA transcripts which

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are controlled by RamA, RamB, and GlxR. J. Biotechnol. 2011, 154, 140-148.

461

(41) Sasaki, M.; Jojima, T.; Inui, M.; Yukawa, H., Xylitol production by recombinant

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Corynebacterium glutamicum under oxygen deprivation. Appl. Microbiol. Biotechnol. 2010,

463

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464

(42) Doo, E. H.; Lee, W. H.; Seo, H. S.; Seo, J. H.; Park, J. B., Productivity of

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cyclohexanone oxidation of the recombinant Corynebacterium glutamicum expressing chnB 20


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466


of Acinetobacter calcoaceticus. J. Biotechnol. 2009, 142, 164-169.

467

(43) Jojima, T.; Omumasaba, C. A.; Inui, M.; Yukawa, H., Sugar transporters in efficient

468

utilization of mixed sugar substrates: current knowledge and outlook. Appl. Microbiol.

469

Biotechnol. 2010, 85, 471-480.

470

471

472

21



473

Page 22 of 39

Figure captions

474 D-tagatose

production from

D-galactose

by recombinant C.

475

Figure 1. Cell growth and

476

glutamicum and E. coli expressing G. thermodenitrificans

477

glutamicum and E. coli cells. Recombinant C. glutamicum (●) and E. coli (□) expressing G.

478

thermodenitrificans D-GaI were cultivated in a 2-L flask containing 500 mL of Riesenberg

479

medium supplemented with 15 μg/mL kanamycin at 30 °C with shaking at 200 rpm for 21 h.

480

At the optical density of 0.6 at 600 nm, IPTG was added at a final concentration of 1.0 mM.

481

(B) Effect of D-galactose concentration on the production of D-tagatose from D-galactose by

482

C. glutamicum and E. coli cells expressing G. thermodenitrificans D-GaI and purified enzyme.

483

After the culture, the reactions of C. glutamicum (□) and E. coli ( ) cells expressing G.

484

thermodenitrificans D-GaI and purified enzyme (■) were performed in 50 mM HEPPS buffer

485

(pH 8.5) containing 25 g/L cells at 60 °C and 50 mM HEPPS buffer (pH 8.0) containing 0.53

486

mg/mL enzyme at 55 °C, respectively, at D-galactose concentrations ranging from 100 g/L to

487

500 g/L for 40 min. Data represent the means of three separate experiments and error bars

488

represent the standard deviations.

D-GaI.

(A) Growth of C.

489

Effects of culture medium and carbon source on the production of D-tagatose

490

Figure 2.

491

from D-galactose by C. glutamicum expressing G. thermodenitrificans D-GaI. (A) Effect of

492

culture medium on the production of

493

expressing G. thermodenitrificans D-GaI. The mass (■), total activity (■) and specific activity

494

(□) of recombinant C. glutamicum. (B) Effect of carbon source on the specific activity of

495

recombinant

496

thermodenitrificans

497

CGXII, A, or Riesenberg medium supplemented with 15 μg/mL kanamycin at 30 °C with

C.

glutamicum D-GaI

cells.

D-tagatose

from

Recombinant

D-galactose

C.

by C. glutamicum

glutamicum

expressing

G.

was cultivated in a 2-L flask containing 500 mL of BHI, MB,

22


Page 23 of 39


498

shaking at 200 rpm for 21 h. At an optical density of 0.6 at 600 nm, IPTG was added at a final

499

concentration of 1.0 mM. After culture, the reactions were performed in 50 mM HEPPS

500

buffer (pH 8.5) containing 15.6 g/L cells and 18 g/L D-galactose at 60 °C for 20 min. Data

501

represent the means of three separate experiments and error bars represent the standard

502

deviations.

503

Effect of detergent treatment on the permeabilization of C. glutamicum

504

Figure 3.

505

expressing G. thermodenitrificans D-GaI for the production of D-tagatose from D-galactose.

506

(A) Effect of detergent treatment. 0% ( ), 2% (■), 5% (■), 0.5% (□), and 1% (w/v) ( ).

507

Effect of Triton X-100 concentration. The reactions were performed in 50 mM HEPPS buffer

508

(pH 8.5) containing 15.6 g/L cells and 18 g/L D-galactose at 60 °C for 20 min. Data represent

509

the means of three separate experiments and error bars represent the standard deviation.

(B)

510

Effects of temperature and pH on the production of D-tagatose from D-galactose

511

Figure 4.

512

by PCG and PICG cells expressing G. thermodenitrificans D-GaI. (A) Effect of temperature.

513

The reactions were performed in temperatures ranging from 45 °C to 70 °C for 20 min in 50

514

mM HEPPS buffer (pH 8.5) containing 15.6 g/L cells (○) and 18 g/L D-galactose for PCG

515

cells, and 50 mM HEPES buffer (pH 8.0) containing 500 g/L alginate beads with cells (●)

516

and 300 g/L D-galactose for PICG cells. (B) Effect of pH. The reactions were performed at

517

60 °C for 20 min in 50 mM HEPES buffer (pH 7.0−8.0), 50 mM EPPS buffer (pH 8.0−8.5),

518

and 50 mM CHES buffer (pH 8.6−9.0) containing 15.6 g/L cells (○) and 18 g/L D-galactose

519

for PCG cells and 500 g/L alginate beads with cells (●) and 300 g/L D-galactose at 65 °C for

520

PICG cells. Data represent the means of three separate experiments and error bars represent

521

the standard deviations.

522 23



523

Figure 5. Thermal inactivation of the D-tagatose producing activity of PCG and PICG cells

524

expressing G. thermodenitrificans D-GaI. PCG and PICG cells were incubated at 50 °C (●),

525

55 °C (○), 60 °C (▼), and 65 °C (△). Data represent the means of three separate experiments

526

and error bars represent the standard deviation. (A) PCG cells. A sample was taken at each

527

time point and assayed at 60 °C for 20 min in 50 mM HEPPS buffer (pH 8.5) containing 15.6

528

g/L cells and 18 g/L D-galactose. (B) PICG cells. A sample was taken at each time point and

529

assayed at 65 °C for 20 min in 50 mM HEPES buffer (pH 8.0) containing 500 g/L alginate

530

beads with cells and 300 g/L D-galactose.

Page 24 of 39

531 532

Figure 6. Time-course reactions for the production of D-tagatose from D-galactose by PICG

533

cells expressing G. thermodenitrificans D-GaI. The reactions by PICG cells to produce D-

534

tagatose (●) from

535

containing 500 g/L alginate beads with cells and 300 g/L D-galactose at 55 °C for 3 h. Data

536

represent the means of three separate experiments and error bars represent the standard

537

deviations.

D-galactose

(■) were performed in 50 mM HEPES buffer (pH 8.0)

538 539

Figure 7. Reuse of PCG and PICG cells expressing G. thermodenitrificans D-GaI for the

540

production of D-tagatose from D-galactose. The reactions containing 300 g/L D-galactose in

541

each recycling batch were performed in 50 mM EPPS buffer (pH 8.5) containing 160 g/L

542

cells for PCG (■) cells, and 500 g/L alginate beads with cells for PICG cells (■) at 55 °C for

543

3 h. Data represent the means of three separate experiments and error bars represent the

544

standard deviations.

545

24


Page 25 of 39


Table 1. Relative Activity of PICG Cells at Various Concentrations of Cells, Ca2+, and Alginate immobilization factor

Cell concentration (g/L)

Ca2+ concentration (M)

Alginate concentration (%)

concentration

relative activity (%)

80

86.0 ± 0.95

100

90.5 ± 0.20

120

93.7 ± 0.08

140

94.1 ± 0.98

160

100.0 ± 0.19

180

98.9 ± 0.33

0.2

71.6 ± 0.13

0.4

96.1 ± 1.01

0.6

100.0 ± 0.83

0.8

95.8 ± 0.06

1

97.1 ± 0.19

2

98.6 ± 0.38

3

100.0 ± 0.72

4

79.3 ± 0.12

25



Page 26 of 39

Table 2. D-Tagatose Production by Cells Expressing D-Galactose and L-Arabinose Isomerases biocatalyst

borate buffer

galactose (g/L)

tagatose (g/L)

conversion (w/w, %)

reaction time (h)

productivity (g/L/h)

L. lactis (expressing B. longum L-AI)25

+

300

186

62.0

24

7.7

B. subtilis (expressing L. fermentum L-AI)27

−

100

75

75.0

24

3.1

Immobilized E. coli (expressing G. stearothermophilus L-AI) a 13

−

300

59

19.5

35

2.9

Immobilized E. coli (expressing T. neapolitana L-AI) a 21

−

180

49

27.0

12

4.0

Immobilized L. fermentum a 16

+

95

57

60.0

24

11

PCG (expressing G. thermodenitrificans D-GaI) (this study)

−

300

144

48.0

3

48

PCG (expressing G. thermodenitrificans D-GaI) (this study)

+

300

180

60.6

3

60

PICG (expressing G. thermodenitrificans D-GaI) (this study)

−

300

165

55.0

3

55

a

Packed-bed bioreactor.

26


Page 27 of 39


A

8

Cell mass (g/L)

6

4

2

IPTG

0 0

5

10

15

Time (h)

Figure 1-continued

27


20


Page 28 of 39

B

Tagatose amount (g/L)

30

20

10

0 100

200

300

D-Galactose

400

concentration (g/L)

Figure 1 28


500

Page 29 of 39


12

1.2

10

10

1.0

8

8

6

6

4

4

2

2

0.2

0

0.0

0 BHI

MB

CGXII

A

Riesenberg

Medium

Figure 2-continued

29


0.8

0.6

0.4

Specific activity (g/g/h)

12

Total activity (g/L/h)

Cell mass (g/L)

A


B

Figure 2

30


Page 30 of 39

Page 31 of 39


A

Figure 3-continued

31



Page 32 of 39

B

250

Relative activity (%)

200

150

100

50

0 0

1

2

3

4

Triton X-100 concentration (%)

Figure 3

32


5

Page 33 of 39


A

120


100

80

60

40

20

0 40

45

50

55

60

Temperature (oC)

Figure 4-continued

33


65

70

75


Page 34 of 39

B

120


100

80

60

40

20

0 6.5

7.0

7.5

8.0

8.5

pH

Figure 4

34


9.0

9.5

Page 35 of 39


A

100 90

Residual activity (%)

80

60

40

20 0

10

20

30

Time (h)

Figure 5-continued

35


40


Page 36 of 39

B

100 90

Residual activity (%)

80

60

40

20 0

10

20

30

Time (h)

Figure 5

36


40

Page 37 of 39


D-Tagatose, D-Galactose

(g/L)

300

250

200

150

100

50

0 0.0

0.5

1.0

1.5

2.0

Time (h)

Figure 6

37


2.5

3.0


Page 38 of 39

180 160

(g/L)

120

D-Tagatose

140

100 80 60 40 20 0 1

2

3

4

5

6

7

Number of recycles

Figure 7

38


8

9

Page 39 of 39


Table of Contents Graphic Increased Production of Food-Grade D-Tagatose from D-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing D-Galactose Isomerase from Geobacillus thermodenitrificans Kyung-Chul Shin, Dong-Hyun Sim, Min-Ju Seo, Deok-Kun Oh Table of Contents Graphic 85x42mm (300 x 300 DPI)


Increased Production of Food-Grade d-Tagatose from d-Galactose by

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