Rational Design of Bacillus coagulans NL01 l-Arabinose Isomerase

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Rational design of Bacillus coagulans NL01 L-arabinose isomerase and using its F279I variant in D-tagatose production Zhaojuan Zheng, Wending Mei, Meijuan Xia, Qin He, and Jia Ouyang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Journal of Agricultural and Food Chemistry

Rational design of Bacillus coagulans NL01 L-arabinose isomerase and using its F279I variant in D-tagatose production

Zhaojuan Zheng1, 2#, Wending Mei2#, Meijuan Xia2, Qin He2, Jia Ouyang1, 2, 3*

1

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest

Resources, Nanjing 210037, People’s Republic of China 2

College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037,

People’s Republic of China 3

Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education,

Nanjing 210037, People’s Republic of China

#

*

These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.O.Y.

([email protected]).

1

ACS Paragon Plus Environment


1

Abstract

2

D-Tagatose

3

isomerase (AI) from D-galactose. To improve the activity of AI toward D-galactose,

4

the AI of Bacillus coagulans was rationally designed based on molecular modeling

5

and docking. After alanine scanning and site-saturation mutagenesis, variant F279I

6

that exhibited improved activity toward

7

temperature and pH of F279I were determined to be 50 °C and 8.0, respectively. This

8

variant possessed 1.4-fold catalytic efficiency compared with the wild-type (WT)

9

enzyme. The recombinant Escherichia coli overexpressing F279I also showed

10

obvious advantages over WT in biotransformation. Under optimal conditions, 67.5 g

11

L-1 and 88.4 g L-1

12

D-galactose,

13

promising alternative for large-scale D-tagatose production.

is a prospective functional sweetener that can be produced by L-arabinose

D-tagatose

D-galactose

was obtained. The optimal

could be produced from 150 g L-1 and 250 g L-1

respectively, in 15 h. The biocatalyst constructed in this study presents a

14 15

Keywords: L-Arabinose isomerase; Rational design; D-Tagatose; Biotransformation

16

2


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17

Introduction

18

D-Tagatose,

19

naturally in gums and dairy products. It shows two obvious advantages over certain

20

common sugars like sucrose and glucose. Firstly, it has only 38% of the calories of

21

sucrose, while the sweetness of the two sugars is almost the same1. Due to this

22

property, it has been used in functional drinks and health foods that are designed for

23

weight loss. Secondly,

24

compared to 100 and 68 for glucose and sucrose, respectively2. As the intake of

25

sugars with high GI values can easily increase the blood sugar level3, D-tagatose can

26

serve as an alternative to these sugars in the treatment of hyperglycemia caused by

27

type 2 diabetes. In addition to the two advantages above, D-tagatose has no laxative

28

effect and has a tooth protection property, which have made it a welcome sweetener

29

in oral hygiene products4. Meanwhile, it can also be used as a synthetic block for

30

other optically active compounds and additives in detergents and cosmetics5.

31

an isomer of D-galactose and D-fructose, is a rare hexoketose that occurs

D-tagatose

The enzymatic production of

shows a very low glycemic index (GI) of 3,

D-tagatose

from

D-galactose

using L-arabinose

32

isomerase (AI, EC 5.3.1.4) as the catalyst has undergone rapid development in recent

33

years. AI can catalyze two reactions, the isomerization of D-galactose to D-tagatose

34

and the isomerization of L-arabinose to L-ribulose, because of the conformational

35

similarity of the two substrates6. A large number of reported AIs are from

36

thermophilic

37

stearothermophilus7, Geobacillus

38

saccharolyticum9,

or

hyperthermophilic

Alicyclobacillus

bacteria,

including

Geobacillus

thermodenitrificans8, Thermoanaerobacterium hesperidum10,

Acetivibrio

3


cellulolytics11,


Page 4 of 32

39

Anoxybacillus flavithermus12, Bacillus stearothermophilus13, Thermotoga maritima14,

40

and Thermotoga Neapolitana15. These AIs show maximal activity when the reaction

41

temperature is above 70 °C. It has been presumed that high temperature shifts the

42

reaction equilibrium to D-tagatose13. Nevertheless, high temperature also aggravates

43

the unwanted browning reaction and the formation of side products, and it is

44

expensive to remove these side products on an industrial scale. To address this

45

problem, AIs derived from mesophilic or acidophilic bacterial strains such as Bacillus

46

halodurans7,

47

pentosaceus18, and Alicyclobacillus acidocaldarius19 have been characterized and

48

used as substitutes for thermoactive AIs. Some of them, such as the AI from L.

49

fermentum CGMCC2921, achieved comparable conversion rates to thermoactive AIs

50

in D-tagatose preparation16.

Lactobacillus

fermentum16,

Lactobacillus

sakei17,

Pediococcus

51

Many studies show that the rational re-design of an available enzyme based on

52

the structure is a practical strategy for constructing a biocatalyst with desired

53

properties20, 21. For AI, the N175H variant of B. stearothermophilus US100 AI was

54

obtained using this method, and it was consequently active at lower temperatures than

55

the wild-type (WT) enzyme22. In addition, the triple-site (F280N-C450S-N475S)

56

variant of G. thermodenitrificans AI was also obtained and exhibited increased

57

specific activity toward D-galactose23.

58

In our recent study, an araA gene (encoding AI) from a mesophilic Bacillus

59

strains, Bacillus coagulans NL01, was cloned and overexpressed in Escherichia coli

60

BL21(DE3)24. The AI from B. coagulans NL01 (BCAI) showed noticeable activity 4


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61

toward D-galactose while AIs from other Bacillus strains gave lower or no detectable

62

activity. Moreover, compared with AIs from other mesophilic strains, BCAI

63

possessed several inherent advantages, such as broad temperature adaptability and

64

low dependency on Mn2+. In this study, residues that might affect the substrate

65

specificity of BCAI were analyzed in detail based on substrate-enzyme docking, and

66

the catalytic efficiency of BCAI against D-galactose was successfully improved based

67

on a rational re-design strategy. The production of D-tagatose employing whole cells

68

of E. coli that expressed the resulting variant was also evaluated.

69

Materials and Methods

70

Bacterial strains, plasmids, primers, and chemicals. Bacterial strains, plasmids,

71

and PCR primers used in this study were listed in Table S1. E. coli BL21(DE3) was

72

grown at 37 °C in Luria-Bertani (LB) medium, and ampicillin was added at a

73

concentration of 100 µg mL–1, if necessary. D-galactose, D-tagatose and L-arabinose

74

were purchased from TCI (Japan), and L-ribulose was purchased from Carbosynth

75

(United Kingdom).

76

Molecular modeling and docking. Homology modeling of WT and F279I was

77

performed out using the Build Homology Models module of Discovery Studio (DS)

78

4.0 (BIOVIA, San Diego). Crystal structures of L. fermentum and E. coli AI (RCSB

79

PDB entry, 4LQL and 4F2D) were used as templates. The generated structures were

80

superimposed on 4LQL and 4F2D to confirm the consistency of the models. Their

81

loop regions were optimized using the Refine Loop module of DS 4.0, and the best

82

loop conformations were selected24. Hydrogen atoms and the CHARMM force field 5



Page 6 of 32

83

were applied to the models. Then, model energies were minimized using 1,000 steps

84

of minimization techniques until the RMS gradient was less than 0.1. The D-galactose

85

molecule was docked to the active sites of the energy-minimized structures using the

86

C-DOCKER module25. The substrate docking poses giving the lowest interaction

87

energy were selected for subsequent visualization and analyses.

88

Site-directed and site-saturation mutagenesis. Both site-directed and site-saturation

89

mutagenesis were conducted using the Fast Mutagenesis System according to the

90

manufacturer’s protocol (TransGen Biotech, Beijing, China). To generate M185A,

91

R186A, F279A, M349A and I370A, GCG was introduced into the corresponding

92

primers for PCR with pETDuet-araA as the template. For site-saturation mutagenesis,

93

NNN was introduced into the corresponding primers for PCR with pETDuet-araA as

94

the template. The PCR products were transformed into E. coli BL21(DE3) to

95

construct mutated plasmids. The mutants were confirmed by DNA sequencing.

96

Initial screening of E. coli transformants obtained by site-saturation mutagenesis.

97

After transformation with the site-saturation mutagenesis reaction mixtures, E. coli

98

BL21(DE3) transformants were grown in 48-well plates at 37 °C and 200 rpm. For

99

expression, the recombinants were grown to a density of OD600

nm

0.6~0.8, then

100

induced by isopropyl-β-D-thiogalactopyranoside (IPTG) for 8 h at 25°C and 200 rpm.

101

The medium was removed by centrifugation at 11,404 g (10,000 rpm) for 10 min. E.

102

coli strains expressing different types (WT or variants) of BCAI were collected for

103

initial screening using a rough activity assay method. The reaction was performed in 1

104

mL of 50 mM Tris-HCl buffer (pH 7.5), 100 mM

D-galactose,

6


1 mM Mn2+, and

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105

bacterial cells with suitable concentration. The D-tagatose formed was determined by

106

the cysteine-carbazole-sulfuric-acid method at 560 nm26. The strains showing higher

107

activities than WT were selected for further study.

108

Crude extraction and purification of F279I. The recombinant E. coli BL21(DE3)

109

harboring pETDuet-araAF279I was used for F279I expression. The detailed method for

110

extraction and purification of F279I was the same as described for WT in our previous

111

report24.

112

Biochemical assays of purified F279I. The activity and protein concentration of

113

F279I were measured as described in our previous study24. To investigate the effect of

114

temperature on F279I toward

115

30 °C~90 °C. Other conditions were the same as for the standard assay. To determine

116

the effect of pH on activity, the pH was varied from 2.2~7.0 using 50 mM disodium

117

hydrogen phosphate-citric acid buffer and 7.0~10.0 using 50 mM Tris-HCl at optimal

118

temperature. Gradient concentrations (from 12.5 to 700 mM) of aldose (L-arabinose

119

or D-galactose) were used to determine the kinetic parameters of F279I. The reaction

120

mixtures were incubated at 50 °C, pH 8.0 for 20 min and then stopped by chilling the

121

tubes in an ice bath to determine L-ribulose or D-tagatose. Km (mM), Vmax (U mg-1)

122

and kcat (min-1) for

123

regression.

124

Biotransformation for

125

overexpressing F279I. The biotransformation was performed with 10 mL reaction

126

mixtures containing 4.8 g DCW L-1 of recombinant E. coli overexpressing F279I in

L-arabinose

D-galactose,

and

D-tagatose

the temperature was varied from

D-galactose

were calculated by non-linear

production by whole cells of E. coli

7



127

50 mM Tris-HCl buffer. The effect of temperature ranging from 40 °C~80 °C on

128

biotransformation was studied using 20 g L-1 D-galactose as the substrate. The effect

129

of D-galactose concentration on biotransformation was investigated between 20 and

130

250 g L-1 at 50 °C. The concentrations of D-galactose and D-tagatose in the reaction

131

mixtures were quantitatively analyzed by high-performance liquid chromatography

132

(HPLC).

133

Analytic methods. The amounts of monosaccharides in the whole-cell biocatalysis

134

system were determined using an HPLC system (Agilent 1200 series, USA) equipped

135

with a Sugar-pak1 column (6.5 × 300 mm) (Waters) and a refractive index detector

136

(SHIMADZU). The column was eluted with deionized water at a flow rate of 0.4 mL

137

min-1 and 80 °C.

138

Results

139

Selection of the modification sites by molecular docking. The structure of BCAI

140

was obtained with the DS 4.0 package using the reported crystal structures of L.

141

fermentum and E. coli AI (RCSB PDB entry 4LQL, 4F2D) as templates. The residues

142

near D-galactose were identified by docking the D-galactose molecule to the active

143

site of the obtained structure. Figure 1 showed that the residues M185, F279, E306,

144

E331, H348, M349, I370 and H447 in the active site were within a docking sphere of

145

6-Å radius around the

146

substrate specificity27. Among these residues, E306, E331, H348 and H447 were the

147

putative catalytic residues determined by multiple sequence alignment and analysis24.

148

In addition, a previous study on B. stearothermophilus US100 AI confirmed that

D-galactose

molecule. These residues might determine the

8


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149

mutations to these residues could cause a serious loss in AI activity27. Therefore, these

150

four residues were not ideal modification targets. The remaining residues, M185,

151

F279, M349, and I370, were selected for alanine scanning to evaluate the effects of

152

crucial residues on enzyme activity and substrate specificity. Moreover, although the

153

residue R186 was not within the 6-Å sphere, it was also selected because it might

154

affect the conformation of M185 and hence affect substrate binding.

155

Alanine scanning on the selected modification sites. The five selected modification

156

sites were replaced separately by alanine residues to obtain five single-site variants,

157

M185A, R186A, F279A, M349A, and I370A. Both the variants and WT were

158

expressed in E. coli BL21(DE3), and their crude extracts were used for enzyme assays.

159

WT showed an activity of 5.07 U mg-1 toward L-arabinose and 0.42 U mg-1 toward

160

D-galactose

(Table 1). All variants gave lower activities than WT. The ratio of

161

L-arabinose

activity to D-galactose activity (A/G) was used to represent the substrate

162

specificity of WT and variants. WT showed an A/G ratio of 12.07, and M185A,

163

R186A, and M394A showed similar A/G ratios to WT. By contrast, F279A and

164

I370A were quite different from WT, as the former exhibited improved substrate

165

specificity toward D-galactose, while the latter gave improved substrate specificity

166

toward L-arabinose (Table 1). Because F279A and I370A produced the most obvious

167

changes to BCAI’s substrate specificity, they were selected as the subsequent

168

site-saturation mutagenesis sites to broaden the mutant library.

169

Site-saturation mutagenesis of F279 and I370. To simplify the workload, variants

170

that might improve activity toward D-galactose were preliminarily screened by color 9



Page 10 of 32

171

reaction using the cysteine-carbazole-sulfuric-acid method. For the site-saturation

172

mutagenesis of F279, six strains were selected by initial screening experiment and

173

further analyzed by DNA sequencing. For the site-saturation mutagenesis of I370,

174

four strains were selected for sequencing. DNA sequencing identified nine types of

175

variants, namely, F279Y, F279A, F279M, F279V, F279I, I370R, I370V, I370S, and

176

I370K. Afterward, their activities toward L-arabinose and D-galactose were assayed

177

quantitatively using crude extracts (Figure 2). Compared with WT, when the aromatic

178

F279 was replaced with neutral alanine (A), valine (V), and isoleucine (I), the A/G

179

ratio decreased to some extent; when aromatic F279 was replaced with tyrosine (Y)

180

and methionine (M), the A/G ratio increased obviously. Regarding their activities

181

against D-galactose, only F279I gave a positive result (Figure 2a). Figure 2b showed

182

that changes to BCAI at position I370 could easily damage its catalytic ability toward

183

D-galactose.

184

production because of its high activity toward D-galactose and weak specificity for

185

L-arabinose.

186

Enzymatic properties and kinetic constants of F279I. Crude extract of the F279I

187

variant was purified using a HisTrap HP 5-mL column. D-galactose activity assays

188

found that F279I achieved maximal activity at 50 °C, which was lower than the

189

optimal temperature of WT24. F279I could maintain its advantage in

190

activity over WT in the temperature range from 30 °C to 50 °C (Figure 3a and [24]).

191

The optimal pH of F279I was shown to be 8.0, which was slightly higher than for WT.

192

Meanwhile, F279I showed better adaptability (higher activity) to a basic environment

Therefore, F279I was the most suitable mutant for

10


D-galactose

D-galactose

Page 11 of 32


193

than WT but lost activity more quickly when the pH decreased from 7 to 2.2 (Figure

194

3b and [24]).

195

F279I exhibited a higher Vmax and lower Km for D-galactose than WT24 (Table 2),

196

which ultimately resulted in a higher catalytic efficiency (based on the kcat/Km value)

197

for D-galactose with 1.4 min-1 mM-1, 1.4-fold higher than WT. The kcat/Km of F279I

198

for

199

demonstrated that F279I is superior to WT for D-tagatose production.

200

Molecular docking studies on D-galactose catalysis of F279I. To better understand

201

the substrate binding difference between WT and F279I, D-galactose was docked into

202

their respective binding pocket. Figure 4a showed that only two hydrogen bonds were

203

formed between the C2/C3 hydroxyl group of D-galactose and the E306 residue of

204

WT. When docked to F279I, in contrast, the D-galactose molecule rotated to a position

205

where its C6 was close to the I279 residue, and three hydrogen bonds were formed

206

between the hydroxyl group of C1 and E306 and the hydroxyl group of C2 and E331.

207

In addition, Figure 4a showed that the F279 residue of WT was contained in the

208

D-galactose

209

outside the same sphere, which indicated that substituting isoleucine for

210

phenylalanine could enlarge the active site space of BCAI and accommodate the C6

211

of D-galactose (Figure 4b). Meanwhile, the CDOCKER energy of D-galactose docked

212

to F279I was -7.91 kcal mol-1, which was lower than for WT (-7.07 kcal mol-1), which

213

also confirmed that F279I was more favorable for D-galactose binding.

214

Biotransformation of

L-arabinose

was 5.2 min-1 mM-1, 40% lower than WT. These data further

docking sphere with a radius of 6 Å, while the I279 residue of F279I was

D-galactose

to

D-tagatose

by recombinant E. coli

11



215

overexpressing F279I. The production of

216

strains overexpressing F279I was investigated and compared with WT. The effect of

217

reaction temperature was firstly examined in the range of 40 °C to 80 °C after 15 h of

218

reaction. As shown in Figure 5a, the conversion rate increased steadily as the

219

temperature was increased from 40 °C to 50 °C, remained stable at approximately 55%

220

at 50 °C~70 °C and decreased sharply at a higher temperature. Between 40 °C and

221

70 °C, F279I achieved a much higher conversion rate than WT24. The effect of

222

D-galactose

223

after 15 h of reaction. The maximal conversion rate (51.6%) was obtained at 20 g L-1,

224

and the maximal

225

(Figure 5b), both of which were obviously higher than for WT24. A time course study

226

of D-tagatose production was performed at 150 g L-1 and 250 g L-1 D-galactose. As

227

shown in Figure 6, after 48 h of biotransformation, the concentrations of D-tagatose

228

were 67.5 g L-1 and 88.4 g L-1, respectively, and the conversion rates were 46.0% and

229

36.7%, respectively. All results were superior to the results using WT (Table 3).

230

Discussion

231

D-tagatose

using recombinant E. coli

concentration was then determined in the range of 20 g L-1 to 250 g L-1

D-tagatose

The production of

concentration (53.7 g L-1) was obtained at 250 g L-1

D-tagatose

employing AI has many advantages over the

232

chemical method, but the weak activity and low conversion rate of AI toward

233

D-galactose

234

novel AI from B. coagulans and applied it to D-tagatose production. In this study, the

235

BCAI was rationally engineered to enhance its activity toward D-galactose and its

236

conversion rate in biotransformation. Our results showed that the substitution of F279

hindered its large-scale application. In previous work, we characterized a

12


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237

with isoleucine lowered the activity of BCAI toward L-arabinose and improved its

238

activity toward D-galactose. Previous studies on the AIs of E. coli and B. licheniformis

239

presumed that the binding of C1 and C2 of the aldose to E331 and E306 was crucial

240

to proton transfer and aldose-ketose interconversion23,

241

hydrogen bonds and shorter bond lengths are beneficial to the isomerization reaction.

242

In this work, for WT, only the hydrogen bond between C2 and E306 (2.35 Å) was

243

helpful to proton transfer (Figure 4a). By contrast, changing F279 to isoleucine

244

apparently enlarged the binding pocket for D-galactose, which allowed D-galactose to

245

rotate to a position in which its C6 side chain was much closer to I279, and both

246

hydroxyl groups of C1/C2 could form more hydrogen bonds with E306/E331. As

247

shown in Figure 4b, all three hydrogen bonds (1.97 Å, 2.02 Å, and 2.05 Å) could play

248

roles in aldose-ketose interconversion. Therefore, the hydrogen bond interactions

249

between D-galactose and F279I were accorded with this presumption.

33

. In other words, more

250

The kinetic constants of WT and F279I also indicated that residue 279

251

determined the substrate specificity of BCAI. Protein sequence alignment showed that

252

the residues at position 279 or corresponding positions were highly conserved as

253

phenylalanine for most AIs, such as the ones from B. halodurans, B. licheniformis, L.

254

fermentum CGMCC2921 and L. sakei 23K7,

255

phenylalanine residue of these AIs by isoleucine might also increase the activity and

256

catalytic efficiency for D-galactose.

17, 28, 34

. Thus, replacement of the

257

Compared with WT, F279I showed an optimal temperature shift from 60 °C to

258

50 °C for maximal enzyme activity (Figure 3a) and possessed a better adaptability 13



Page 14 of 32

259

between 50 °C~70 °C in biotransformation (Figure 5a). The productivity achieved by

260

E. coli containing F279I at 50 °C was higher than the productivity obtained at

261

temperatures

262

surface-displayed AI (Table 3). As mentioned above, most reported AIs came from

263

thermophilic and hyperthermophilic bacteria whose optimal temperatures were above

264

60 °C (also shown in Table 3), and operation temperatures above 60 °C caused

265

unwanted browning reaction and by-products. Therefore, the optimal temperature at

266

50 °C would be an advantage of F279I on the industrial scale.

267

above

60

°C

by

immobilized

cells,

immobilized

AI,

and

In summary, a model of BCAI-galactose complex was obtained by homology

268

modeling and molecular docking. Five residues near

269

mutation sites, and one variant, F279I, was obtained that showed higher activity and

270

specificity toward D-galactose. F279I had advantages over WT not only in enzymatic

271

properties (optimal temperature and catalytic efficiency) but also in conversion rate

272

and

273

economically valuable in industrial D-tagatose production.

274

ASSOCIATED CONTENT

275

Supporting Information

276

The Supporting Information is available free of charge on the ACS Publications

277

website.

278

D-tagatose

D-galactose

were selected as

concentration in a whole-cell biocatalysis system, which would be

Bacterial strains, plasmids, and PCR primers used in this study were listed in the

279

supporting information。

280

AUTHOR INFORMATION 14


Page 15 of 32


281

Corresponding Author

282

* Corresponding author. Address: College of Chemical Engineering, Nanjing Forestry

283

University, Nanjing 210037, People’s Republic of China, Tel.: 86-025-85427129, Fax:

284

86-025-83587587, E-mail: [email protected].

285

Funding

286

We acknowledge the financial support from the Major Program of the Natural Science

287

Foundation of Jiangsu Higher Education of China (16KJA220004), the National

288

Natural Science Foundation of China (51561145015, 31300487), and the Priority

289

Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

290

We also thank Dr. Bingfang He from Nanjing Tech University for offering Discovery

291

Studio Package 4.0 software.

292

Notes

293

The authors declare no competing ﬁnancial interest.

294

ABBREVIATIONS USED

295

AI, L-arabinose isomerase; WT, wild-type; GI, glycemic index; BCAI, B. coagulans

296

NL01

297

isopropyl-β-D-thiogalactopyranoside;

298

chromatography.

299

REFERENCES

300

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DS, HPLC,

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high-performance

IPTG, liquid

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Tables

Table 1 Enzyme assay of protein crude extract. Table 2 Kinetic constants of F279I. Table 3 Comparison of various reported efficient processes for D-tagatose production using AIs.

21



Page 22 of 32

Figure captions

Figure 1 Structure of the active site of BCAI complexed with

D-galactose

molecule. The solid line represents the docking sphere of 6-Å radius. The hydrogen bonds are represented by green dotted lines. D-galactose molecules and amino acid residues are displayed in stick form and colored according to elemental types. Carbon atoms of D-galactose were colored pink for a better visualization. Figure 2 Comparison of activities toward L-arabinose and D-galactose. (a) WT and F279 variants; (b) WT and I370 variants. Enzyme assay was carried out at 60 oC, pH 7.5. Figure 3 Effects of temperature (a) and pH (b) on D-galactose activity of F279I. Figure 4 Docking of D-galactose to WT (a) and F279I (b). The hydrogen bonds are represented by green dotted lines. D-galactose molecules and amino acid residues are displayed in stick form and colored according to elemental types. Carbon atoms of D-galactose

were colored pink for a better visualization.

Figure 5 Effects of temperature (a) and substrate concentration (b) on D-galactose

bioconversion by whole cells of E. coli expressing F279I. (■),

Conversion rate; (□), D-Tagatose concentration. Figure 6 Time course of whole cell catalysis of 150 g L-1 (a) and 250 g L-1 (b) D-galactose.

(■), D-Galactose concentration; (□), D-Tagatose concentration.

22


Page 23 of 32


Table 1 Enzyme assay of protein crude extract. L-arabinose

activity

D-galactose

activity

Enzyme

L-arabinose

activity D-galactose actvity

（U mg-1）

（U mg-1）

WT

5.07±0.08

0.42±0.04

12.07

M185A

1.46±0.06

0.13±0.01

11.23

R186A

3.38±0.13

0.36±0.03

9.39

F279A

0.53±0.03

0.14±0.01

3.79

M349A

2.62±0.38

0.13±0.01

20.15

I370A

2.36±0.16

0.04±0.01

59.00

Enzymatic activity was measured at 60oC and pH 7.5.

23



Table 2 Kinetic constants of F279I. Substrate

Vmax (U mg-1)

Km (mM) kcat (min-1) kcat /Km (min-1 mM-1)

D-galactose

7.4

292.8

395.5

1.4

L-arabinose

18.9

194.1

1010.2

5.2

All experiments were carried out at 50 oC and pH 8.0.

24


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Page 25 of 32


Table 3 Comparison of various reported efficient processes for D-tagatose production using AIs. Conversion

Galactose

Tagatose

-1

-1

Productivity

Biocatalyst

Reference conditions

a

(g L )

-1

-1

(g L )

(g L h )

65 oC, pH 6.5

100

57.1

2.4

[28]

75 oC, pH 7.5

18

7.9

1.9

[29]

Immobilized AI from hot-spring bacteria

60 oC, pH 8.0

100

58

0.64

[30]

Surface-displayed AI from L. fermentum

70 oC, pH 6.5

100

75

3.13

[31]

a

60 oC, pH 8.5-9.0

500

370

15.4

[32]

150

48.1

1.5

250

55.5

1.7

150

67.5

4.5

Immobilized L. fermentum

b

Immobilized AI from Thermoanaerobacter mathranii

Purified mutant AI from G. thermodenitrificans

Whole cells of E. coli BL21 expressing AI from B. o

60 C, pH 7.5

[24]

coagulans

Whole cells of E. coli BL21 expressing F279I

50 oC, pH 8.0

This study 250

a

88.4

Borate was added to the bioconversion system; b MnCl2 was added to the bioconversion system.

25


5.9


Figure 1

26


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Page 27 of 32


Figure 2

27



Figure 3

28


Page 28 of 32

Page 29 of 32


Figure 4

29



Figure 5

30


Page 30 of 32

Page 31 of 32


Figure 6

31



TOC graphic: L-arabinose isomerase was rationally designed to F279I and used for D-tagatose

production.

32


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Rational Design of Bacillus coagulans NL01 l-Arabinose Isomerase

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