Engineering of Yersinia Phytases to Improve Pepsin and Trypsin

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Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry Canfang Niu, Peilong Yang, Huiying luo, Huoqing Huang, Yaru Wang, and Bin Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02116 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Journal of Agricultural and Food Chemistry

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Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and

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Thermostability and Application Potential in the Food and Feed Industry

3 4

Canfang Niu*, Peilong Yang*, Huiying Luo, Huoqing Huang, Yaru Wang, Bin Yao*

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National Engineering Research Center of Biological Feed, Key Laboratory for Feed

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Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese

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Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China

9 10 11 12 13 14 15

Corresponding authors. Address: National Engineering Research Center of

*

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Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of

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Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No.

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12 Zhongguancun South Street, Beijing 100081, P. R. China. Tel.: +86 10 62169913;

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fax: +86 10 82106054.

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E-mail addresses: [email protected] (PY); [email protected] (CN); [email protected] (BY).

ACS Paragon Plus Environment


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Abstract

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Susceptibility to proteases usually limits the application of phytase. We sought to

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improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and

25

YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The

26

predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA

27

and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant

28

YkAPPA were substituted with S and H, respectively. Six variants were produced in

29

Pichia pastoris for catalytic and biochemical characterization. F89S, E226H and

30

F89S/E226H elevated pepsin resistance and thermostability and K226H and

31

F89S/K226H improved pepsin and trypsin resistance and stability at 60°C and low pH.

32

All the variants increased the ability of the proteins to hydrolyze phytate in corn meal

33

by 2.6–14.9-fold in the presence of pepsin at 37°C and low pH. This study developed

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a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can

35

improve enzyme tolerance against proteases and heat and benefit the food and feed

36

industry in a cost-effective way.

37

Key words: Phytases, Pepsin resistance, Trypsin resistance, Thermostability, Stability

38

at low pH, Hydrolysis of corn meal phytate, optimizing the polarity and charge of the

39

amino acids

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Introduction

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Efficient utilization of phytate phosphorus catalyzed by phytase has been attracting

42

extensive attention for sustainable development and eco-efficiency1. But insufficient

43

rresistance to gastric protease and high temperature may affect the in vivo activity and

44

efficacy of phytases and decrease nutrient utilization efficiency, resulting in economic

45

and environmental problems2.

46

Phytases specifically act on phosphomonoester bonds linked to inositol ring or

47

some other specific compounds and generate various functional molecules (e.g.,

48

myo-inositol pentakis-, tetrakis-, tris-, bis- and monophosphate and inorganic

49

phosphates)3. Some bacterial phytases are highly specific for phytate4. Other fungal

50

phytases from Aspergillus sp. show a broad substrate specificity and high enzyme

51

activity for AMP, ATP, ADP, tripolyphosphate, pyrophosphate, acetyl phosphate,

52

p-nitrophenyl phosphate and sodium phytate5,6. In contrast to phytase, phosphatases,

53

e.g. alkaline phosphatases nonspecifically hydrolyze almost all phosphate monoesters

54

in varios moleculars including nucleotide, protein and alkaloids, etc7.

55

The enzymes are classified into three families, 3-, 5-, or 6-phytases, based on the

56

position of the first removed phosphate in the inositol ring8. Generally, phytases from

57

bacteria and fungi are 3-phytases, while those from plants are 6-phytases9. Phytases

58

are also categorized into the following four groups based on their protein structure and

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catalytic properties: (1) histidine acid phosphatase (HAP), (2) β-propeller phytase

60

(BPP), (3) cysteine phosphatase (CP) and (4) purple acid phosphatase (PAP)10,11.

61

The susceptibility of phytases to proteases usually decreases their activity and

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stability in the monogastric gut and limits their use in commercial applications. Pepsin

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and trypsin are important digestive enzymes with different substrate specificities.

64

Pepsin preferentially cleaves peptide bonds on the carboxylic side of the F, L, E and K

65

residues, while trypsin is specific for K and R12–14. Moreover, protease resistance and

66

thermostability are not necessarily coupled. Examples have been presented wherein

67

yeast and E. coli phosphoglycerate kinase possess similar global stabilities but

68

different proteolytic susceptibilities15. Using metagenomic technology, some phytases

69

was identified with thermostability or stability under acidic plus pepsin conditions16,17.

70

Many efforts have been made to ameliorate the thermostability, pH tolerance and

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catalytic properties, including increasing conformational rigidity, optimizing protein

72

surface properties and introducing disulfide bridges, hydrogen bonds and ionic

73

interactions by rational protein design18–22. key residue replacement was performed to

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shift the pH profile of the thermostable phytase from A. niger NII 08121 to match

75

conditions in the stomach (from pH 2.5 to 3.2)23. A counter-selectable mazF marker

76

and high-fidelity fusion PCR method were used to engineer a no-scar-leaving Bacillus

77

subtilis genome24. Site-directed mutagenesis was used for improving the neutral

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phytase activity from Bacillus amyloliquefaciens25.

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The crystal structures of various phytases have been determined26,27 ,and some

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conserved substrate specificity sites have been verified, including K91, K94, E228,

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D262, K300 and K301 in Aspergillus ficuum phytase and D75 and E272 in A. niger

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pH 2.5 acid phosphatase280,29. Structure-based rational design is beneficial for the

83

improvement of inferior phytase enzymatic features for industrial applications30,31.

84

We have previously cloned two Yersinia phytases of the HAP type: YeAPPA from

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Yersinia enterocolitica and YkAPPA from Y. kristensenii32,33. Their biochemical

86

characterization showed YeAPPA was sensitive to pepsin and trypsin after expression

87

in Pichia pastoris, and P. pastoris-produced YkAPPA was sensitive to pepsin but

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resistant to trypsin34. The protease resistance can be improved by disturbing the

89

interaction of the preference residue with protease35. In this study, modeled structures

90

of Yersinia phytases showed two theoretical pepsin cleavage sites: F89 conserve in the

91

two phytases and E226 in YkAPPA, corresponding to K in YeAPPA. Site-directed

92

mutagenesis and comparison of the enzyme properties of wild-type and mutant

93

proteins showed that the F89S, E226H and F89S/E226H variant phytases are more

94

resistant to pepsin digestion and are more stable at 60°C and the K226H and

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F89S/K226H variants increased the thermostability and resistance to pepsin and

96

trypsin. The decreased hydrophobicity or increased surface positive charge may

97

improve the fitness of an enzyme in processing temperature and gastric protease by

98

increasing its thermostability and its pepsin and trypsin resistance.

99 100

Materials and methods

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Strains, vectors and chemicals

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Escherichia coli Trans1-T1 cells (TransGen, Beijing, China) were used for DNA

103

cloning and sequencing. The DNA purification kit, restriction endonucleases, T4 DNA

104

ligase and Pfu DNA polymerase were purchased from TaKaRa (Tsu, Shiga, Japan),

105

New England Biolabs (Beverly, MA, USA) and TransGen (Beijing, China),

106

respectively. Pichia pastoris GS115 and the pPIC9γ vector were purchased from

107

Invitrogen (Carlsbad, CA, USA) as the host and vector for protein expression

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in eukaryotes, respectively. Phytate (sodium salt), pepsin (P0685) and trypsin (T0458)

109

were supplied by Sigma-Aldrich (St Louis, Mo, USA). All the chemicals were the

110

highest grade available commercially.

111

Phytase mutagenesis 5



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Sequence alignment and predicted solvent surface accessibility were used for

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mutation site selection. The sequence alignment of the phytases from Y. kristensenii

114

and Y. enterocolitica was analyzed with the ClustalW program (European

115

Bioinformatics Institute, Cambridge, MA, USA). Phytase numbering starts at the

116

initiating M in the signal sequence. The tertiary structures of Yersinia phytases were

117

homology-modeled using the Accelrys Discovery Studio v2.5 software with the

118

crystal structure of E. coli phytase (PDB: 1DKL) as the template. The solvent surface

119

accessibility of each residue in the two phytases was determined using the I-TASSER

120

program (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) and assigned solvent

121

accessibility scores range from 0 (buried residue) to 9 (highly exposed residue). Six

122

variants were constructed by substitution of F89 and E/K226 (solvent accessibility

123

values of 3 and 4, respectively) with S and H using overlap-extension PCR with

124

specific primers as described previously (Table S1)36. The YkAPPA and YeAPPA

125

phytase genes (GenBank accession no. EU203664 and GU936684, respectively) were

126

used as the DNA template32,33. The desired variant genes were inserted into the

127

pEASY-T3 vector (TransGen, Beijing, China) for DNA sequencing.

128

Enzyme production and purification

129

The wild type and variants harboring the correct mutations were digested with

130

restriction endonucleases, fused in-frame with the pPIC9γ vector, linearized with

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BglII and individually expressed in P. pastoris GS115 competent cells. The crude

132

phytase solutions were purified using a Vivaflow ultrafiltration membrane

133

(Vivascience) with a 5-kDa molecular weight cut-off and a fast protein liquid

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chromatography system consisting of nickel-nitrilotriacetic acid (Ni-NTA) (Qiagen,

135

Hilden, NRW, Germany) and diethylaminoethyl (DEAE) columns (GE Healthcare,

136

Munich,

137

N-deglycosylated by Endo H. The N-deglycosylated and untreated enzymes were

138

subjected to 10% SDS-PAGE analysis and stained with Coomassie brilliant blue

139

R-250 (CBR-250)38. Total protein concentration was determined by a Bio-Rad assay

140

with bovine serine albumin as a standard (Hercules, CA, USA).

141

Phytase activity assay

142

The phytase activity was determined by the amount of released inorganic phosphate

143

using the ferrous sulfate-molybdenum blue method30. The enzyme solution was

144

diluted in 0.25 M sodium acetate at optimum pH (pH 4.0  5.0). The appropriately

145

diluted enzyme at a concentration of 0  1 mg/mL was mixed with 1.5 mM sodium

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phytate as the substrate at a 1:19 proportion and then incubated at 37°C for 30 min.

147

The reaction was stopped by adding an equal volume of 10% (w/v) trichloroacetic

148

acid. The color was developed with a double volume of color reagent [1% (w/v)

149

ammonium molybdate, 3.2% (v/v) sulfuric acid and 7.2% (w/v) ferrous sulfate]. The

150

absorbance at 700 nm was determined to calculate the enzyme activity. One unit (U)

151

of activity was defined as the amount of enzyme that liberates 1 μmol of phosphate

152

per minute under assay conditions.

153

Determination of pH characteristics

154

The optimal pH for phytase activities was determined over a pH range from 1.0–7.0 at

155

37°C for 30 min. After the enzymes were pre-incubated at pH 1.0–11.0 for 1 h at

Bavaria,

Germany)37.

The

purified

glycoenzyme

YkAPPA

was

7



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37°C without substrate, the pH stability was evaluated by measuring their residual

157

activities. The biochemical properties of the wild-type and mutant phytases were

158

assayed in the following 250 mM buffer systems: (1) glycine-HCl at pH 1.0–3.5, (2)

159

sodium acetate-acetic acid at pH 3.5–6.0, (3) Bis-Tris-HCl at pH 6.0 to 7.3, (4)

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Tris-HCl at pH 7.0–8.5 and (5) glycine-NaOH at pH 8.5–12.0.

161

Determination of thermal characteristics

162

The optimal temperature was determined at each optimal pH by performing the

163

enzyme activity assays over the temperature range from 30–90°C for 30 min. The

164

thermostability was assessed by measuring the residual enzyme activities and the

165

half-life of enzyme inactivation after preincubation at 60°C and optimal pH without

166

substrate for the desired times. The untreated enzyme was considered the control

167

(100%).

168

Pepsin and trypsin resistance assays

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Protease resistance was assessed by incubating the wild-type and variant phytases

170

YkAPPA (2655 U/mg, 0.1 U/mL) and YeAPPA (3.2 U/mg, 0.1 U/ml) with either

171

pepsin (2827 U/mg) in 0.25 M glycine-HCl (pH 2.0) or trypsin (2839 U/mg) in 0.25

172

M Tris-HCl (pH 7.0) at 37°C at various protease/phytase ratios ranging from 3 to 141

173

U/mg. After treatment with pepsin and trypsin for 2 h, the enzyme solutions were

174

aliquoted for the residual activity assay. The time course of proteolysis with pepsin

175

was determined at a ratio of 28 U/mg and 37°C for different durations ranging from 0

176

to 30 min. The half-life of enzyme proteolysis is the time needed to keep half of the

177

phytase after pepsin/trypsin treatment at a ratio of 28 U/mg and 37°C. The proteolysis

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mixtures with pepsin were also subjected to SDS-PAGE, stained with Coomassie

179

Brilliant Blue and quantified by densitometric scanning using the ImageJ software

180

(http://rsbweb.nih.gov/ij/) after denaturation by boiling in SDS-β-mercaptoethanol.

181

One unit (U) of pepsin was defined as a 0.001 change in absorbance at 280 nm/min at

182

37°C and pH 2.0 with hemoglobin as the substrate. One unit (U) of trypsin was

183

defined as a 0.001 change in absorbance at 253 nm/min at 37°C and pH 7.0 with

184

Na-Benzoyl-L-arginine ethyl ester (BAEE) as the substrate. The phytase activity was

185

determined as described above.

186

Phytase kinetic measurements

187

Kinetic parameters for wild-type and variant phytases were measured using

188

0.06251.5 mM sodium phytate as the substrate at optimal pH and 37°C for 10 min.

189

The phytase Km, Vmax and kcat values were determined by non-linear regression. The

190

catalytic efficiency (kcat/Km) of the phytases towards sodium phytate was determined

191

using the Matsui equation as previously described39.

192

Enzymatic hydrolysis of corn meal

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The corn meal was pretreated under simulated gastric conditions of pH 1.5 to 5.5 at

194

37°C for 30 min40. The wild-type and mutant phytases, each at 1.0 U/g, were

195

incubated in a 10% (w/v) corn meal solution at 37°C for 1 h with or without pepsin at

196

a pepsin/phytase ratio of 28 U/mg. The released inorganic phosphorus was determined

197

as described previously41.

198 199

Results and discussion

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Designing the mutants

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The YeAPPA phytase from Y. enterocolitica and YkAPPA phytase from Y. kristensenii

202

have a high amino acid sequence identity of 88.7%. Homology-modeled structures of

203

Yersinia phytases showed two theoretical pepsin cleavage sites on the protein surface:

204

F89 (numbering from the initiator M of premature proteins) was conserved in both

205

phytases and E226 in YkAPPA, corresponding to K226 in YeAPPA (Fig. 1B). F89 in

206

YeAPPA and YkAPPA, E226 in YkAPPA and K226 in YeAPPA were selected for

207

mutagenesis at different locations and predicted solvent accessibility scores (3 and 4,

208

respectively, Fig. 1A). Proteolytic resistance can be improved by disturbing the

209

interaction of substrate residues with protease. Decreasing the hydrophobicity of the

210

substrate residue could reduce its interaction with the pepsin hydrophobic cavity and

211

increase the enzyme stability42. Optimizing the surface charge-charge interaction

212

improved the stability of an enzyme42,43. The substitutions F89S and E/K226/H could

213

decrease the hydrophobicity and change the surface charge polarity and distribution,

214

respectively, which were hypothesized to play a role in the enzyme stability. Thus,

215

substitutions of F89 and E/K226 with S and H, respectively, were constructed.

216

Production and purification of wild-type and variant phytases

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The wild-type and variant phytases without the signal peptide-coding sequences were

218

heterogeneously expressed in P. pastoris. YeAPPA and its F89S, K226H and

219

F89S/K226H variants showed a molecular mass of ~46 kDa, which is similar to the

220

theoretical value of 45.9 kDa (Fig. 2A, lanes 912), while YkAPPA and its variants

221

showed a single band of ~52 kDa (Fig. 2A, lanes 14). After treatment with endo H, 10


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YkAPPA and its F89S, E226H and F89S/E226H variants showed a single band with

223

an expected theoretical mass of ~46 kDa (Fig. 2A, lanes 58). These results indicated

224

that the same levels of N-glycosylation occurred in YkAPPA and its variants after its

225

production in P. Pastoris. In native-PAGE, the wild type and F89S, E/K226H,

226

F89S/E226H and F89S/K226H variants of YkAPPA (Fig. 2B line 1-4) and YeAPPA

227

(Fig. 2B line 5-8) in the active forms exhibited a single band with an apparent mass of

228

~52 and ~46 kDa, respectively, similar to that in its denatured form by SDS-PAGE

229

(Fig. 2B line 1-4 and 9-12) . The results indicated that YkAPPA, YeAPPA and their

230

variants are monomeric proteins.

231

The pH profiles of wild-type and variant phytases

232

Effect of pH on the enzymatic properties of the wild-type and variant phytases was

233

determined by using phytate as the substrate (Table 1). All the tested phytases are

234

active at acidic pH and showed maximum activities at pH 4.05.0. After incubation

235

for 1 h, the YkAPPA-E226H and YkAPPA-F89S/E226H substitution mutants

236

displayed increased acid tolerance, retaining ≥91.3% activity at pH 1.0  10.0, while

237

the activity of YkAPPA and YkAPPA-F89S dropped to approximately 64% of the

238

initial value at pH 1.0. The results indicated that substitution of E226 with H plays a

239

role in terms of improving the pH stability. This conclusion was further verified by

240

YeAPPA and its YeAPPA-K226H and YeAPPA-F89S/K226H variants, which retained

241

more activity, with a residual value of ≥77.3%, at pH 2.09.0 versus ≤12.3% activity

242

compared with YeAPPA and YeAPPA-F89S at pH 2.0. In contrast, A. ficuum NTG-33

243

phytase had an optimal activity at pH 1.3 but lost almost all activity at pH 5.043. The

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YeAPPA and YkAPPA variants were more acid-stable, with higher activities ≥88.7%

245

at pH 3.06.0, versus commercial Peniophora lycii-derived phytase, which has YkAPPA-E226H (43.9%) > YkAPPA-F89S (34.3%)

253

> YeAPAP-F89S/K226H (27.3%) > YeAPPA-K226H (21.6%) > YeAPPA-F89S

254

(14.4%) > YeAPPA (0.5%) after 30 min of incubation at 60°C (Table 1; Fig. 3A).

255

When incubated at 50°C for various periods up to 30 min, YeAPPA-F89S,

256

YeAPPA-K226H and YeAPPA-F89S/K226H lost up to 79.3, 72.1 and 67.9% activity,

257

respectively, but YeAPPA lost all activity (Fig 3B). The thermostability of YkAPPA

258

and

259

YkAPPA-E226H (35.2%) > YkAPPA-F89S (25.8%) > YkAPPA (4.3%) after 30 min

260

of incubation at 70°C (Fig 3B). The results indicated that F89S and E/K226H in

261

Yersinia phytases play a major role in enzyme thermostability and the combination of

262

the two single mutations had an additive effect on the improvement in enzyme

263

performance.

their

variants

followed

the

order: YkAPPA-F89S/E226H

(48.3%)

>

264

Further half-life analysis of YkAPPA, YeAPPA and their variants confirmed a

265

functional role against thermal inactivation for the residues at positions 89 and 226.

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266

The

single

variants

YeAPPA-F89S,

YeAPPA-K226H,

YkAPPA-F89S

and

267

YkAPPA-E226H displayed greater thermostability, showing longer half-life of

268

inactivation at 60°C, 3.3, 4.9, 19.7 and 26.6 min, respectively, versus 1.1 and 7.3 min

269

for YeAPPA and YkAPPA (Table 1; Fig 3A). The thermostability was higher for

270

double variants YkAPPA-F89S/E226H and YeAPPA-F89S/K226H than for their

271

single variants. YkAPPA-F89S/E226H and YeAPPA-F89S/K226H showed a longer

272

half-life time than the single variants, respectively, with an increased half-life value

273

up to 4.4 and 9.2-fold (Table 1; Fig 3A).

274

Protein engineering by single or multiple mutations has been utilized to construct

275

various thermostable enzymes21,22,45. Removing the bulky and hydrophobic side chain

276

of F89S may increase the thermostability by decreasing residual size and

277

hydrophobicity42. Increasing the positive charge of E226H and enlarging the

278

positively-charged surface of K226H may improve the thermostability by optimizing

279

the surface charge-charge interaction. The high thermostability of phytases enables

280

the enzyme to withstand the transient high temperatures used in the feed-pelleting

281

process and facilitate the development of new enzymes of industrial importance.

282

Pepsin and trypsin resistance of wild-type and variant phytases

283

Various proteases are commonly produced in the stomach, the functional site of food

284

and feed phytases46. Wild types and YkAPPA and YeAPPA variant pepsin resistance

285

were determined over a broad range of pepsin/phytase (U/mg) ratios at 37°C for 2 h

286

(Fig. 4A  B). After 2 h incubation with pepsin at pH 2.0, the F89S, E226H and

287

F89S/E226H YkAPPA variants displayed improved pepsin resistance, retaining

288

greater residual activity than the wild-type enzyme at pepsin/phytase ratios ranging 13



Page 14 of 34

289

from 3 to 141 U/mg (Fig. 4A). Moreover, the YkAPPA-F89G/E226H double variant

290

retained higher activity (≥57.4% versus ≥21.4% activity of YkAPPA-F89S and

291

≥47.8% activity of YkAPPA-E226H) at all the tested ratios after 2 h of pepsin

292

treatment (Fig. 4A). These results indicated that the substitutions of S and H at the

293

sites F89 and E226, respectively, could confer pepsin resistance and they had an

294

additive effect on improving pepsin resistance. This conclusion was further verified

295

by the F89S, K226H and F89S/K226H YeAPPA variants, which had the lowest to

296

highest residual activities after pepsin treatment at ratios ranging from 3 to 141 U/mg

297

(Fig. 4B). Compared with the pepsin-sensitive YeAPPA, all the YeAPPA variants had

298

increased resistance to pepsin, with residual activities increased up to 228- to 588-fold

299

at a ratio of 71 U/mg (Fig. 4B). Thus, the pepsin resistance of Yersinia phytases could

300

be regulated by the residues at positions 89 and 226.

301

After incubation with trypsin in 0.25 M Tris-Cl (pH 7.0) at 37°C for 2 h, the

302

activity of YkAPPA and its variants remained unchanged at trypsin/phytase ratios of 3

303

to 141 U/mg (Fig. 4A). The activity of YeAPPA-K226H and YeAPPA-F89S/K226H

304

after 2 h of trypsin treatment decreased to 88.7  90.6% of the initial value at ratios

305

ranging from 71 to 141 U/mg, while trypsin-treated YeAPPA and YeAPPA-F89S lost

306

40-46% activity at the ratio of 141 U/mg (Fig. 4B). These results indicated that the

307

K226H substitution may account for the resistance of YeAPPA to pepsin and trypsin.

308

Pepsin resistance was also monitored on a time-course basis for all the tested

309

phytases at a pepsin/phytase ratio of 28 U/mg. All the phytase activities decreased

310

time-dependently after pepsin treatment. The YkAPPA-F89S, YkAPPA-E226H,

311

YeAPPA-F89S and YeAPPA-K226H single substitution variants lost activity over

312

time, but at a lower rate than the wild-type enzymes (Fig. 4C and 4D). The

313

YkAPPA-F89S/E226H and YeAPPA-F89P/K226H combination variants showed

14


Page 15 of 34


314

unchanged activity or lost the least activity among all the tested enzymes (Fig. 4C and

315

4D). The results confirmed the role and additive effects of the F89S and E/K226H

316

substitutions in pepsin resistance.

317

SDS analysis further revealed a higher resistance against pepsin degradation for the

318

variant phytases than for the wild-type enzymes (Fig. 4E and 4F). Pepsin treatment

319

resulted in decreased degradation of the YkAPPA-F89S, YkAPPA-E226H,

320

YeAPPA-F89S and YeAPPA-K226H single variants than the wild-type enzymes at a

321

pepsin/phytase ratio of 141 U/mg for 2 h and over time at a pepsin/phytase ratio of 28

322

U/mg. The pepsin-treated YkAPPA-F89S/E226H and YeAPAP-F89S/K226H double

323

variants retained more protein than the corresponding single variants after incubation

324

with pepsin (Fig. 4E and 4F).

325

The proteolytic half-lives against pepsin and trypsin digestion confirmed the

326

functional role of the F89S and K/E226H residues in the pepsin and trypsin resistance

327

(Table 1). After treatment with pepsin at the ratio of 28 U/mg at 37°C and pH 2.0, the

328

single mutants YeAPPA-F89S, YeAPPA-K226H, YkAPPA-F89S and YkAPPA-E226H

329

revealed greater tolerance to pepsin digestion, with the proteolytic half-life time

330

increased 4.8, 22.6, 3.3 and 1.9-fold, respectively, versus 1.7 and 31.2 min for

331

YeAPPA and YkAPPA (Table 1). A combination of F89S and E/K226H had an

332

additive effect on the increase of pepsin digestion half-life. Proteolytic half-life

333

against pepsin digestion was higher for F89S/K226H and F89S/E226H than for its

334

single mutants (Table 1). When treated with trypsin at the ratio of 28 U/mg at 37°C

335

and pH 7.0, YkAPPA and its variants were highly stable with the proteolytic half-life

336

value of about 24 h (Table 1). Although YeAPPA and YeAPPA-F89S were sensitive to

15



Page 16 of 34

337

trypsin at the ratio of 28 U/mg, losing activity rapidly with the proteolytic half-life of

338

125 min, YeAPPA-K226H and YeAPPA-F89S/K226H improved the trypsin resistance,

339

with their half-lives of trypsin degradation increased up to 4.3-fold (Table 1).

340

In the present study, the thermostable F89S and E/K226H variants conferred pepsin

341

resistance to YkAPPA and YeAPPA and the thermostable E226H and F89S/E226H

342

variants conferred pepsin and trypsin resistance to YeAPPA. The F89S variants could

343

remove the hydrophobic interactions between the bulky phenyl F group and pepsin or

344

promote a space between the enzyme and pepsin, thereby decreasing the affinity of

345

pepsin for the enzyme and producing resistance47  49. The E/K226H variants likely

346

confer pepsin and trypsin resistance by reducing the number of hydrophobic

347

methylene groups in E/K226H, which increases the positive surface charge of E226H

348

and enlarges the positively charged-surface of the K226H imidazole ring. The

349

protease resistance of a phytase can increase its functioning time when high protease

350

activity is present and benefit animal production by promoting lower costs.

351

Kinetic parameters

352

High-efficiency removal of phytate would alleviate the negative effects caused by

353

phytates in animal production. The kinetic parameters of the wild-type and variant

354

phytases were assayed using phytate as a substrate at concentrations ranging from

355

0.0625 to 1.5 mM (Table 2). Residue substitution at the protease cleavage sites had

356

little or no effect on substrate affinity. The F89S and E/K226H single mutations in

357

YkAPPA and YeAPPA increased the catalytic efficiency, reaction velocity and

358

turnover rate by less than 1.1-fold. The F89S and E/K226H combination mutations

359

caused the most significant effect of all the mutations on the reaction velocity and

16


Page 17 of 34


360

turnover rate (up to 1.7-fold change). As a result, the catalytic efficiencies of

361

YkAPPA-F89S/E226H and YeAPPA-F89S/K226H were improved by 1.2- and

362

1.8-fold, respectively.

363

Enzymatic hydrolysis of corn meal

364

The in vitro hydrolysis of phytate in corn meal by Yersinia phytases was performed

365

under simulated gastric conditions of 37°C, pH 1.5  5.5 and high pepsin activity to

366

analyze the efficiency of the phytases to hydrolyze phytate into phosphorus in the

367

digestive tracts of monogastric animals. After incubation at pH 1.55.5 and 37°C for 2

368

h, the F89S, E226H and F89S/E226H YkAPPA and YeAPPA variants released more

369

inorganic phosphorus than the wild-type enzymes, with an increased maximum up to

370

1.31.7-fold at pH 4.04.5 and 2.02.8-fold at pH 4.55.0, respectively, versus 1640

371

μg and 19.6 μg of inorganic phosphorus for YkAPPA at pH 4.5 and YeAPPA at pH 5.0,

372

respectively (Fig. 5A and 5B). The addition of pepsin at a pepsin/phytase ratio of 28

373

U/mg decreased the hydrolysis efficacy of the YeAPPA and YkAPPA variants at a

374

lower rate than the wild-type enzyme, which increased the maximal values of released

375

inorganic phosphorus by 2.64.2- and 9.114.9-fold, respectively (Fig. 5A and 5B).

376

The increased hydrolysis of corn meal phytate by Yersinia phytase improves its

377

cost-effectiveness application in animal production.

378 379

ASSOCIATED CONTENT

380

Supporting Information

381

Table S1 Primers for YkAPPA and YeAPAP and their mutants. (PDF)

17



Page 18 of 34

382

Funding

383

This work was supported by the National Science Fund for Distinguished Young

384

Scholars of China (31225026), the National Natural Science Foundation of China

385

(31402110) and the Special Fund for Agro-Scientific Research in the Public Health of

386

China (201403047).

387

Notes

388

The authors declare no competing financial interest.

18


Page 19 of 34


389

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390

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Page 27 of 34


544

Figure legends

545

FIG 1. Sequence alignment of predicted pepsin and trypsin cleavage sites (A) and

546

their location in the modeled structures of YeAPPA (pink) and YkAPPA (blue) using E.

547

coli phytase (1DKP) as the template (B). For panel (A), Identical residues are shaded

548

in black. The solvent accessibilities of the mutated sites F89 and E/K226 were

549

indicated. YeAPPA from Yersinia enterocolitica (GU936684) and YkAPPA from Y.

550

kristensenii (EU203664). For panel (B), conserved residues in two phytases are shown

551

in black.

552 553

FIG 2. SDS-PAGE (A) and native-PAGE (B) analysis of the wild-type and variant

554

phytases produced in P. pastoris. Lanes: M, the protein markers; 1 to 8 in A, wild-type

555

(WT) YkAPPA and its variants F89S, E226H, F89S/E226H with (+) or without

556

N-deglycosylation with Endo H treatment, respectively; 9 to 12 in A, YeAPPA and its

557

variants F89S, K226H and F89S/K226H in SDS-PAGE; 1 to 4 and 5 to 8 in B,

558

YkAPPA, YeAPPA and their variants F89S, E/K226H, F89S/E226H and F89S/K226H

559

in native-PAGE.

27



Page 28 of 34

560 561

FIG 3. Thermostability of YkAPPA, YeAPPA and their variants. (A) Thermostability

562

of YkAPPA, YeAPPA and their variants at 60C. (B) Thermostability of YeAPPA and

563

its variants at 50C and YkAPPA and its variants at 70C.

564 565

FIG 4. Proteolytic resistance of YkAPPA, YeAPPA and their variants. (A and B)

566

Evaluation of resistance to pepsin at pH 2.0 and trypsin at pH 7.0 for 2 h at 37C and

567

various protease/phytaseratios of 3 to 141 U/mg. (C and D) Time course of pepsin

568

resistance over 30 min at a pepsin/phytase ratio of 28 U/mg. For A to D, the residual

569

phytase activity was determined using 1.5 mM sodium phytate as the substrate at

570

37  C for 30 min and indicated as a percentage of activity of untreated enzymes. (E

571

and F), SDS-PAGE analysis of the proteolytic products of YkAPPA and YeAPAP

28


Page 29 of 34


572

variants degraded by pepsin at different ratios and durations. The phytase band

573

intensity was assessed by using ImageJ software.

574 575

FIG 5. The hydrolysis ability of wild types and variants of YkAPPA (A) and YeAPPA

576

(B). The hydrolysis of corn meal phytate by each phytase was measured after

577

incubation for 2 h at 37  C and pH 1.5 to 5.5 with or without pepsin at a

578

pepsin/phytase ratio of 28 U/mg. The inorganic phosphorus released from corn meal

579

phytate was quantified to calculate the hydrolysis efficacy of the phytases.

29



Page 30 of 34

580

581

30


Page 31 of 34


Table 1 The pH and temperature optima and stability parameters of wild type and mutant phytasesa

a

The phytase activity towards sodium phytate (1.5 mM) at 37C

for 30 min was

regarded as 100%. The residual activity (%) was indicated as percentage of activity of untreated enzyme.

31



Page 32 of 34

Table 2 Kinetic analysis of wild type and variant phytases a

a

Enzyme kinetic parameters were assayed using 0.625-1.5 mM sodium phytate as

substrate at each optimal pH and 37  C for 10 min and determined by non-linear regression. Data were presented as means from triplicates.

32


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TOC graphic

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Engineering of Yersinia Phytases to Improve Pepsin and Trypsin

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