Characterization of a Novel Maltose-Forming Î±-Amylase from

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Characterization of a Novel Maltose-forming #-amylase from Lactobacillus plantarum subsp. plantarum ST-III Hye-Yeon Jeon, Na-Ri Kim, Hye-Won Lee, Hye-Jeong Choi, WooJae Choung, Ye-Seul Koo, Dam-Seul Ko, and Jae-Hoon Shim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05892 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 7, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Journal of Agricultural and Food Chemistry

1 2 3

Characterization of a Novel Maltose-forming α-amylase from Lactobacillus

4

plantarum subsp. plantarum ST-III

5 6

7

Hye-Yeon Jeon, Na-Ri Kim, Hye-Won Lee, Hye-Jeong Choi, Woo-Jae Choung, Ye-Seul Koo,

8

Dam-Seul Ko, and Jae-Hoon Shim*

9

10

11

Department of Food Science and Nutrition, and Center for Aging and Health Care, Hallym

12

University, Hallymdaehak-gil 1, Chuncheon, Gwangwon-do, 200-702, Korea

13

14

*Author to whom correspondence should be addressed

15

E-mail: [email protected], Phone: 82-33-248-2137, FAX: 82-33-248-2146

16

17

ACS Paragon Plus Environment


18

ABSTRACT

19

A novel maltose (G2)-forming α-amylase from Lactobacillus plantarum subsp. plantarum

20

ST-III was expressed in Escherichia coli and characterized. Analysis of conserved amino-acid

21

sequence alignments showed that Lactobacillus plantarum maltose–producing α-amylase

22

(LpMA) belongs to glycoside hydrolase family 13. The recombinant enzyme (LpMA) was a

23

novel G2-producing α-amylase. The properties of purified LpMA were investigated following

24

enzyme purification. LpMA exhibited optimal activity at 30°C and pH 3.0. It produced only

25

G2 from the hydrolysis of various substrates, including maltotriose (G3), maltopentaose (G5),

26

maltosyl β-cyclodextrin (G2-β-CD), amylose, amylopectin, and starch. However, LpMA was

27

unable to hydrolyze cyclodextrins. Reaction pattern analysis using 4-nitrophenyl-α-D-

28

maltopentaoside (pNPG5) demonstrated that LpMA hydrolyzed pNPG5 from the non-

29

reducing end, indicating that LpMA is an exo-type α-amylase. Kinetic analysis revealed that

30

LpMA had the highest catalytic efficiency (kcat/Km ratio) to G2-β-CD. Compared with β-

31

amylase, a well-known G2-producing enzyme, LpMA produced G2 more efficiently from

32

liquefied corn starch due to its ability to hydrolyze G3.

33

Key words:

-amylase, Lactobacillus plantarum, maltose, glycoside hydrolase (GH) 13

34


Page 2 of 31

Page 3 of 31


35

INTRODUCTION

36

Numerous α-amylases (EC 3.2.1.1) from bacteria, fungi, mammals, and plants have been

37

characterized, and their genes have been cloned.1 These amylolytic enzymes catalyze the

38

hydrolysis of internal α-1,4 glucan bonds in substrates at random. They are among the most

39

important and well-known industrial enzymes, having various applications in the starch

40

processing, alcohol production, brewing, textile, and paper industries.2-6 These enzymes are

41

mainly members of glycoside hydrolase family 13 (GH13), also known as the α-amylase

42

superfamily. GH13 consists of various hydrolases, isomerases, and transglycosidases (e.g., α-

43

amylase, α-glucosidase, cyclomaltodextrin glucanotransferase, and maltogenic amylase

44

[MAase]).7, 8

45

Maltose (G2) is an important and valuable product, which is generated as a result of

46

carbohydrate hydrolysis.9 G2 is mildly sweet and has good thermal stability in solution.3 Due

47

to these properties, G2 is widely used in the food, biomedical, pharmaceutical, and chemical

48

industries.10, 11

49

Some G2-producing carbohydrate enzymes are members of GH13, GH14, and GH57.4, 11-15

50

Among them, β-amylase (EC 3.2.1.2), a member of GH14, is a well-known exo-type enzyme

51

that liberates β-G2 from starch.16 MAase (EC 3.2.1.133), also a member of GH13, is known

52

for its G2 production. MAase has the unique ability to hydrolyze both α-1,4 and α-1,6

53

linkages in starch, producing G2 as the primary product. This reaction occurs through an

54

endo-type attack that retains the anomeric configuration.17-20 Recently, novel G2-forming

55

exo-type α-amylases from Thermococcus sp. CL1 and Pyrococcus sp. were characterized as

56

members of GH57.4, 7 These G2-forming enzymes are capable of hydrolyzing α-1,4 and α-1,6

57

linkages, which has not been observed previously by any GH57 member.



Page 4 of 31

58

In this study, we report on a novel G2-forming α-amylase of GH13 from Lactobacillus

59

plantarum subsp. plantarum ST-III (Lactobacillus plantarum maltose–producing α-amylase

60

[LpMA]). We investigated its reaction patterns using various substrates. In addition, we

61

evaluated the ability of this enzyme to produce G2 by comparison with the G2 production of

62

β-amylase, a well-known G2-producing enzyme utilized widely in the food industry.

63 64

MATERIALS AND METHODS

65 66

Chemicals and reagents

67

Escherichia coli MC1061 (F±, araD139, recA13, D [araABCleu] 7696, galU, galK,

68

∆lacX74, rpsL, thi, hsdR2, and mcrB) was used as a parent strain for DNA manipulation and

69

transformation. We purchased Luria–Bertani (LB) medium from Becton Dickinson (BD;

70

Franklin Lakes, NJ) and soluble starch from Showa Chemical (Showa, Japan). β-Amylase

71

from barley, G2, maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose

72

(G6), maltoheptaose (G7), glycogen from bovine liver, and p-nitrophenyl α-D-maltopentaose

73

(pNPG5) were purchased from Sigma-Aldrich (St. Louis, MO). Branched β-CDs, including

74

G2-, G5-, and G6-β-CDs were purchased from CarboExpert Inc. (Daejeon, Korea). Liquefied

75

corn starch (LCS; 23%) was kindly provided by Samyang Genex Co. (Incheon, Korea).

76 77

Cloning and nucleotide sequence analysis

78

The gene encoding lpst_c0146 was amplified from Lactobacillus plantarum subsp.

79

plantarum ST-III (KCTC 3015) genomic DNA. The gene was amplified by polymerase chain

80

reaction

81

AAAAGTCGACAGCACGCGATACGCAAACG -3’) and a reverse primer (lpst_c0146 R,

(PCR)

using

a

forward

primer


(lpst_c0146

F,

5’-

Page 5 of 31


82

5’- AAAACTCGAGATTGGACTGGTCAGCAAC -3’), containing SalI and XhoI restriction

83

sites, respectively. The PCR was performed as follows: 1 min at 98°C for denaturation; 30

84

cycles of 10 s at 98°C for denaturation, 30 s at 55°C for annealing, and 1 min 28 s at 72°C for

85

extension; and 5 min at 72°C for final extension.

86

The amplified fragment (1.3 kbp) was digested with SalI/XhoI and ligated into the

87

kanamycin-resistant pTKNd119 vector containing the maltogenic amylase (BLMA) promoter

88

from Bacillus licheniformis.21 The resulting plasmid was transformed into E. coli MC1061

89

and spread onto LB agar plates containing kanamycin (50 µg/mL).

90 91

Expression and purification of recombinant protein

92

E. coli MC1061 transformants with pTKNdlpst in LB medium (1% bacto-tryptone, 0.5%

93

yeast extract, 0.5% NaCl) supplemented with 50 µg/mL kanamycin were incubated for 20 h

94

at 150 rpm and 30°C. Cells were harvested by centrifugation (7,000 × g, 20 min, 4°C) and

95

suspended in lysis buffer (300 mM NaCl, 50 mM Tris–HCl buffer [pH 7.5], and 10 mM

96

imidazole). Following cell disruption using a sonicator (XL-2000; QSonica, LLC., Newtown,

97

CT; output 12, 5 min, four times), the cell extract was centrifuged (7,000 × g, 20 min, 4°C)

98

and the supernatant was collected. The target enzyme was purified using nickel-

99

nitrilotriacetic acid (Ni-NTA) affinity chromatography, washed (300 mM NaCl, 50 mM Tris-

100

HCl [pH 7.5], and 20 mM imidazole), and then eluted using elution buffer (300 mM NaCl, 50

101

mM Tris-HCl [pH 7.5], and 250 mM imidazole). Purified enzyme, referred to as LpMA, was

102

visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

103 104

Phylogeny and sequence analysis

105

Several α-amylase, maltogenic amylase, and β-amylase sequences from members of GH13

106

and GH14 were obtained from the Carbohydrate-Active enZymes (CAZy) database



107

(http://www.cazy.org/). The amino-acid sequences were then used to construct a neighbor

108

joining tree from 1000 bootstrap replicates using MEGA6 software.22

109 110

Enzyme assay

111

The amount of reducing sugars produced by LpMA during enzymatic activity in a 0.5%

112

soluble starch solution in 50 mM sodium acetate (pH 3.0) at 30°C was measured. After the

113

mixture was preheated for 5 min without LpMA, enzyme activity was measured by 3,5-

114

dinitrosalicylic acid (DNS).23 The reaction mixture with LpMA was boiled for 5 min to stop

115

the reaction. The standard curve consisted of 0.01–0.1% G2 solution, and 1 unit of LpMA

116

activity was defined as the amount of enzyme required to generate 1 µmol of maltose per

117

minute. Measurements were made using a spectrophotometer (Multiskan FC; Thermo

118

Scientific, Whatman, MI) at 570 nm. Protein concentration was measured using the Bradford

119

method with bovine serum albumin (BSA) as the standard.24

120 121

Effects of temperature and pH on enzyme activity and stability

122

The optimal temperature of LpMA activity was determined using soluble starch as substrate

123

in 50 mM sodium acetate (pH 6.0) buffer, and DNS to measure activity at various

124

temperatures (10–50°C). Similarly, the effect of pH on LpMA activity was measured at

125

various pHs (2.5–6) using 50 mM citric-NaOH (pH 2.5–4.0) and 50 mM sodium acetate (pH

126

4.0–6.0).

127 128

Effects of metal ions on enzyme activity

129

The effects of various metal ions on LpMA enzymatic activity were determined by adding the

130

metal ions to a final concentration of 5 mM in the reaction mixture, which was then incubated

131

for 10 min at 20°C. The following metal ions were used: Mg2+, Mn2+, Ca2+, Cu2+, Fe3+, Co2+,


Page 6 of 31

Page 7 of 31


132

and ethylenediaminetetraacetic acid (EDTA). The activity of LpMA was measured under

133

standard conditions (pH 3.0 at 30°C), and enzyme activity in the absence of metal ions was

134

considered to be 100%.

135 136

Kinetic parameters

137

The enzyme kinetics of LpMA were studied using a diverse set of substrates, including G2-β-

138

CD, G3, G5, glycogen, corn starch, potato starch, soluble starch, amylose, and amylopectin.

139

Kinetic analysis of LpMA activity was measured at 30°C in 50 mM citric-NaOH buffer (pH

140

3.0), and determined using high-performance anion-exchange chromatography (HPAEC).

141

Concentrations of potato starch, amylose, and amylopectin used in the determination of

142

kinetic parameters were 0.05–3.5 times the Km values of the hydrolyzing activity. LpMA

143

enzyme kinetics were calculated by fitting a Michaelis-Menten equation in GraFit (Ver. 7.0;

144

Erithacus Software Ltd, Staines, UK). When saturation was not detected, the enzymatic

145

reaction was performed with a lower substrate concentration than Km and the apparent kcat/Km

146

values were determined from the slope of the plot of Vo versus “S” (from the equation: Vo =

147

kcatEo[S]/Km), which measured several substrates at a range of concentrations (2.5–20

148

mg/mL).21, 25, 26

149

150

HPAEC and thin-layer chromatographic analysis

151

Enzyme products were analyzed by HPAEC and thin-layer chromatography (TLC). HPAEC

152

was performed using a Dionex CarboPacTM PA1 column (4 × 250 mm; Thermo Scientific). A

153

20 µL sample was initially injected and eluted with 600 mM sodium acetate in 150 mM

154

NaOH solution, which was gradually increased. The flow rate was 1.0 mL/min. TLC was

155

performed using a K5F silica-gel plate (Whatman, Maidstone, UK). Samples were spotted on



156

the silica-gel plate, dried, and then added to developing solvent (n-butanol-ethanol-water,

157

5:5:3, v/v/v). To analyze the reaction products, the silica-gel plate was dried, soaked in

158

dipping solution (0.3% [w/v] N-[1-naphthyl]-ethylenediamine and 5% [v/v] H2SO4 in

159

methanol), and incubated for 10 min at 110°C.

160

161

Comparison of LpMA and plant β-amylase action patterns

162

In the presence of 1% G3, G5, G6, and 23% LCS, enzyme reactions with LpMA (0.9 U/mg of

163

substrate) or commercial β-amylase (0.9 U/mg of substrate, barley originating β-amylase;

164

Sigma, St. Louis, MO) were performed for 24 h using 50 mM citric-NaOH buffer (pH 3.0) at

165

30°C and 50 mM sodium acetate buffer (pH 5.0) at 20°C, respectively. The reaction products

166

from LpMA and β-amylase were analyzed using TLC and HPAEC.

167

168

RESULTS

169 170

Cloning, expression, and purification of recombinant LpMA from Lactobacillus plantarum

171

subsp. plantarum ST-III

172

The encoding gene, lpst-c0146, from genomic DNA (KCTC 3105) was cloned and amplified

173

using PCR (see Figure S1 in the supplemental data). The amplified gene (approximately 1.3

174

kbp) was ligated into the pTKNd119 vector. This recombined plasmid, pTKNdlpst, was

175

transformed into E. coli (MC1061) and the expressed protein was purified from whole-cell

176

extracts using Ni-NTA affinity chromatography. The purified protein consisted of 462 amino

177

acids and had an expected weight approximately 49-50 kDa. The expression of LpMA was

178

determined by a single band using SDS-PAGE analysis. Expression of the recombinant clone


Page 8 of 31

Page 9 of 31


179

was greatest when incubated at 30°C for 20 h with shaking (150 rpm) and oxygenation (see

180

Figure S2, Figure S3, and Table S1 in the supplemental data).

181 182

Characterization of LpMA

183

To determine the optimal conditions for LpMA enzymatic activity, enzyme reactions were

184

performed at various temperatures and pHs. The optimal temperature and pH were measured

185

using soluble starch as a substrate, and all experiments were performed in triplicate. The

186

optimal conditions for LpMA enzymatic activity were 30°C and pH 3.0 (Figure 1). LpMA

187

showed catalytic activity on α-1,4 linked substrates, including G3, G4, G7, amylose,

188

amylopectin, and soluble starch, producing only G2. However, LpMA could not hydrolyze α-

189

CD, β-CD, or γ-CD, suggesting that it is an exo-type glucan hydrolase (Figure 2A).4, 27 To

190

examine the reaction patterns more precisely, pNPG5 was added to LpMA reactions. LpMA

191

hydrolyzed the α-1,4 glucosidic bonds of pNPG5, resulting in production of G2 and pNPG3,

192

indicating that the α-amylase hydrolyzed glucosidic linkages from the non-reducing end

193

(Figure 2B). LpMA requires at least one G2 unit at the substrates’ working sites (Figure 2C).

194

When the substrate’s branch was longer than G2, LpMA primarily attacked α-1,4–glycosidic

195

linkages first, and then cleaved off the G2 unit until it was able to reach the final G2. After

196

the final G2, LpMA performed a debranching reaction by acting on α-1,6–glycosidic bonds at

197

branching points. In the case of G5-β-CD, LpMA released G2 units until the substrate was

198

hydrolyzed to G1-β-CD, with few compounds being degraded into β-CD and glucose (G1).

199

The released β-CD was not hydrolyzed.

200

The specific activity of LpMA indicated that it had the highest enzymatic activity for G2-β-

201

CD, followed by G5, soluble starch, G4, G3, and glycogen. For potato starch, corn starch,

202

amylose, and amylopectin, little activity was detected. For soluble starch and glycogen,



203

LpMA’s specific activity was higher than that of other polysaccharides. Other substrates,

204

including potato starch, corn starch, amylose, and amylopectin were cleaved by LpMA at low

205

levels (Table 1).

206

Multiple amino acid sequence alignments revealed that the LpMA enzyme contained five

207

conserved regions and three catalytic sites that belong to the GH13 family (Figure 3). Based

208

on the analysis of phylogenetic trees, LpMA is distinct from the other G2 forming α-

209

amylases (α-maltohydrolases) from Lactobacillus sp. that were identified previously (Figure

210

4).28

211

212

Effects of metal ions on enzyme activity

213

Among the various metal ions assessed, enzymatic activity was not affected significantly by 5

214

mM Ca2+ or EDTA. Enzymatic activity was highest in the presence of 5 mM Mg2+. In

215

contrast, α-amylase activity was slightly reduced in the presence of 5 mM Mn2+. However,

216

some divalent and trivalent metal ions at 5 mM, including Co2+, Cu2+, and Fe3+, completely

217

inhibited LpMA activity (Figure 5).

218

219

Kinetic parameters of LpMA

220

The kinetic parameters of LpMA were analyzed using various substrates at a range of

221

concentrations. The catalytic efficiency (kcat/Km) of LpMA for G2-β-CD hydrolysis was

222

highest among all maltooligosaccharide substrates, followed by G5 and G3 (Table 2). When

223

polysaccharides were used as the substrates, LpMA had higher kcat/Km values in amylose than

224

that in amylopectin. Among the starch substrates, soluble starch demonstrated the highest


Page 10 of 31

Page 11 of 31

225


catalytic efficiency, which may result from its lower Km value.

226

227

Comparison of G2 production between LpMA and plant β-amylase

228

To evaluate the industrial applicability of LpMA, the same amounts of LpMA and

229

commercial β-amylase were used in reactions with 3% LCS for 24 h under their respective

230

optimal conditions. The reaction products from both enzymes were analyzed using TLC and

231

HPAEC (Figure 6). LpMA showed catalytic activity similar to that of β-amylase, with both

232

producing G2 as the main product of LCS. However, G3 hydrolysis differed notably. LpMA

233

hydrolyzed G3 to G2 and G1, whereas β-amylase did not (Figure 6A). To verify the practical

234

application of LpMA, the yield of G2 production from 3% LCS in a reaction with both

235

enzymes was evaluated. Final G2 yields from LpMA and β-amylase were 74% and 64%,

236

respectively (Figure 6B).

237

238

DISCUSSION

239

Phylogenetic tree analysis suggests that LpMA is a member of the α-amylase GH13 and has a

240

long, distant evolutionary relationship with maltogenic amylases in GH13 and G2-forming α-

241

amylases in GH57 (Figure 4). Amino-acid sequence alignment analysis revealed that LpMA

242

has five conserved regions and three amino-acid residues necessary for GH13 α-amylase

243

activity (Asp171, Glu200, and Asp277; Figure 3).29,

244

amino-acid sequences of LpMA and other α-amylases was detected within the third

245

conserved region. In this region, each His (H) and Glu (E) residue is conserved in bacterial α-

246

amylases and maltogenic amylases, respectively. In maltogenic amylase, the E residue is

247

related to the extra sugar-binding space and transglycosylation activity.31 In α-amylases, the

30

Interestingly, dissimilarity in the



248

H residue, at the same position as the H residue in maltogenic amylase, acts as a Ca2+-binding

249

ligand, CD ring-opening, and +1 substrate binding site.32-35 However, the corresponding

250

residue for LpMA is Leu-175, suggesting that LpMA is unique among GH13 members.

251

To date, many α-amylases from lactobacilli have been studied.26, 28, 36-38 Transcriptional and

252

enzymatic analyses showed that extracellular α-amylases hydrolyze starch into small

253

oligosaccharides then, other amylases, including neopullulanases, matogenic amylases, and

254

α-maltohydrolyase catalyze further hydrolysis reactions. Among the α-amylases from

255

lactobacilli, FERMENTA, an extracellular enzyme isolated from Lactobacillus fermentum,

256

primarily produced G2 and G3.26, 37 Also, 1,4-α-maltohydrolyases that generate maltose were

257

discovered from L. plantarum,28,

258

oligosaccharides like LpMA, the activity of LpMA differed from that of maltogenic amylases

259

and other α-amylases in GH13 (Figure 2). Both maltogenic amylase and LpMA belong to

260

GH13 and hydrolyze α-1,4 and α-1,6 glycosidic linkages to generate G2 as the main product.

261

However, LpMA is not able to hydrolyze CDs and acts on the non-reducing ends of

262

substrates. This reaction is similar to that of G2-forming amylases in GH57 rather than that of

263

maltogenic amylases in GH13 (Table 3).

39

and although these GH13 α-amylases produce small

264

According to the kinetic data, LpMA prefers the α-1,6 linkage to the α-1,4 linkage for

265

hydrolysis. The kcat/Km value of G2-β-CD was higher than that of G3 or G5 (Table 2).

266

However, the activity of LpMA is different from that of debranching enzymes that hydrolyze

267

α-1,6–glycosidic linkages, since LpMA primarily hydrolyzed the α-1,4–glycosidic linkages in

268

sequence, and only cleaved the α-1,6–glycosidic linkage when it reached the final G2 (Figure

269

2C). Thus, these results indicate the distinctions between LpMA, MAases, and other types of

270

α-amylase. The comparisons among G2-forming amylases are summarized in Table 3.

271

The optimal temperature and pH for LpMA enzymatic activity were 30°C and 3.0,


Page 12 of 31

Page 13 of 31


272

respectively (Figure 1). A low pH offers advantages, including enhanced bacterial

273

decontamination.40 Addition of heavy metal ions (Co2+, Cu2+, and Fe3+) to LpMA activity

274

completely inhibited enzyme activity. However, LpMA activity was not affected by EDTA.

275

These results suggest that some metal ions (heavy metals) available for industrial processing

276

could be utilized to control LpMA enzyme reactions.

277

In the beverage fermentation industry, β-amylase is usually used to produce G2.12, 41-43 Our

278

experiments showed that LpMA is more effective than commercial β-amylase in G2

279

production, which is a result of G3 hydrolysis (Figure 6A). Interestingly, LpMA hydrolyzed

280

G3, which is not readily hydrolyzed by β-amylase.44 Furthermore, during LpMA reactions,

281

the amount of G3 decreased, which may have increased the yield and purity of G2. In

282

addition, G1 released from G3 hydrolysis would be beneficial for ethanol fermentation

283

because G1 is the preferred energy source of budding yeast (Saccharomyces cerevisiae).45-47

284

In this study, we evaluated the properties and industrial applicability of LpMA. Our results

285

indicated that LpMA is a novel α-amylase GH13 member that produces G2 as a main

286

product. Furthermore, its properties suggest that it would be highly applicable in the food

287

industry.

288 289

Acknowledgment

290

This study was supported by the Basic Science Research Program (NRF-2014-0009695) of

291

the National Research Foundation, funded by the Korean Government.

292 293

Supporting Information available

294

Table S1 and Figure S1-S3

295

The Supporting Information is available free of charge on the ASC Publication website.

296



297

REFERENCES

298 299

1.

Dong, G.; Vieille, C.; Savchenko, A.; Zeikus, J. G., Cloning, sequencing, and expression of t

300

he gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical c

301

haracterization of the recombinant enzyme. Appl. Environ. Microb. 1997, 63, 3569-3576.

302

2.

stry - a review. Braz. J. Microbiol. 2010, 41, 850-861.

303 304

de Souza, P. M.; de Oliveira Magalhaes, P., Application of microbial alpha-amylase in indu

3.

Srivastava, G.; Singh, K.; Talat, M.; Srivastava, O. N.; Kayastha, A. M., Functionalized graphe

305

ne sheets as immobilization matrix for Fenugreek beta-amylase: enzyme kinetics and stabi

306

lity studies. PLoS One 2014, 9, e113408.

307

4.

Jung, J. H.; Seo, D. H.; Holden, J. F.; Park, C. S., Maltose-forming alpha-amylase from the

308

hyperthermophilic archaeon Pyrococcus sp. ST04. Appl. Microbiol. Biotechnol. 2014, 98, 21

309

21-2131.

310

5.

Giraud, E.; Cuny, G., Molecular characterization of the alpha-amylase genes of Lactobacillu

311

s plantarum A6 and Lactobacillus amylovorus reveals an unusual 3' end structure with dir

312

ect tandem repeats and suggests a common evolutionary origin. Gene 1997, 198, 149-15

313

7.

314

6.

biotechnological perspective. Process Biochem. 2003, 38, 1599-1616.

315 316

Gupta, R.; Gigras, P.; Mohapatra, H.; Goswami, V. K.; Chauhan, B., Microbial α-amylases: a

7.

Jeon, E. J.; Jung, J. H.; Seo, D. H.; Jung, D. H.; Holden, J. F.; Park, C. S., Bioinformatic and

317

biochemical analysis of a novel maltose-forming alpha-amylase of the GH57 family in the

318

hyperthermophilic archaeon Thermococcus sp. CL1. Enzyme Microb. Tech. 2014, 60, 9-15.

319

8.

r. Med. 2014, 7, 773-777.

320 321 322

Koibuchi, E.; Suzuki, Y., Exercise upregulates salivary amylase in humans (Review). Exp. The

9.

Upadhyay, C. M.; Shah, N. K.; Kothari, R. M., A protocol for optimal extraction and stabiliz ation of barley β-amylase. Biotechnology Tech. 1990, 4, 117-120.


Page 14 of 31

Page 15 of 31

323


10. Doylea, E. M.; Noone, A. M.; Kellya, C. T.; Quigleya, T. A.; Fogarty, W. M., Mechanisms of

324

action of the maltogenic α-amylase of Byssochlamys Fulva. Enzyme Microb. Tech. 1998, 2

325

2, 612-616.

326 327 328 329 330 331 332 333 334

11. Ammar, Y. B.; Matsubara, T.; Ito, K.; Iizuka, M.; Limpaseni, T.; Pongsawasdi, P.; Minamiura, N., New action pattern of a maltose-forming alpha-amylase from Streptomyces sp. and its possible application in bakery. J. Biochem. Mol. Biol. 2002, 35, 568-575. 12. Hopkins, R. H.; Murray, R. H.; Lockwood, A. R., The beta-amylase of barley. Biochem. J. 19 46, 40, 507-512. 13. Oh, K. W.; Kim, M. J.; Kim, H. Y.; Kim, B. Y.; Baik, M. Y.; Auh, J. H.; Park, C. S., Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli. FEMS Microbiol. Lett. 2005, 252, 175-181. 14. Todorov, S. D.; van Reenen, C. A.; Dicks, L. M., Optimization of bacteriocin production by

335

Lactobacillus plantarum ST13BR, a strain isolated from barley beer. J. Gen. Appl. Microbiol

336

. 2004, 50, 149-157.

337 338

15. Blesak, K.; Janecek, S., Sequence fingerprints of enzyme specificities from the glycoside hy drolase family GH57. Extremophiles 2012, 16, 497-506.

339

16. Oyama, T.; Miyake, H.; Kusunoki, M.; Nitta, Y., Crystal structures of beta-amylase from Baci

340

llus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagent

341

s. J. Biochem. 2003, 133, 467-474.

342

17. Kim, I. C.; Cha, J. H.; Kim, J. R.; Jang, S. Y.; Seo, B. C.; Cheong, T. K.; Lee, D. S.; Choi, Y. D

343

.; Park, K. H., Catalytic properties of the cloned amylase from Bacillus licheniformis. J. Biol.

344 345

Chem. 1992, 267, 22108-22114. 18. Derde, L. J.; Gomand, S. V.; Courtin, C. M.; Delcour, J. A., Characterisation of three starch

346

degrading enzymes: thermostable beta-amylase, maltotetraogenic and maltogenic alpha-a

347

mylases. Food Chem. 2012, 135, 713-721.

348

19. Kim, T. J.; Kim, M. J.; Kim, B. C.; Kim, J. C.; Cheong, T. K.; Kim, J. W.; Park, K. H., Modes o



349

f action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltog

350

enic amylase, the gene for which was cloned from a Thermus strain. Appl. Environ. Micro

351

biol. 1999, 65, 1644-1651.

352

20. Kim, T. J.; Park, C. S.; Cho, H. Y.; Cha, S. S.; Kim, J. S.; Lee, S. B.; Moon, T. W.; Kim, J. W.;

353

Oh, B. H.; Park, K. H., Role of the glutamate 332 residue in the transglycosylation activit

354 355

y of Thermus maltogenic amylase. Biochemistry 2000, 39, 6773-6780. 21. Kim, Y. W.; Lee, S. S.; Warren, R. A.; Withers, S. G., Directed evolution of a glycosynthase

356

from Agrobacterium sp. increases its catalytic activity dramatically and expands its substra

357

te repertoire. J. Biol. Chem. 2004, 279, 42787-42793.

358

22. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S., MEGA6: Molecular evolutionary

359

genetics analysis version 6.0. Molecular biology and evolution 2013, 30, 2725-2729.

360

23. Miller, G. L., Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal.

361 362

Chem. 1959, 31, 426-428. 24. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantiti

363

es of protein utilizing the principle of protein-dye binding. Analytical biochemistry 1976,

364

72, 248-254.

365

25. Byun, D. H.; Choi, H. J.; Lee, H. W.; Jeon, H. Y.; Choung, W. J.; Shim, J. H., Properties and

366

applications of β-glycosidase from Bacteroides thetaiotaomicron that specifically hydrolyse

367 368

s isoflavone glycosides. Int. J. Food Sci. Tech. 2015, 50, 1405-1412. 26. Talamond, P.; Noirot, M.; de Kochko, A., The mechanism of action of alpha-amylase from

369

Lactobacillus fermentum on maltooligosaccharides. Journal of chromatography. B, Analytica

370

l technologies in the biomedical and life sciences 2006, 834, 42-47.

371

27. Park, K. M.; Jun, S. Y.; Choi, K. H.; Park, K. H.; Park, C. S.; Cha, J., Characterization of an e

372

xo-acting intracellular alpha-amylase from the hyperthermophilic bacterium Thermotoga ne

373

apolitana. Appl. Microbiol. Biot. 2010, 86, 555-566.

374

28. Humblot, C.; Turpin, W.; Chevalier, F.; Picq, C.; Rochette, I.; Guyot, J. P., Determination of


Page 16 of 31

Page 17 of 31


375

expression and activity of genes involved in starch metabolism in Lactobacillus plantarum

376

A6 during fermentation of a cereal-based gruel. International journal of food microbiology

377 378 379 380

2014, 185, 103-111. 29. Janeček, S.; Svensson, B.; MacGregor, E. A., α-Amylase: an enzyme specificity found in vari ous families of glycoside hydrolases. Cell. Mol. Life Sci. 2014, 71, 1149-1170. 30. Xu, B.; Yang, F.; Xiong, C.; Li, J.; Tang, X.; Zhou, J.; Xie, Z.; Ding, J.; Yang, Y.; Huang, Z., Cl

381

oning and characterization of a novel alpha-amylase from a fecal microbial metagenome.

382

J. Microbiol. Biotechn. 2014, 24, 447-452.

383

31. Park, K. H.; Kim, T. J.; Cheong, T. K.; Kim, J. W.; Oh, B. H.; Svensson, B., Structure, specifici

384

ty and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase fami

385

ly. Biochim. Biophys. Acta 2000, 1478, 165-185.

386

32. van der Veen, B.; Uitdehaag, J.; Dijkstra, B.; Dijkhuizen, L., Engineering of cyclodextrin glyc

387

osyltransferase reaction and product specificity. Biochim. Biophys. Acta 2000, 1543, 336-36

388

0.

389

33. Strokopytov, B.; Knegtel, R. M.; Penninga, D.; Rozeboom, H. J.; Kalk, K. H.; Dijkhuizen, L.;

390

Dijkstra, B. W., Structure of cyclodextrin glycosyltransferase complexed with a maltononaos

391

e inhibitor at 2.6 angstrom resolution. Implications for product specificity. Biochemistry 19

392

96, 35, 4241-4249.

393

34. Lawson, C. L.; van Montfort, R.; Strokopytov, B.; Rozeboom, H. J.; Kalk, K. H.; de Vries, G.

394

E.; Penninga, D.; Dijkhuizen, L.; Dijkstra, B. W., Nucleotide sequence and X-ray structure of

395

cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent

396

crystal form. J. Mol. Biol. 1994, 236, 590-600.

397

35. Koo, Y. S.; Lee, H. W.; Jeon, H. Y.; Choi, H. J.; Choung, W. J.; Shim, J. H., Development an

398

d characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzym

399

e using site-directed mutagenesis. Protein Eng. Des. Sel. 2015, 28, 531-537.

400

36. Ganzle, M. G.; Follador, R., Metabolism of oligosaccharides and starch in lactobacilli: a revi



401

ew. Front. Microbiol. 2012, 3, 340.

402

37. Talamond, P.; Desseaux, V.; Moreau, Y.; Santimone, M.; Marchis-Mouren, G., Isolation, char

403

acterization and inhibition by acarbose of the alpha-amylase from Lactobacillus fermentu

404

m: comparison with Lb. manihotivorans and Lb. plantarum amylases. Comparative bioche

405

mistry and physiology. Part B, Biochemistry & molecular biology 2002, 133, 351-360.

406

38. Turpin, W.; Humblot, C.; Guyot, J. P., Genetic screening of functional properties of lactic a

407

cid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starc

408

hy foods. Appl. Environ. Microbiol. 2011, 77, 8722-8734.

409

39. Zhang, Z. Y.; Liu, C.; Zhu, Y. Z.; Zhong, Y.; Zhu, Y. Q.; Zheng, H. J.; Zhao, G. P.; Wang, S.

410

Y.; Guo, X. K., Complete genome sequence of Lactobacillus plantarum JDM1. Journal of b

411

acteriology 2009, 191, 5020-5021.

412

40. Li, D.; Park, J. T.; Li, X.; Kim, S.; Lee, S.; Shim, J. H.; Park, S. H.; Cha, J.; Lee, B. H.; Kim, J.

413

W.; Park, K. H., Overexpression and characterization of an extremely thermostable maltoge

414

nic amylase, with an optimal temperature of 100 degrees C, from the hyperthermophilic a

415

rchaeon Staphylothermus marinus. N. Biotechnol. 2010, 27, 300-307.

416

41. Halima, N. B.; Khemakhem, B.; Fendri, I.; Ogata, H.; Baril, P.; Pichon, C.; Abdelkafi, S., Ident

417

ification of a new oat beta-amylase by functional proteomics. Biochim. Biophys. Acta. 201

418

5, 1864, 52-61.

419 420 421 422

42. Olufunke, F. O. T.; Azeez, I. I., Purification and characterization of beta-amylase of bacillus

subtilis isolated from Kolanut weevil. J. Biol. Life Sci. 2013, 4. 43. Yosigi, N.; Okada, Y.; Sahara, H.; Koshino, S., PCR cloning and sequencing of the beta-am ylase cDNA from barley. Biochem. J. 1994, 115, 47-51.

423

44. Ishikawa, K.; Nakatani, H.; Katsuya, Y.; Fukazawa, C., Kinetic and structural analysis of enzy

424

me sliding on a substrate: multiple attack in beta-amylase. Biochemistry 2007, 46, 792-79

425

8.

426

45. Crabtree, H. G., Observations on the carbohydrate metabolism of tumours. Biochem. J. 19


Page 18 of 31

Page 19 of 31

427 428 429 430 431


29, 23, 536-545. 46. Kim, J. H.; Roy, A.; Jouandot, D., 2nd; Cho, K. H., The glucose signaling network in yeast.

Biochim. Biophys. Acta 2013, 1830, 5204-5210. 47. Lagunas, R., Energetic irrelevance of aerobiosis for S. cerevisiae growing on sugars. Mol. C

ell Biochem. 1979, 27, 139-146.

432 433



434

FIGURE CAPTIONS

435

Figure 1. Temperature and pH affect LpMA enzyme activity. Optimal temperature (A)

436

and pH (B) were measured using 0.5% soluble starch as the substrate in 50 mM citric-NaOH

437

buffer (pH 2.5–4.0) and sodium acetate buffer (pH 4.0–6.0) at 30°C.

438

439

Figure 2. LpMA action patterns using various substrates. (A) Lanes: S, standard (G1–

440

G7); 1, maltose; 2, maltotriose; 3, maltotetraose; 4, maltoheptaose; 5, α-CD; 6, β-CD; 7, γ-

441

CD; 8, amylose; 9, amylopectin; 10, soluble starch; a, before reaction; b, LpMA reaction. (B)

442

Lanes: S, standard (G1–G7); 1, pNPG5 control; 2, 0.5 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7,

443

overnight. (C) Lanes: S, standard (G1–G7); 1, β-CD; 2, G5-β-CD; 3, G6-β-CD; a, control

444

(before reaction); b, 5 min; c, 10 min; d, 30 min; e, 1 h; f, 3 h; g, overnight.

445

Figure 3. Comparison of amino-acid sequences from LpMA and related α-amylases.

446

Multiple sequence alignments of known α-amylases were analyzed using MEGA6 software.

447

Conserved sequences were marked with gray boxes.

448

Figure 4. Phylogenetic tree analysis of LpMA and related enzymes. Phylogenetic tree of

449

recombinant LpMA and α-amylases from various subfamilies, including α-amylases (GH13

450

and GH57), maltogenic amylases (GH13), and β-amylases (GH14). A novel phylogenetic

451

tree was constructed using the bootstrap method (1,000 iterations) with MEGA6 software.

452

Figure 5. Effects of metal ions on enzymatic activity. The effects of metal ions on LpMA

453

activity were evaluated by the addition of 5 mM Mg2+, Mn2+, Ca2+, Cu2+, Fe3+, Co2+, or

454

ethylenediaminetetraacetic acid (EDTA).

455

Figure 6. Comparison of maltose production by LpMA and plant β-amylase. The

456

enzymatic activities of LpMA and β-amylase were measured using maltooligosaccharides


Page 20 of 31

Page 21 of 31


457

and LCS as the substrates. (A) Maltooligosaccharides, lanes: S, standard (G1–G7); 1,

458

maltotriose; 2, maltopentaose; 3, maltohexaose; a, control (prior to the reaction); b, LpMA

459

reaction; c, barley β-amylase reaction. (B) LCS, lanes: a, LCS control; b, LpMA reaction; c,

460

barely β-amylase reaction. LCS: liquefied corn starch.



Table 1. Specific activities with various substrates Substrate

Specific activity (U/mg)

Maltosyl β-CD (G2-β-CD)

30.09 ± 3.24

Maltopentaose (G5)

3.15 ± 0.11

Maltotetraose (G4)

2.29 ± 0.11

Maltotriose (G3)

2.03 ± 0.05

Soluble starch

2.88 ± 0.01

Glycogen

1.40 ± 0.01

Potato starch

0.0360 ± 0.0044

Corn starch

0.0305 ± 0.0015

Amylose

0.0191 ± 0.0007

Amylopectin

0.0145 ± 0.0002


Page 22 of 31

Page 23 of 31


Table 2. Kinetic parameters of LpMA Substrate

kcat

Km

kcat/Km

(min-1)

(mM)

Maltosyl β-CD (G2β-CD)

244.4 ± 11.9

1.9 ± 0.3

117.58

Maltotriose (G3)

128.5 ± 25.9

23.5 ± 9.6

5.47

Maltopentaose (G5)

n.da

n.d

8.03

Glycogen

n.d

n.d

0.37

Corn starch

n.d

n.d

0.11

Potato starch

4.45 ± 0.22

12.95 ± 1.59

0.34

Soluble starch

4.66 ± 0.21

2.05 ± 0.33

2.06

Amylose

0.94 ± 0.06

0.56 ± 0.19

1.44

Amylopectin

1.17 ± 0.04

2.59 ± 0.36

0.45

a

(mg/mL)

n.d: not determined


(mM-1 × min-1)

(mL/mg) × min-1


Page 24 of 31

Table 3. Comparisons among G2-forming enzymes LpMA

MAase

β-amylase

PSMA

Action type

exo

endo

exo

exo

Final product

G2

G2

G2

G2

α-1,4 < α-1,6

α-1,4 > α-1,6

α-1,4

α-1,4 < α-1,6

CD hydrolysis

×

O

×

×

GH

13

13

14

57

Refs

this study

31

12

7

Hydrolysis linkage

LpMA: maltose-forming α-amylase from Lactobacillus plantarum subsp. plantarum ST-III; MAase: maltogenic amylase; PSMA: Pyrococcus sp. ST04 maltose-forming α-amylase; CD: cyclodextrin; GH: glycoside hydrolase family; Refs: references


Page 25 of 31


Fig. 1



Fig. 2


Page 26 of 31

Page 27 of 31


Fig. 3



Fig. 4


Page 28 of 31

Page 29 of 31


Fig. 5



Fig. 6


Page 30 of 31

Page 31 of 31


TOC GRAPHIC


Characterization of a Novel Maltose-Forming Î±-Amylase from

Recommend Documents