Characterization of thermostable and chimeric enzymes via isopeptide

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Biotechnology and Biological Transformations

Characterization of thermostable and chimeric enzymes via isopeptide bond-mediated molecular cyclization De-Ying Gao, Xiao-Bao Sun, Ming-Qi Liu, Yan-Ni Liu, Hui-En Zhang, XinLei Shi, Yang-Nan Li, Jia-kun Wang, Shang-Jun Yin, and Qian Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01459 • Publication Date (Web): 04 Jun 2019 Downloaded from http://pubs.acs.org on June 9, 2019

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Journal of Agricultural and Food Chemistry

1

Characterization of thermostable and chimeric enzymes via

2

isopeptide bond-mediated molecular cyclization

3 4

De-Ying Gao†, Xiao-Bao Sun†, Ming-Qi Liu‡, Yan-Ni Liu†, Hui-En Zhang†, Xin-Lei

5

Shi†, Yang-Nan Li†, Jia-Kun Wang§, Shang-Jun Yin†, *, Qian Wang†, *

6 7 8 9

†

College of Biological and Environmental Sciences, Zhejiang Wanli University,

10

Ningbo 315100, Zhejiang, China

11

‡

12

Instrumentation for Marine Food, College of Life Science, China Jiliang University,

13

Hangzhou 310018, Zhejiang, China

14

§ College

National and Local United Engineering Lab of Quality Controlling Technology and

of Animal Science, Zhejiang University, Hangzhou 310058, Zhejiang, China

15

ACS Paragon Plus Environment


16

ABSTRACT

17

Mannooligosaccharides are released by mannan-degrading endo-β-1,4-mannanase and

18

are known as functional additives in human and animal diets. To satisfy demands for

19

biocatalysis and bioprocessing in crowed environments, in this study, we employed a

20

recently developed enzyme-engineering system, isopeptide bond-mediated molecular

21

cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results

22

revealed that the cyclized enzymes showed enhanced thermostability and ion stability,

23

resilience to aggregation and freeze-thaw treatment by maintaining their

24

conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we

25

generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic

26

activity in substrate deconstruction without compromising substrate affinity.

27

Interestingly, the dual-enzyme ring conformation was observed to be more robust than

28

the linear enzyme but inferior to the single-enzyme ring conformation. Taken

29

together, these findings provided new insights into the mechanisms of molecular

30

cyclization on stability improvement and will be of useful in the production of new

31

functional oligosaccharides and feed additives.

32 33

KEYWORDS ： mannanase, isopeptide bond-mediated ligation, molecular

34

cyclization, thermostability, chimeric enzyme, synergy

35


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INTRODUCTION

37

Hemicelluloses, mainly composed of mannan and xylan, are the second most

38

abundant renewable lignocellulosic biomass in nature. Complete deconstruction of

39

mannan

40

endo-β-1,4-mannanase

41

α-L-arabinosidase (EC 3.2.1.55), α-galactosidase (EC 3.2.1.22), and acetylmannan

42

esterase (EC 3.1.1.6).1 Among them, endo-β-1,4-mannanase is the key enzyme that

43

randomly catalyzes the cleavage of β-1,4- glycosidic bonds of the mannan backbone

44

to release mannooligosaccharides and facilitate initial degradation.2 Mannanases are

45

classified into the glycoside hydrolase (GH) families 5, 26, and 113 in the CAZy

46

database (http://www.cazy.org/) and mainly follow a preserving catalytic mechanism.3

47

Feed enzymes, including mannanase and xylanase, are known to degrade

48

non-starch polysaccharides (NSPs) in plant-derived feed diets. Oligosaccharides

49

produced by feed enzymes were believed to promote beneficial bacteria, such as

50

Bifidobacterium and Lactobacillus species, and inhibit harmful bacteria in the colon

51

by oligosaccharides released from NSPs.4,5 The studies on mannose- and

52

mannooligosaccharide-producing enzymes and microorganisms started in the late

53

1970s. Preliminary statistics have found more than 100 species of enzyme-producing

54

microorganisms, including bacteria (e.g., Bacillus subtilis and Pseudomonas

55

aeruginosa), fungi (e.g., Aspergillus spp.) and Actinomycetes (e.g., Streptomyces

56

spp.).6 However, poor tolerance of native mannanases to heat and inhibitors leads to

57

inferior stability and thus inefficient catalysis, limiting their applications in industrial

requires

the

activities (EC

of

a

3.2.1.78),

number

of

β-mannosidase


enzymes, (EC

such

as

3.2.1.25),


58

processing.

59

Protein engineering approaches, such as site-directed mutagenesis and directed

60

evolution, have been used to improve enzyme thermostability and/or pH stability.7

61

However, these strategies could not be adopted directly, when the target enzyme lacks

62

a well-characterized catalytic mechanism or an efficient activity assay method for

63

high-throughput screening. The correlation between thermostability and structural

64

rigidity was previously discussed in depth,8 while the activity was generally regarded

65

as closely related to flexibility.9 Therefore, the delicate balance between rigidity and

66

flexibility is crucial for protein design.10 Importantly, due to the limitation of

67

condition-based screening methods, mutants obtained by enzyme evolution might

68

show improved stability (rigidity) but depressed the activity (flexibility). Compared

69

with proteins in a linear conformation, cyclic proteins exhibit higher thermostability

70

and structural stability and are widely observed in bacteria, plants, fungi and

71

animals.11 Protein cytoskeleton cyclization is a process in which the C- and N-termini

72

of linear peptides are combined via amide bonds to form cyclic molecules. This cyclic

73

structure enables the target enzyme to be more stable than the native enzyme in

74

adverse environments, such as those at high temperature or having strong ions or

75

denaturing agents.12 Protein trans-splicing, ligation of two proteins with a peptide, and

76

sortagging mediated by transpeptidase are commonly utilized strategies for molecular

77

cyclization. However, these techniques suffer from several limitations, including

78

incontrollable splicing, the requirement for specific termini for crosslinking, and low

79

efficiency. It was reported that pilins from some Gram-positive bacteria such as


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Streptococcus pyogenes contain spontaneous isopeptide bond.13 Subsequently, a

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molecular cyclization system “superglue” was developed from Spy0128 and other

82

pilins.14-17 The cyclization reaction was achieved via the spontaneously formed

83

isopeptide bond between Lys and Asn/Asp. Importantly, the system is versatile and

84

efficient, and industrial enzymes such as phytase18 and xylanase19 were engineered to

85

improve thermostability.

86

Although the effects and mechanisms of single-enzyme cyclization on

87

thermostability have been addressed, resilience to crowded environments needs to be

88

elucidated. In this study, β-mannanase from B. subtilis was engineered as a cyclized

89

enzyme and a mannanase-xylanase dual-enzyme ring conformation was developed.

90

We investigated the stability and activities of the linear and the cyclized enzymes in

91

adverse conditions, such as alkaline pH, high temperature, and freeze-thaw treatment.

92

Additionally, the thermostability and catalytic efficiency of the bifunctional enzymes

93

were evaluated.

94

MATERIALS AND METHODS

95

Materials. Plasmids pET-30a(+) (Novagen, Madison, WI, USA) and pETTC19

96

were used as the heterologous expression vectors in Escherichia coli BL21 (DE3).

97

Locust bean gum and beechwood xylan were purchased from Sigma (St. Louis, MO,

98

USA). Restriction endonucleases and T4 ligase were obtained from Promega

99

(Madison, WI, USA). Ni-NTA 6× His-tag agarose was purchased from Qiagen

100

(Shanghai, China). Pfu polymerase was purchased from TransGen Biotech (Beijing,

101

China). The oligonucleotides used in this study were synthesized by Sangon



102

(Shanghai, China) (Table 1). The synthetic fragment SnoopTag/SnoopCatcher (PDB:

103

2WW8) was designed according to the sequence of GenBank No. AP017971 and

104

synthesized by Genscript (Nanjing, China). Recombinant plasmid pUCm-T/TFX

105

carrying a xylanase gene from Thermobifida fusca20 and pUCm-T/LPMO carrying a

106

lytic polysaccharide monooxygenase (LPMO) gene from B. subtilis were constructed

107

in our previous studies.

108

Plasmid Construction and Protein Expression. To locate the SnoopTag and

109

SnoopCatcher at the N- and C-termini of the target gene, the synthetic fragment

110

SnoopTag/SnoopCatcher was digested with BamHI and NotI and inserted into

111

pET30a(+) to obtain pETSn. The BSM gene (GenBank No.: DQ269473) was

112

amplified from the B. subtilis genome, and the PCR fragments were inserted into

113

pET30a(+), pETTC and pETSn; the resulting plasmids were designated

114

pET30a(+)/BSM, pETTC/BSM and pETSn/BSM, respectively. To locate the SpyTag

115

at both the N- and C-termini of the target gene, the SpyTag fragment was amplified

116

and inserted into pETTC between the HindIII and NotI sites to replace the

117

SpyCatcher peptide downstream of the multiple cloning sites (MCS). To locate the

118

SpyCatcher at both the N- and C-termini of the target gene, the SpyCatcher fragment

119

was amplified and inserted into pETTC between the BglII and EcoRV sites to

120

replace the SpyTag peptide upstream of MCS; the resulting plasmids were

121

designated pETTT and pETCC, respectively. BSM/TFX were introduced into

122

pETCC/pETTT to obtain pETCC/BSM (CC/BSM) and pETTT/TFX (TT/TFX),

123

respectively. All recombinant plasmids were confirmed by sequencing (Sangon) and


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transformed into E. coli BL21 (DE3) competent cells by heat shock. All recombinant

125

E.

126

isopropyl-β-D-thiogalactopyranoside (IPTG)-induced, and sonicated as previously

127

described.19

coli

stains

harboring

linear

or

cyclized

enzymes

were

cultured,

128

Protein Purification and In Vitro Assembly. Supernatants collected after

129

centrifugation at 15,294×g for 15 min at 4°C were each subjected to affinity

130

purification using a HisTrapTM FF column (GE Healthcare Bio-Sciences, Pittsburgh,

131

PA, USA) as previously described19 with minor modifications. The imidazole gradient

132

in the elution buffer ranged from 20 mM to 1 M. Recombinant bovine enterokinase

133

(EK) (BBI, Shanghai, China) was used to relinearize TC/BSM and Sn/BSM (1 U per

134

50 μg protein). Reactions were performed in a digestion buffer (25 mM Tris-HCl, pH

135

7.6, 50 mM NaCl, and 2 mM CaCl2) at 25°C for 12 h. The mannanase-xylanase

136

chimera was achieved by mixing the purified CC/BSM (~1 μmol), TT/TFX (~1 μmol)

137

and TT/LPMO (~1 μmol) followed by assembly reaction was performed at 16°C with

138

gentle rotation at 100 rpm for 10 min.

139

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

140

and Zymogram Analyses. All native, cyclized, relinearized, and chimeric enzymes

141

were analyzed with SDS-PAGE (12% running gel and 4% stacking gel).21 For

142

SDS-PAGE, the gels were stained with Coomassie Brilliant Blue G250 and destained

143

with 15% methanol and 5% acetic acid. For zymogram analysis, all samples were run

144

on gels containing locust bean gum (0.5%) or beechwood xylan (0.5%). After

145

electrophoresis, the gels were soaked in 25% isopropanol twice for 20 min each and



146

washed extensively in 1× phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM

147

KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) at 4°C overnight. The gels were

148

stained with 1% Congo red for 20 min and further destained with 1 M NaCl until

149

transparent bands appeared.

150

Activities and Protein Concentration Assays. The catalytic activities of

151

mannanase and xylanases were measured using the 3,5-dinitrosalicylic acid (DNS)

152

method.22 Briefly, 15 μL of enzyme solution was incubated with 60 μL of 0.5% locust

153

bean gum or beechwood xylan at various temperatures for 8 min, followed by the

154

addition of 75 μL of DNS and boiling for 5 min. After cooling to room temperature,

155

the absorbance was determined spectrophotometrically at 540 nm. One unit of

156

catalytic activity (U) was defined as the amount of enzyme that released 1 μmol of

157

reducing sugar equivalent to mannose or xylose per minute. Kinetic parameters were

158

determined using 0.5-12 mg/mL locust bean gum or 2-18 mg/mL beechwood xylan as

159

substrate. Protein concentration was measured using the method described by

160

Bradford (1976) with bovine serum albumin as the standard. Approximately 0.2 μg of

161

purified protein was used in all these assays, and all assays were performed in

162

quadruplicate unless otherwise noted.

163

Effects of pH and Temperature. The optimal pH was determined by assaying

164

the mannanase activity in various pH buffers (citrate/phosphate buffer for pH 2.2-8.0

165

and glycine-NaOH buffer for pH 9.0-10.0) at 50°C for 8 min. The optimal

166

temperature was determined by assaying the mannanase activity at pH 6.0 and

167

30-80°C for 8 min.


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To evaluate the pH stability, all linear and cyclized enzymes were preincubated

169

in various pH buffers (citrate/phosphate buffer for pH 2.2-8.0 and glycine-NaOH

170

buffer for pH 9.0-10.0) at room temperature (25°C) for 1 h, and the residual activities

171

were determined under the optimal conditions (pH 6.0, 50°C). The initial activities

172

before preincubation were regarded as 100%.

173

To assess the thermostability, all linear, cyclized, and relinearized enzymes were

174

preincubated in citrate/phosphate buffer (pH 6.0) at 50-70°C for 1 h. Aliquots were

175

taken at different time intervals (2, 5, 10, 20, 30, and 60 min) and subjected to activity

176

assays under the optimal conditions (pH 6.0, 50°C). The initial activities before

177

preincubation were taken as 100%.

178

Protein Aggregation. All linear, cyclized, and relinearized enzymes (~25 μg

179

each) were preincubated at various temperatures (25, 37, 50, 60, 70, 80, 90 and

180

100°C) for 15 min. The supernatants were collected after centrifugation at 15,294×g

181

and 4°C for 15 min and then subjected to SDS-PAGE, followed by optical density

182

analysis using the Image Lab v.5.2.1 software (Bio-Rad, Hercules, CA, USA). Data

183

for native enzymes were set as 100%. Further, residual enzymatic activity in the

184

supernatant was assayed under the optimal conditions (pH 6.0, 50°C). Protein

185

aggregation were monitored by turbidity measurement at 400 nm using a Shimadzu

186

UV-1800 (Shimadzu, Kyoto, Japan) spectrophotometer at 25°C.

187

Circular dichroism (CD). All linear and cyclized enzymes were prepared by

188

dialysis in 2 mM HEPES buffer to a final concentration of 0.075 mg/mL. Enzymes

189

were preincubated at 50°C, 70°C, and 90°C for 10 min and subjected to CD analysis



190

using a MOS-500 spectropolarimeter (BioLogic Science Instruments, Claix, France)

191

and a 0.1-cm path-length cuvette at wavelengths ranging from 190 nm to 250 nm. The

192

percentage of α helix, β strand, and random coil were calculated using CDPro

193

(http://sites.bmb.colostate.edu/sreeram/CDPro/).

194

Intrinsic fluorescence measurement. The intrinsic fluorescence spectra of all

195

linear and cyclized enzymes were measured with an F-2500 spectrofluorometer

196

(Hitachi, Japan) and a 1-cm path-length cuvette. The enzymes were pretreated by

197

heating at 50℃, 60℃ or 80℃ for 1 h. Subsequently, fluorescence emission spectra

198

were determined with excitation at 280 nm and the emission in the range of 300-500

199

nm. Native enzymes were used as controls. All spectra were collected at 25 ℃ in

200

1×PBS buffer (pH 7.4).

201

Freeze-thaw stability. All linear and cyclized enzymes (~10 μg each) were snap

202

frozen in liquid nitrogen for 1 min and then warmed to 37°C for 3 min. A total of 20

203

cycles of freeze-thaw treatment were conducted, and aliquots were taken at the end of

204

the 5th, 10th, 15th, and 20th cycles and subjected to residual enzymatic activity assay as

205

described above. The initial activity before freeze-thaw treatment was regarded as

206

100%. Protein aggregation was monitored by turbidity measurement as above.

207

Effects of metal ions on stability. All linear and cyclized enzymes were

208

preincubated with 0.5, 1 or 5 mM various metal ions (Cr2+, Pb2+, Cu2+ and Ca2+) or

209

EDTA for 1 h. Then, the enzymes were subjected to residual enzymatic activity assay

210

as described above. The initial activity before preincubation was considered as 100%.

211

Synergy of chimeric enzyme on substrate degradation. The chimeric enzymes


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were achieved by simply incubating a mixture of equal amount (~0.1 mol) of

213

CC/BSM and TT/TFX or TT/LPMO at 16

214

mannanase-xylanase chimera (MXC) or mannanase-LPMO chimera (MLC). Then,

215

0.5% locust bean gum and beechwood xylan substrates were treated with MXC or

216

MLC for 10 min, followed by spectrometrically determining the released reducing

217

sugars at 540 nm as described above. To determine the synergism on natural

218

lignocellulose, substrate was prepared from corn husk, which was milled to pass

219

through a mesh of 1.40 mm and subjected for 2% NaOH treatment at 121℃ for 30

220

min.23 Then, the milled and pretreated corn husk was rinsed using ddH2O to PH 7.0

221

and then further rinsed using 50% ethanol and dried. Finally, the treated corn husk

222

samples were used to DNS assay to verify complete removal of residual reducing

223

sugars. To conduct natural lignocellulose deconstruction assay, 0.1 g alkaline-treated

224

corn husk was subjected to hydrolysis by MXC or MLC for 12 h, followed by

225

spectrometrically determining the released reducing sugars at 540 nm as described

226

above.

℃

for 10 min to obtain

227

Statistical analysis.

228

Kinetic parameters were calculated by GraphPad prism 7.0 (San Diego, CA)

229

using Michaelis-Menten. Statistical significance was analyzed using a two-tailed

230

Student’s t-test. Multiple comparisons were carried out by IBM SPSS Statistics v.

231

21.0 (IBM, Armonk, NY, USA) using a one-way ANOVA with Tukey’s test.

232

RESULTS AND DISCUSSION

233

Protein Expression and Purification of Recombinant Mannanase. The



234

mannanase gene BSM was directed cloned from the B. subtilis genome (GenBank:

235

DQ269473). According to the CAZy classification, BSM belongs to the GH26 family,

236

and its amino acid sequence shares high similarity with that of the endo-mannanase of

237

other species of the same genus: 99% identity, B. velezensis (GenBank:

238

WP_088056375); 99% identity, B. amyloliquefaciens (GenBank: WP_115996828);

239

75% identity, B. licheniformis (GenBank: WP_075749790.1); and 74% identity, B.

240

sonorensis (GenBank: WP_029418987.1). The mature peptide of BSM was used for

241

molecular cyclization, and both linear and cyclized enzymes were heterologously

242

expressed in E. coli. The molecular weights of 30a/BSM, TC/BSM and Sn/BSM were

243

approximately 45, 60, and 60 kDa (Figure 2), respectively, which were consistent

244

with their theoretical molecular weights (MW) of 44.3, 57.6 and 56.1 kDa. Notably,

245

both cyclized enzymes, especially Sn/BSM, were observed to have multiple bands on

246

SDS-PAGE, probably indicating insufficient cyclization by isopeptide bond-mediated

247

ligation.24 To validate the topology of the cyclized enzymes, both TC/BSM and

248

Sn/BSM were further digested with EK (Figure 2) or subjected to KA mutations

249

(Figure S1).

250

Optimal pH and pH Stability. The enzymes 30a/BSM, TC/BSM and Sn/BSM

251

all shared an optimal pH of 6.0 (Figure S2), which is consistent with the reported

252

optimal pH for most mannanases under neutral or weakly acidic pH conditions. 25-27 In

253

terms of pH stability, 30a/BSM, TC/BSM and Sn/BSM were all stable under neutral

254

and weakly acidic conditions (pH 5.0-7.0). Interestingly, compared with 30a/BSM,

255

TC/BSM showed significantly higher residual activity at pH 4.0, 8.0 and 9.0 (P
50°C (Figure 3B-D). The TC/BSM and

275

Sn/BSM showed half-lives of 88.8±16.1 and 73.1±17.6 min at 60°C, significantly

276

higher than 30a/BSM of 6.91±0.72 min. After pretreatment at 60°C for 1 h, the

277

TC/BSM and Sn/BSM retained 66.59% and 65.88% of their initial activities,

CtManT and 60°C CtManF from Clostridium



278

respectively (Figure 3C and 3D). However, only negligible activities of 30a/BSM or

279

KA mutants were retained (Figure 3B and S3). Interestingly, the relinearized TC/BSM

280

(L-TC/BSM) and Sn/BSM (L-Sn/BSM) were again almost inactive after the

281

pretreatment under the same conditions (Figure 3E and F). It was inferred that

282

cyclized protein conformation, rather the SpyTag or SpyCatcher peptides located at

283

their flanking regions, contributed to the considerably enhanced thermostability,

284

which was in agreement with previous studies

285

50°C for 1 h, the relinearized enzymes, particularly L-Sn/BSM, retained 38.65%

286

residual activity, which was even more depressed than that of 30a/BSM. It is

287

speculative, but the enzyme might have underdone partial degradation during the 12 h

288

digestion at room temperature by EK. Thus, it is of considerable interest to investigate

289

the effects and mechanisms of molecular cyclization on stability against crowded

290

adverse conditions such as the presence of trypsin34 and metal ions.19

18, 19, 33

Notably, after preincubation at

291

The kinetic analysis showed that when using locust bean gum as a substrate, the

292

Vmax and Km of 30a/BSM were 1238±55.54 μmol/min/mg and 3.74±0.40 mg/ (Table

293

2), respectively, indicating that BSM was a typical endo-mannanase with moderate

294

catalytic ability2. Though thermostability was impressively improved, the Km was not

295

altered by molecular cyclization.28 However, the Kcat/km values significantly increased

296

(P < 0.05) by molecular cyclization, corroborating the finding of several previous

297

works.19, 2 4 The reason for this effect needs to be further elucidated.

298

To evaluate the effects of molecular cyclization on heat-induced aggregation, all

299

linear, cyclized and relinearized enzymes were preheated at temperatures ranging


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300

from 25 to 100°C for 15 min and subjected to SDS-PAGE analysis. The enzyme

301

30a/BSM was stable below 50°C, which was in good agreement with the

302

thermostability assay result (Figure 3A). The linear enzyme started to precipitate

303

when the temperature reached 60°C and aggregated severely at higher temperatures

304

(Figure 4A). On the other hand, both TC/BSM and Sn/BSM remained soluble, even

305

when pretreated at 100°C for 15 min (Figure 4B and C). However, the relinearized

306

enzymes (Figure 4D and E) or KA mutants again precipitated, even at 50°C (Figure

307

S4). The cyclized enzymes were still robust, whereas the linear and relinearized

308

enzymes could hardly maintain their activities (Figure 4F and G). To understand the

309

conformational change during heat challenge, CD was employed to analyze the

310

secondary structure of the linear and cyclized enzymes. The native BSM mainly

311

comprised a (β/α)8 barrel, typical of the structure characteristics of GH26 family. Heat

312

treatment greatly altered the secondary structure pattern of 30a/BSM (Figure 5A and

313

D), but not that of TC/BSM or Sn/BSM (Figure 5B and C). After incubation at >

314

70°C, the α-helix (the major composition of the native protein) was disrupted as a

315

random coil. Moreover, a considerable loss of fluorescence intensity and red shift of

316

maximum emission wavelength (Figure 5G) were also observed, suggesting a

317

complete structural breakdown in the linear enzyme. However, the cyclized enzymes

318

were generally capable of maintaining their structure even after incubation at 90°C,

319

which was also illustrated by analyzing solvent exposure of inner hydrophobic

320

residues, particularly tryptophan and tyrosine (Figure 5H and I).19

321

We further performed freeze-thaw cycle treatment with linear and cyclized



322

enzymes. After 5 freeze-thaw cycles, the majority of 30a/BSM aggregated and

323

precipitated out (Figure 6A). The residual activity of 30a/BSM in the supernatant

324

declined significantly (P < 0.05), whereas the activities of both TC/BSM and Sn/BSM

325

were mostly retained (Figure 6B-E). When compared to that of the control that did not

326

undergo freeze/thaw cycles, all the enzymes showed significantly degraded activity as

327

the number of cycles increased. Notably, after 20 freeze-thaw cycles, the cyclized

328

enzymes were still more robust than the linear enzymes, with TC/BSM retaining

329

71.93% of its initial activity, which was 1.73-fold that of Sn/BSM. We also

330

determined the effects of metal ions on the activities of both the linear and the

331

cyclized enzymes. As seen in Figure 7, Cr2+, Pb2+, Cu2+ and Ca2+, as well as EDTA,

332

were found to inhibit mannanase activity. Interestingly, molecular cyclization

333

conferred the linear enzyme with improved ion stability. The cyclized enzymes can be

334

used in pulp bleaching, in which many metal ions, such as Pb2+ Cr2+, and Cu2+, are

335

included as the crowded environment.35, 36

336

NSPs-active enzymes, such as mannanase and xylanase, are known to produce

337

functional oligosaccharides from agricultural lignocellulosic substrates. During many

338

industrial processes, thermal tolerance is required for commercial enzymes to

339

function. Thus, enzymes with good thermostability are desirable in probiotics,37

340

breadmaking38 and the brewing industry.39 During the past decade, genetic

341

engineering approaches, including site mutation, domain shuffling and directed

342

evolution, have been used to improve the thermostability of many enzymes. For

343

example, Wang et al40 mutated a critical amino acid with the lowest mutation energy,


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344

Ala 336 Pro, and obtained Mutant 336, which had an increased dynamic transition

345

temperature and half-life at 60°C but a decreased irreversible thermal denaturation

346

constant. In another study, an optimal mutant with the apparent melting temperature

347

increased by 14°C was generated using in silico design. Importantly, the mutant

348

increased xylooligosaccharide production at 70°C by 10-fold.7 Moreover, the addition

349

of a carbohydrate-binding module (CBM)32 or the deletion41 or replacement42 of key

350

distal residues also improved enzyme thermostability. Collectively, rational,

351

semi-rational and irrational designs are useful tools to modify enzymes. However,

352

these strategies generally require detailed stereostructures, known catalytic

353

mechanisms or high-throughput screening techniques.43 Moreover, stability

354

enhancement by truncating one or more key amino acid residues may not be used

355

extensively due to the diversity of disordered residues in the distal region and/or

356

catalytic mechanism. Recently, molecular cyclization based on isopeptide

357

bond-mediated ligation was established as an effective method for protein

358

engineering.44 For example, the SpyTag/SpyCatcher cyclization not only increased

359

thermostability18, 28, 45 but also contributed to improved catalytic efficiency.19, 24 More

360

importantly, this approach is universal for enzyme engineering and compatible with

361

various enzymes, such as phytase,18 lichenase,24 xylanase,19 and luciferase.28 Enzymes

362

are commonly stored at low temperature (mainly at -20°C or -80°C) and later thawed

363

for biochemical reactions. Such storage of enzymes without a cryoprotectant can

364

decrease their activity rapidly. In this study, the cyclized enzymes were found to be

365

more robust after freeze-thaw treatment. These enzymes can be better suited to



366

applications in both industrial processes and research because they are more stable

367

during prolonged storage.46 Interestingly, Schoene et al18 proposed a better stability

368

improvement using SpyRing cyclization than using SnoopRing. However, no

369

significant difference between SpyRing and SnoopRing was observed in our study

370

(Figure 3-5), though SpyRing cyclization showed slightly higher residual activity after

371

freeze-thaw treatment for 5 to 20 cycles (Figure 6). The reason for this result remains

372

unclear, and further study needs to be performed.

373

Assembly and Characterization of Chimeric Enzymes. To extend the

374

substrate spectrum of BSM, we generated a mannanase-xylanase chimeric enzyme

375

(MXC) as shown in Figure 1. The enzymes TT/TFX and CC/BSM showed clear

376

bands of approximately 32 and 68 kDa, which is consistent with the calculated MWs

377

of 30.3 and 67.5 kDa, respectively (Figure 8A). However, though a major band of

378

approximately 120 kDa could be distinguished, several fainter bands were also

379

observed with the MXC. We speculated that both the MW and conformation of the

380

MXC, rather than MW alone, contributed to the altered electrophoretic velocity in the

381

gel.45 Insufficient and over-cyclization might also lead to the presence of multiple

382

bands.18, 47 Zymogram analysis revealed that CC/BSM and TT/TFX showed specific

383

degradation to locust bean gum and beechwood xylan, respectively, whereas the MXC

384

showed bifunctional activities towards both substrates (Figure 8B and C).

385

To determine whether SpyTag/SypCatcher could stabilize the dual-enzyme ring

386

conformation, we compared the thermostability of CC/BSM, TT/TFX, and MXC to

387

that of single-enzyme cyclization. After incubation at 60°C for 1 h, the MXC retained


Page 18 of 44

Page 19 of 44


388

55.36% of its initial activity against locust bean gum, whereas CC/BSM retained

389

43.39% activity (Figure 9A and B). Interestingly, the residual activity of the MXC

390

was less than that of TC/BSM (66.59%) (Figure 3C) or Sn/BSM (61.61%) (Figure

391

3D) when treated under the same conditions. Similarly, the same results of residual

392

xylanase activities were observed using beechwood xylan as the substrate (Figure 9C

393

and D).19 Collectively, the dual-enzyme ring conformation was more stable than the

394

linear enzyme after heat challenge but inferior when compared to the single-enzyme

395

ring conformation. Our previous study19 and other works18,

396

molecular

397

conformation. Thus, we speculated that compared with the single-enzyme ring, the

398

dual-enzyme ring was loosened, which probably expose its hydrophobic regions to

399

solvent, leading to the breakdown of the secondary and tertiary structures. In other

400

words, a compact conformation may contribute to a more stable structure without

401

compromising catalytic efficiency. To our knowledge, this is the first study to

402

investigate the effects of dual- or more-ring cyclization generated via isopeptide

403

bond-mediated ligation on enzymatic catalysis and stability. However, further study

404

should be performed to elucidate the mechanism.

cyclization

enhanced

protein

thermostability

28

demonstrated that

by

stabilizing

its

405

Generally, natural plant biomass comprises complicated NSPs, such as xylanase,

406

mannan, and cellulose. Therefore, deconstruction of lignocellulosic biomass requires

407

the combined activities of multiple NSP-degrading enzymes.4,

408

CC/BSM, the MXC showed comparable Km but increased Kcat by 36.67% (P < 0.05)

409

and Kcat/km by 25.00% (P = 0.0589) when locust bean gum served as the substrate


48

Compared with


410

(Table 2), indicating cooperation of mannanase and xylanase on substrate breakdown.

411

LPMO, previously classified into the GH61 family and recently reclassified into an

412

auxiliary activity family, is a novel lignocellulose-active enzyme with oxidation

413

activity.49 In this study, we further evaluated synergistic activities of chimeric

414

enzymes towards locust bean gum, beechwood xylan and corn husk degradation.

415

Compared with mannanase alone, MXC did not exhibit higher hydrolysis (Figure

416

10A), and TFX was inactive against locust bean gum (Figure 8). However, MXC

417

showed improved catalysis to beechwood xylan (P < 0.05) (Figure 10C), though BSM

418

showed no measurable activity against beechwood xylan. We speculated that BSM

419

likely acted on the side chains of the substrate and negligible reducing sugars were

420

released by itself. Interestingly, MXC showed significantly higher hydrolysis of corn

421

husk compared with BSM and TFX alone (P < 0.05) (Figure 10D). Moreover, LPMO

422

was found to boost BSM either on locust bean gum or corn husk (P < 0.01) (Figure

423

10B and E), indicating chimeric enzyme complexes were more robust for

424

lignocellulose deconstruction.50

425

In summary, a mesophilic mannanase from B. subtilis was cloned, cyclized, and

426

heterologously expressed. Cyclized enzymes, both TC/BSM and Sn/BSM, showed

427

improved thermostability and ion stability, resilience to aggregation and freeze-thaw

428

treatment. Moreover, two mannanase-xylanase or mannanase-LPMO chimeric

429

enzyme complexes were generated. The dual-enzyme ring was observed to be more

430

robust than linear enzyme but inferior to single-enzyme ring. The results of this study

431

can used in production of functional oligosaccharides and feed additives production


Page 20 of 44

Page 21 of 44

432


from agricultural lignocellulosic biomass.



433

ASSOCIATED CONTENT

434

Supporting Information

435

SDS-PAGE, thermostability and protein aggregation analysis of KA mutants.

436

Optimum pH and pH stability of linear and cyclized enzymes. The Supporting

437

Information is available free of charge on the ACS Publications website.

438

AUTHOR INFORMATION

439

Corresponding Author

440

*Tel: +86 574 88222391; fax: +86 574 88222957; e-mail: [email protected]

441

(S.J. Yin), [email protected] (Q. Wang).

442

Funding

443

This work was supported by the Ningbo Public Service Platform for High-Value

444

Utilization of Marine Biological Resources (NBHY-2017-P2), General Project

445

Supported by Department of Education of Zhejiang Province (Y201840329) and

446

Zhejiang Provincial Top Key Discipline (CX2018004 and CX2018031).

447

Notes

448

The authors declare no competing financial interest.

449

ACKNOWLEDGEMENTS

450

The authors thank professor Zhongtang Yu from The Ohio State University for

451

language revision.

452

ABBREVIATIONS USED

453

BSM, Bacillus subtilis mannanase; 30a/BSM, linear BSM expressed in pET30a(+);

454

TC/BSM, BSM cyclized by SpyTag/SpyCathcer; Sn/BSM, BSM cyclized by


Page 22 of 44

Page 23 of 44


455

SnoopTag/SnoopCatcher; L-TC/BSM, relinearized TC/BSM; L-Sn/BSM, relinearized

456

Sn/BSM; CC/BSM, linear BSM expressed in pETCC; TT/TFX, linear TFX expressed

457

in pETTT; CBM, carbohydrate-binding module; CD, circular dichroism; DNS,

458

3,5-dinitrosalicylic acid; GH, glycoside hydrolase; LPMO, lytic polysaccharide

459

monooxygenase; MCS, multiple cloning sites; MXC, mannanase-xylanase chimeric

460

enzyme; MLC, mannanase-LPMO chimeric enzyme; MW, molecular weight; NSP,

461

non-starch

462

enterokinase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

polysaccharide;

IPTG,

isopropyl-thio-β-D-galactopyranoside;


EK,


463

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Page 30 of 44

Page 31 of 44


FIGURES AND TABLES Figure 1. Schematic diagram of enzymes in this study. 30a/BSM, Linear B. subtilis mannanase; TC/BSM, Cyclized mannanase using SpyTag/SpyCatcher; Sn/BSM, Cyclized mannanase using SnoopTag/SnoopCatcher; CC/BSM, Linear mannanase with SpyCatcher located at both N- and C-termini; TT/TFX, Linear T. fusca xylanase with SpyTag located at both N- and C-termini; TT/LPMO, Linear B. subtilis LPMO with SpyTag located at both N- and C-termini; MXC, Mannanase-xylanase chimeric enzyme; MLC, Mannanase-LPMO chimeric enzyme; EK, enterokinase. Figure 2. SDS-PAGE analysis. Lanes: 1, 30a/BSM; 2, TC/BSM; 3, Linearized TC/BSM achieved by enterokinase digestion; 4, Sn/BSM; 5, Linearized Sn/BSM achieved by enterokinase digestion; M, Standard protein marker. Figure 3. Optimum temperature and thermostability. (A) Optimum temperature; (B-F) thermostability of 30a/BSM (B), TC/BSM (C), Sn/BSM (D), Relinearized TC/BSM (L-TC/BSM) (E) and relinearized Sn/BSM (L-Sn/BSM) (F). The highest activities under the optimum conditions (pH 6.0, 50°C) or initial activities before preincubation were taken as 100%. Data represent the mean ± SD (n=4). Figure 4. Heat-induced protein aggregation. (A) 30a/BSM; (B) TC/BSM; (C) Sn/BSM; (D) L-TC/BSM; (E) L-Sn/BSM; M, Standard protein marker; 1, Native enzyme control; 2, 25°C; 3, 37°C; 4, 50°C; 5, 60°C; 6, 70°C; 7, 80°C; 8, 90°C; 9, 100°C. (F) Residual activities after heat challenge; (G) Optical densities of supernatant proteins. Data represent the mean ± SD (n=4). Figure 5. Circular dichroism and intrinsic fluorescence analysis. (A-C) CD spectra of 30a/BSM, TC/BSM and Sn/BSM. (D-F) The percentages of 30a/BSM, TC/BSM and Sn/BSM

were

calculated

using

CDPro

(http://sites.bmb.colostate.edu/

sreeram/CDPro/). Data represent the mean ± SD (n=4). (G-I) Intrinsic fluorescence analysis of 30a/BSM, TC/BSM and Sn/BSM. Inset was the maximum peak wavelength. Figure 6. Freeze-thaw stability. (A-C) SDS-PAGE analysis after free-thaw treatment of 30a/BSM, TC/BSM and Sn/BSM. M, Standard protein marker; 1, Control; 2, 5 cycles; 3, 10 cycles; 4, 15 cycles; 5, 20 cycles. (D) Protein aggregation monitored by



spectrometry at 400 nm. (E) Residual activities of linear BSM and cyclized BSM after freeze-thaw treatment. Data represent the mean ± SD (n=4). Statistical significance was analyzed using a two-tailed Student’s t-test and is indicated by asterisks. *, P

Characterization of thermostable and chimeric enzymes via isopeptide

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