Construction of a Rapid Feather-Degrading Bacterium by

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Construction of a Rapid Feather-Degrading Bacterium by Overexpression of a Highly Efficient Alkaline Keratinase in its Parent Strain Bacillus amyloliquefaciens K11 Lian Yang, hui wang, Yi Lv, Yingguo Bai, Huiying Luo, Pengjun Shi, Huoqing Huang, and Bin Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04747 • Publication Date (Web): 16 Dec 2015 Downloaded from http://pubs.acs.org on December 17, 2015

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Page 1 of 34

Journal of Agricultural and Food Chemistry 1

Construction of a Rapid Feather-Degrading Bacterium by Overexpression of a Highly Efficient Alkaline Keratinase in its Parent Strain Bacillus amyloliquefaciens K11 Lian Yang, Hui Wang, Yi Lv, Yingguo Bai, Huiying Luo, Pengjun Shi, Huoqing Huang*, Bin Yao*

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China

Running title: An extreme alkaline keratinase from B. amyloliquefaciens.

* Corresponding authors. Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China. Tel.: +86 10 82106053; fax: +86 10 82106054. E-mail addresses: [email protected] (B. Yao), [email protected] (H. Huang). 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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2

ABSTRACT: Keratinase is essential to degrade the main feather component, keratin, and is

3

of importance for wide industrial applications. In this study, Bacillus amyloliquefaciens strain

4

K11 was found to have significant feather-degrading capacity (completely degraded whole

5

feathers within 24 h). The keratinase encoding gene, kerK, was expressed in the Bacillus

6

subtilis SCK6. The purified recombinant KerK showed optimal activity at 50 °C and pH 11.0

7

and degraded whole feathers within 0.5 h in the presence of DTT. The recombinant plasmids

8

harboring kerK were extracted from B. subtilis SCK6 and transformed into B.

9

amyloliquefaciens K11. As results, the recombinant B. amyloliquefaciens K11 exhibited

10

enhanced feather-degrading capacity with shortened reaction time within 12 h and increased

11

keratinolytic activity (1500 U/ml) by 6-fold. This efficient and rapid feather-degrading

12

character makes the recombinant strain of B. amyloliquefaciens K11 potential for applications

13

in feather meal preparation and waste feather disposal.

14

Keywords: Bacillus amyloliquefaciens K11, Keratinase, Extreme alkaline, Gene expression

15


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16

INTRODUCTION

17 18

Keratin is a family of insoluble structural proteins that represent the major component of

19

mammalian hair, nails, wool, hoof and horn, poultry feathers, and so on.1,2 Keratinous wastes

20

constitute a serious environmental contaminant that are mainly from poultry and leather

21

industries, for instance, large quantities of feathers, approximately 8.5 million tons annually,

22

are produced worldwide as a by-product of chicken poultry.3 The feathers are composed of

23

over 90 % of keratin protein that mainly consists of small and essential amino acid residues

24

such as glycine, valine, serine and cysteine. These and other residues are cross-linked by

25

disulfide bonds, hydrogen bonds and hydrophobic bonds and tightly packed into a super

26

coiled polypeptide, forming an insoluble, highly stable structure.1,4 The mechanical stability

27

makes keratin highly resistant to proteolytic degradation of trypsin, pepsin and papain.5,6 As

28

results, degradation of waste feathers is very slow in nature and causes serious environmental

29

pollution.7 How to dispose waste feathers has been a major concern of poultry industry. A

30

current value-added approach is to convert feathers into digestible dietary protein, i.e. feather

31

meal, by using conventional physical and chemical treatments.8 However, this process not

32

only destroys keratin amino acids and decreases protein quality, but also consumes large

33

amounts of energy. 9

34

Alternatively, microbial degradation or enzymolysis of feathers is becoming an attractive

35

approach to manage keratinous wastes.9-11 To date, a number of keratinolytic microorganisms

36

have been reported, including some species of Bacillus, Actinomycetes and fungi.12−16 These

37

organisms can produce proteases specific for insoluble keratin substrates, i.e. keratinase.10



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38

Processing waste feathers by these keratinolytic microorganisms and related enzymes

39

represents an alternative method, as it offers a specific, cost-effective, and environmentally

40

benign solution to produce valuable products.9,17

41

Keratinase has application potentials in many fields. In feed industry, poultry feathers are

42

degraded by keratinase into feather meal, which supplementation into animal diets can

43

provide essential amino acids and replace soybean meal at 7 % dietary level.18 Besides,

44

keratinolytic enzymes might be used in agriculture, pharmaceutical, biomedical fields and

45

leather industry. KERUS from Brevibacillus brevis US575, has been reported, could

46

accomplish the whole process of dehairing by oneself.19 Moreover keratinase also can be

47

applied in the cosmetics industry for high purity. Currently, there are only a few commercial

48

keratinases from Bacillus licheniformis PWD-1 under the trade names Versazyme, Valkerase,

49

Prionzyme and PURE100. Due to the slow feather-degrading efficiency and high production

50

cost, keratinase, unlike other feed enzymes (phytases, xylanase, mannanase, etc), is not

51

applied widely. Thus it is of importance to obtain a super feather-degrading microorganism

52

and achieve low-cost production of highly active keratinase for large-scale industrial

53

processes.

54

Bacillus subtilis is a highly efficient system that has been used for the production of

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heterologous keratinase.20−22 Its non-pathogenic characteristic and free of endotoxins make it

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earn a “generally recognized as safe” (GRAS) status by the American Food and Drug

57

Administration (FDA). The recombinant B. subtilis system not only secretes extracellular

58

proteins directly into the culture medium and simplifies the downstream processing procedure,

59

but also has non-biased codon usage.23,24


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In this article, a highly efficient feather-degrading bacterium, Bacillus amyloliquefaciens

61

K11, was reported, which was able to disintegrate chicken feathers completely within 24 h.

62

By cloning and heterologous expression of the coding gene, kerK, the keratinase was

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produced in B. subtilis SCK6, and exhibited substantial keratinase activity. The recombinant

64

plasmid pUB110-kerK was then extracted from B. subtilis SCK6 and transformed its parent

65

strain. As results, the feather-degrading ability of B. amyloliquefaciens K11 was enhanced

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significantly, shortening the feather disintegration duration to 12 h and increasing the

67

keratinase production by 6-fold as compared with that of strain K11.

68

69

MATERIALS AND METHODS

70 71

Strains, Plasmids, Materials and Chemicals. B. amyloliquefaciens K11 with high

72

neutral protease-producing capacity was isolated and deposited at Agricultural Culture

73

Collection of China, Beijing under the Registration No. ACCC19735. B. subtilis SCK6

74

(BGSC 1A976) and plasmid pUB110 were gifts from Dr. Daniel Zeiglerat of the BGSC. The

75

pGEM-T Easy vector was purchased from TransGen (Beijing, China). Chicken feathers were

76

collected from a farm in Beijing suburb, washed with tap water, soaked in 70 % ethanol for 1

77

h, and air-dried.25 Folin-Ciocalteu’s phenol reagent, casein, and other chemicals were of

78

analytical grade and commercially available.

79 80

Feather-degrading Capacity Assessment and Isolation of the Native Keratinase. B.

81

amyloliquefaciens K11 grown in 20 ml of LB medium at 37°C for 12 h at the agitation speed



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of 200 rpm was used as seed culture. 1% of the seed culture was inoculated into 150 ml of

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fresh feather medium containing (per litre) 0.5 g of NaCl, 0.4 g of KH2PO4, 0.3 g of K2HPO4,

84

and 4 g of feather (the sole source of carbon and nitrogen) at 37 °C. The feather degrading

85

degree was visualized at different time intervals. After 36-h incubation, the culture broth was

86

centrifuged at 4 °C and 12,000 ×g for 10 min, and the supernatant was concentrated by a

87

Vivaflow 200 membrane of 3-kDa molecular weight cutoff (Vivascience, Hannover,

88

Germany). The crude enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel

89

electrophoresis (SDS-PAGE) according to the method of King and Laemmli (1971).26 The

90

most obvious band in the SDS-PAGE gel was excised and identified using liquid

91

chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) by Tianjin

92

Biochip Co. Ltd. (Tianjin, China).

93 94

Cloning

of

the

Keratinase-encoding

Gene

(kerK).

Genomic

DNA of

B.

95

amyloliquefaciens K11 was extracted using TIANprep Midi Bacteria DNA kit (TIANGEN,

96

Beijing, China). The primer pair kerKF and kerKR (Table 1) was designed according to the

97

sequence alignment of the peptide fragments from LC-ESI-MS/MS and the putative

98

keratinase gene of B. amyloliquefaciens Y2 (Accession No. CP003332.1). The keratinase

99

gene of B. amyloliquefaciens K11, designated kerK, was amplified with an annealing

100

temperature of 55 °C, and the PCR product was purified and sequenced.

101 102

Sequence Analysis. The sequence assembly was carried out using the DNAMAN6.0

103

software. Homology analysis of the nucleotide and amino acid sequences was performed


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104

using the BLAST program available from the National Center for Biotechnology Information.

105

The putative signal peptide was predicted online (http://www.cbs.dtu.dk/services/SignalP/).

106 107

Construction of the Expression Vector pUB110-kerK. The expression vector

108

pUB110-kerK was constructed by using the simple cloning method.27 Briefly, the linear

109

backbone of plasmid pUB110 without the mob gene and the gene fragment including the

110

native promoter, kerK and the terminal sequence of B. amyloliquefaciens K11 were amplified

111

by using the primers pUB110F and pUB110R and kerKF and kerKR (Table 1), respectively.

112

The PCR products were gel purified and used as templates for the prolonged overlap

113

extension (POE)-PCR using the high fidelity KOD-Plus-Neo enzyme (TOYOBO, Osaka,

114

Japan; KOD-401) without primers. The POE-PCR conditions were denaturation at 95 °C for 1

115

min; 30 cycles of 95 °C denaturation for 20 s, 60 °C annealing for 40 s, and 72 °C extension

116

for 3 min; followed by 72 °C extension for 10 min.

117 118

Transformation of pUB110-kerK into B. subtilis SCK6. The POE-PCR product

119

multimers (5 µl) were added to 100 µl of freshly prepared B. subtilis SCK6 competent cells,27

120

and agitated at 37 °C and 200 rpm for 90 min. The cultures were then incubated at 37 °C

121

overnight, followed by cell plating on LB plates with kanamycin (20 µg/ml). Colonies were

122

then transferred to LB medium for 12-h growth at 37 °C and 200 rpm. Recombinant plasmids

123

were then extracted from B. subtilis SCK6 and were verified by restriction digest and

124

sequencing.

125



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Functional Characterization of kerK Gene in B. subtilis SCK6. To determine the

127

function of kerK, colonies of recombinant B. subtilis SCK6 containing pUB100-kerK were

128

grown on LB plates containing 2 % skimmed milk and in the feather medium at 37 °C for 24

129

h, respectively. Cells of the recombinant B. subtilis SCK6 harboring the empty vector pUB110

130

were cultured under the same conditions as controls. The feather-degrading capacity and

131

keratinase activity were assessed as described above and below, respectively.

132 133

Purification of Recombinant KerK. Recombinant B. subtilis SCK6 harboring

134

pUB100-kerK was grown at 37 °C for 36 h in the feather medium, and the culture supernatant

135

was collected by centrifugation at 12,000 ×g for 10 min to remove cell debris. The culture

136

supernatant was concentrated by a hollow fiber (cutoff 3 kDa; Motianmo, Tianjin, China)

137

followed by vacuum freeze-drying. The crude enzyme was precipitated by solid ammonium

138

sulfate up to 85 % saturation level, stayed intact for 12 h, and centrifuged at 8000 ×g for 10

139

min. The pellet was then dissolved in 50 mM Tris-HCl (pH 8.0) and dialyzed overnight

140

against the same buffer at 4 °C.

141

The SDS-PAGE analysis was performed according to the method of King and Laemmli

142

(1971)26 with some modifications. Briefly, before enzyme loading, 10 µl extra 20 % SDS was

143

added, and then was mixed with the loading buffer. The mixture was boiled for approximately

144

10 min.

145 146

Enzyme Activity Assay. Keratinolytic activity was assayed following Vermelho et al.

147

(2009)28 with some modifications. Briefly, feather keratin powder was prepared according to


Page 9 of 34


148

the method of Wawrzkiewicz et al. (1991).29 A reaction mixture (4.4 ml) containing 0.01 g of

149

feather keratin powder and 200 µl of crude enzyme sample in glycine-NaOH buffer (pH 11.0)

150

with 2 mM dithiothreitol (DTT) was incubated at 50 °C for 1 h, and the reaction was

151

terminated by addition of 2 ml of 20 % trichloroacetic acid (TCA). The mixture was

152

centrifuged at 12,000 ×g for 5 min, followed by the measurement of supernatant absorbance

153

at 280 nm (A280) in a 1-cm cell. One unit of keratinolytic activity was defined as the amount

154

of enzyme required to increase the A280 by 0.01 under the standard assay conditions (pH 11.0

155

and 50 °C for 1 h).

156

The protease activity was determined by using the Folin-phenol method of the People's

157

Republic of China GB/T 23527-2009. Brieﬂy, 0.5 ml of 1 % (w/v) casein solution was

158

preheated at 40 °C for 10 min, followed by addition of 0.5 ml of 40 °C-preheated

159

appropriately diluted enzyme solution. The reaction mixture was incubated at 40 °C for 10

160

min, and 1 ml of 400 mM TCA was added to terminate the reaction. The reactions with

161

enzyme addition after TCA were used as controls. After centrifugation at 13,000 ×g for 5 min,

162

1 ml of the supernatant was added into a test tube containing 5 ml of 400 mM Na2CO3 and 1

163

ml of Folin-phenol reagent, followed by incubation at 40 °C for 20 min. The absorbance was

164

measured at 680 nm. One unit of protease activity was defined as the amount of enzyme that

165

hydrolyzed casein to produce 1 µg of tyrosine per minute under the standard conditions (pH

166

11.0 and 40 °C for 10 min).

167 168

Characterization of Purified Recombinant KerK. Feather keratin powder was used as

169

the substrate for biochemical characterization of recombinant KerK. The pH-activity profile



Page 10 of 34 10

170

of recombinant KerK was determined at 50 °C for 1 h in different buffers of pH 7.0−12.0. For

171

pH stability assay, the enzyme was incubated at 37 °C in different buffers of pH 5.0−12.0 for 3

172

h without substrate, and the residual enzyme activities were measured under standard

173

conditions (pH 11.0 and 50 °C for 1 h). The buffers used were 50 mM of McIlvaine buffer

174

(pH 5.0−6.0), 50 mM of Tris-HCl (pH 7.0−8.0), and 50 mM of glycine-NaOH (pH 9.0−12.0).

175

The temperature-activity profile was determined by measuring the keratinase activity at

176

different temperatures (30−70 °C) and optimal pH for 1 h. Thermal stability of KerK was

177

determined by measuring the residual enzyme activities under standard conditions after

178

incubation of the enzyme at 50 °C for 2, 5, 10, 20, 30 or 60 min without substrate.

179

The effect of metal ions and chemical reagents on the activity of purified recombinant

180

KerK was determined by adding 1 or 5 mM of various metal ions (Ca2+, Mg2+, Mn2+, Cr3+,

181

Fe3+, Ni2+, Zn2+, and Co2+) and chemical reagents (5 mM of EDTA and PMSF and 1 %

182

β-mercaptoethanol, SDS, Triton X−100, Tween-80, and Tween-20) to the assay system. The

183

system without any addition of extra metal ions or chemical reagents mentioned above was

184

treated as a control. Each reaction was run in triplicate.

185 186

Chicken Feather Degradation by Purified Recombinant KerK in Vitro. Enzymatic 25

187

degradation of feathers was conducted as described by Liang et al. (2010)

188

modifications. Briefly, sterilized chicken feather (~5 cm) was incubated with 150 U of

189

purified enzyme in 8 ml of 50 mM glycine-NaOH (pH 11.0) containing 2 mM DTT. The

190

degradation degrees of feathers were observed at different time intervals, and enzymatic

191

hydrolysis products were analyzed by HPLC-Chip/ESI-QTOF-MS in Institute of Apicultural


with some

Page 11 of 34


192

Research, CAAS.

193 194

Construction

of

B.

amyloliquefaciens

K11

Harboring

pUB110-kerK.

The

195

electrocompetent cells of B. amyloliquefaciens K11 were prepared as described by Zhang et al.

196

(2013).30 The plasmid pUB110-kerK was extracted from recombinant B. subtilis SCK6 using

197

the TIANprep Midi Plasmid kit (Tiangen). Eighty microgram of competent cells were mixed

198

with 5 µl of recombinant plasmid pUB110-kerK (~ 250 ng), kept on ice for 5 min, followed

199

by electroporatation via a Bio-Rad Gene Pulser. The cells were then plated on LB plates with

200

kanamycin (20 µg/ml). The positive transformants were verified by PCR and plated on 2 %

201

(w/v) skim milk plates for preliminary screening of the protease activity.

202 203

Degradation of Chicken Feathers and Production of Extracellular Protease. The

204

engineered B. amyloliquefaciens K11 containing pUB110-kerK and parent strain B.

205

amyloliquefaciens K11 were both grown in the feather medium with chicken feathers as the

206

sole source of carbon and nitrogen. Their feather-degrading efficiency and halo sizes around

207

the colonies were measured as described above for comparison. Meanwhile the keratinolytic

208

and proteolytic activities of the culture supernatants were measured, respectively, as described

209

above at different time intervals.

210 211

Nucleotide Sequence Accession Number. The nucleotide sequence for the B.

212

amyloliquefaciens K11 keratinase gene (kerK) was deposited in the GenBank database under

213

accession no. KR868996.



Page 12 of 34 12

214 215

RESULTS AND DISCUSSION

216 217

The Feather Degrading Capacity of B. amyloliquefaciens K11. The keratinolytic

218

potential of B. amyloliquefaciens K11 was assessed by growing the cells at 37 °C in the

219

medium with chicken feathers as the only source of carbon and nitrogen. As results, visible

220

degradation of chicken feathers was observed after cultivation for 12 h, and complete feather

221

degradation was achieved after 24 h (Fig. 1a). Usually, chicken feather degradation is a rather

222

slow process, which takes most of strains at least 3−7 days for complete degradation. For

223

example, B. subtilis SLC degraded feather completely at room temperature after 7 days,31 B.

224

licheniformis PWD-1 degraded the feather keratin completely after 7 to 10 days at 50 °C,32

225

and Thermoactinomyces sp. CDF took 72 h to degrade feather keratin completely.33 In

226

comparison

227

feather-degradation capacity and great potential in the disposal of waste feathers for industrial

228

purposes.

with

these

feather-degrading

strains,

strain

K11 showed

significant

229 230

Isolation of the Keratinase from B. amyloliquefaciens K11. The culture supernatants of

231

B. amyloliquefaciens K11 were collected after 36-h incubation at 37 °C. SDS-PAGE analysis

232

showed the presence of a major band of 27 kDa in the culture supernatants (Fig. 2a). The

233

absence of other proteins might be ascribed to their very low expression levels or other

234

proteins were degraded by the protease in the feather medium. Further LC-ESI-MS/MS

235

analysis identified peptide fragments, VAVIDSGIDSSHPDLK, YPSVIAVGAVDSSNQR, and


Page 13 of 34


236

HPNWTNTQVR, which were 100 % identical to the putative protein sequence of B.

237

amyloliquefaciens Y2.

238 239

Gene Cloning, Sequence analysis and Recombinant Plasmid Construction. The

240

full-length keratinase gene (kerK) of B. amyloliquefaciens K11, 1149 bp in length, showed

241

high identity (99 %) with that of B. amyloliquefaciens Y2. The Keratinase showed significant

242

feather-degrading activity, and its encoding gene kerK was first reported in B.

243

amyloliquefaciens. Sequence analysis showed that the kerK encoded a polypeptide of 382

244

amino acids consisting of a signal peptide of 30 amino acids residues, a pro-sequence of 77

245

residues and a mature protein of 275 residues, which shares a 63.7 % sequence identity with

246

the commercial keratinase from Bacillus licheniformis PWD-1.34 The kerK gene including the

247

native promoter and terminal sequence was then cloned into pUB110 vector by POE-PCR27

248

and transforming into B. subtilis SCK6 to construct the recombinant plasmid pUB110-kerK.

249 250

Expression and Purification of kerK in B. subtilis SCK6. Due to the favorable

251

characteristics of secretion pathway, nonpathogenic and non-biased codon usage,22,24

252

protease-deficient B. subtilis SCK6 was selected as the expression host for the kerK gene. The

253

cells of recombinant B. subtilis SCK6 harboring pUB110-kerK produced halo zones when

254

grown on LB plates containing 2 % skimmed milk, while those of B. subtilis SCK6 harboring

255

the empty vector (the control) didn’t (Fig. 3a). This indicated that kerK encodes an enzyme

256

which has protease activity as reported by Wang et al. (2015).33 When grew the recombinant

257

cells at 37 °C in the feather medium, feathers were completely degraded at 24 h by B. subtilis



Page 14 of 34 14

258

SCK6 harboring pUB110-kerK while the control didn’t (Fig. 1b). These results indicated that

259

kerK is the key gene for feather degradation. The successful expression of kerK in B. subtilis

260

SCK6 endowed the strain SCK6 capacity to degrade feather efficiently. Recombinant KerK

261

was then purified to eletrophoretic homogeneity through ammonium sulfate precipitation. The

262

purified protein migrated as a single band on SDS-PAGE with the molecular mass of about

263

27.0 kDa (Fig. 2b).

264 265

Biochemical Characterization of Recombinant KerK. Most microbial keratinases are

266

neutral or alkaline proteases with the pH optima ranging from 7.5 to 9.0, except for a few

267

alkaliphilic enzymes from Kocuria rosea35 and Nocardiopsis sp. TOA-1.36 The purified

268

recombinant KerK is an extreme alkaline keratinase. Using feather keratin powder as the

269

substrate, purified recombinant KerK showed the maximum activity at pH 11.0 (Fig. 4a), and

270

retained stable over a wide pH range from 6.0 to 12.0 (Fig. 4b). These alkaline characteristics

271

make KerK suitable for industrial applications in textile processing enzymatic depilation and

272

detergent37. The optimum temperature of recombinant KerK at pH 11.0 was 50 °C (Fig. 4c).

273

And the enzyme lost 30 % enzyme activity after pre-treatment at 50 °C for 30 min (Fig. 4d),

274

which was similar to that of commercial keratinase from B. licheniformis PWD-1.38

275

The effects of different metal ions and chemical reagents on the enzyme activity were

276

shown in Table 2. The activity of recombinant KerK was enhanced by Ca2+, Fe3+ and

277

β-mercaptoethanol, partially inhibited by Cr3+ and Mg2+, and strongly inhibited by Mn2+, Ni2+,

278

Co2+, SDS, PMSF and EDTA. The stimulatory effect of Ca2+ might be ascribed to its

279

formation of salt or ion bridges, thus stabilizing the enzyme under its active conformation and


Page 15 of 34


280

protecting the enzyme against denaturation.39 The increased activity by β-mercaptoethanol

281

may own to its reducing power for disruption of the disulfide bonds. However, the possible

282

mechanism of Fe3+ stimulation needs further study. The results suggested that the activity of

283

recombinant KerK will be enhanced by adding reducing agents and some metal ions in

284

industrial applications. Moreover, the strong inhibition of recombinant KerK by PMSF and

285

EDTA indicated that KerK belongs to serine-metallo-proteases10,33,40 and deduced KerK share

286

a 97 % sequence identity with B. amyloliquefaciens subtilisin (1ST2_A), showing that KerK

287

belongs to subtilisin family of serine proteases.

288 289

Degradation of Feathers by Purified KerK. Most purified keratinases cannot degrade

290

keratin by themselves.41 The process of feather degradation by keratinase is presumed to

291

consist of two basic steps, i.e. reduction of disulfide bonds42 and proteolysis that releases

292

short peptides and amino acids.5,43,11 Microbial feather degradation requires the cooperation of

293

reducing power provided by the cell themselves and extracellular secretion of proteolytic

294

enzymes.33,39,44,45 The feather-degrading capacity of purified recombinant KerK was assessed

295

in the presence of reducing agent DTT or not in this study. When DTT was included in the

296

enzyme reaction system, the chicken feather was completely degraded by purified

297

recombinant KerK within 0.5 h at 50 °C (Fig. 5). Without DTT, the degradation of chicken

298

feather was unobvious. This result indicated that the presence of reducing agent is necessary

299

for the efficient hydrolysis of feathers by purified recombinant KerK.

300 301

Analysis of Enzymatic Hydrolysis Products. The peptides with molecular masses



Page 16 of 34 16

302

below 3 kDa possess reduced allergenicity and are rich in many high-value bioactive

303

components.46 HPLC-Chip/ESI-QTOF-MS analysis indicated that the peptides obtained from

304

feather hydrolysis have molecular masses of 1 to 3 kDa and have major cleavage sites at

305

serine

306

IQPSPVVVTLPGPILSS, and ILSEEGVPISSGGF as the main feather hydrolysis products

307

contained many essential amino acids, therefore would be applied for recycling utilization of

308

feathers, especially in feed industry as feed components.

and

hydrophobic

amino

acids.

Polypeptides

VVIQPSPVVVTLPGPILSS,

309 310

Comparison of the Feather-degrading Capacity and Keratinase Production by

311

Native and Recombinant B. amyloliquefaciens K11. Keratin-degrading microorganisms

312

have attracted much attention due to their extensive industrial applications.40 When incubated

313

in the feather medium, native B. amyloliquefaciens K11 completely degraded the feathers at

314

24 h (Fig. 1a). Under the same conditions, complete feather degradation was achieved by

315

recombinant B. subtilis SCK6 and recombinant B. amyloliquefaciens K11 at 24 h and 12 h

316

(Fig. 1b, 1c), respectively. Besides the highest efficiency of feather degradation, recombinant

317

B. amyloliquefaciens K11 was found to produce the biggest zone on skimmed milk plates (Fig.

318

3b), suggesting that it has the highest proteolytic activity.

319

During fermentation, the keratinolytic activity of recombinant B. amyloliquefaciens K11

320

harboring multiple copies of kerK increased gradually and reached the maximum (1500 U/ml)

321

at 60 h. This yield is significantly higher than that of the parent strain B. amyloliquefaciens

322

K11 (240 U/ml) (Fig. 3c and Table 3) and Thermoactinomyces sp. CDF (400 U/ml).33

323

Meanwhile it is noteworthy that the recombinant B. amyloliquefaciens K11 containing


Page 17 of 34


324

pUB110-kerK only took 12 h to degrade feathers completely and released many essential

325

amino acids. Therefore, the recombinant B. amyloliquefaciens K11 has great potential for

326

applications in waste feather disposals for poultry industry, additional, which has higher

327

keratinase expression level than others.

328

In conclusion, a B. amyloliquefaciens strain with significant feather-degrading capacity

329

was reported in this study. The functional gene for feather degradation, kerK, was then cloned

330

and expressed in B. subtilis SCK6. The purified recombinant KerK showed the maximum

331

activity at pH 11.0 and 50 °C, and degraded whole feathers within 0.5 h in the presence of

332

DTT. In comparison with native K11, recombinant B. amyloliquefaciens K11 containing the

333

plasmid pUB110-kerK had a 6-fold increase in keratinase production and degraded feathers

334

completely within 12 h. The efficient and rapid feather-degrading capacity of recombinant

335

strain K11 makes it valuable in waste feather disposal and recycling.

336 337

ACKNOWLEDGEMENTS

338 339

This research was supported by the “Twelfth Five-Year” National Science and

340

Technology Project in Rural Areas of China (2013BAD10B01-2) and the National High

341

Technology Research and Development Program of China (863 program, 2013AA102803).

342 343

Competing Interests:

344

The authors declare no competing financial interest.

345



Page 18 of 34 18

346

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473

A. Influence of chemical modification of whey protein conformation on hydrolysis with

474

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475



Page 24 of 34 24

476 477

Figure legends

478

Fig. 1 Visual observation of the chicken feather degradation by B. amyloliquefaciens K11 at

479

24 h (a), recombinant B. subtilis SCK6 at 24 h (b) and recombinant B. amyloliquefaciens K11

480

at 12 h (c).

481 482

Fig. 2 SDS-PAGE analysis of native and purified recombinant KerK. Lane M shows the

483

molecular mass standards, lane 1 shows the native KerK from the culture supernatants of B.

484

amyloliquefaciens K11, and lane 2 shows the purified recombinant KerK.

485 486

Fig. 3 Proteolytic and keratinolytic activities of parent B. amyloliquefaciens K11 and

487

recombinant B. subtilis and B. amyloliquefaciens K11 strains. a The halo zones of B. subtilis

488

SCK6 harboring empty vector (1) and pUB100-kerK (2) on LB agar containing 2 % skimmed

489

milk at 37 °C for 24 h. b The halo zones of parent strain B. amyloliquefaciens K11 (3),

490

recombinant B. subtilis SCK6 containing pUB100-kerK (4) and recombinant B.

491

amyloliquefaciens K11 (5). c Extracellular keratinolytic activities. Each value in the panel

492

represents the means ± SD (n = 3).

493 494

Fig. 4 Enzymatic properties of purified KerK. a Effect of pH on enzyme activities. The

495

enzyme assay was performed at 50 °C for 60 min. b pH stability. The enzyme was

496

pre-incubated without substrate at 37 °C for 3 h, and then subjected to residual activity assay

497

under standard conditions (pH 11.0 and 50 °C for 60 min). c Effect of temperature on enzyme

498

activities determined at pH 11.0 for 60 min. d Thermostability. The residual enzyme activities


Page 25 of 34


499

were measured under standard conditions after pre-incubation of the enzyme without

500

substrate in glycine-NaOH (pH 11.0) for various periods. Each value in the panel represents

501

the means ± SD (n = 3).

502 503

Fig. 5 In vitro degradation of chicken feathers by purified KerK (150 U). Chicken feathers (5

504

cm) were incubated in 8 ml of 50 mM glycine-NaOH (pH 11.0) at 50 °C in the presence (+)

505

or absence (−) of 2 mM DTT. Images of feather degradation were captured at 0.5 h.



Page 26 of 34 26

Table 1. Oligonucleotide primers used in this study. Primers

Sequences (5´→3´)

kerKF

CTGACCGAGATTTTTTTGAGCAACTCGGGTTCCTATTAAACGAAAGA GAG

kerKR

CTTAGTGCTTTCATAGATTAAACTCTAAAAAAACCGGCGGGGCCA TGGCC

pUB110F

GGCCATGGCCCCGCCGGTTTTTTTAGAGTTTAATCTATGAAAGCACTA AG

pUB110R

CTCTCTTTCGTTTAATAGGAACCCGAGTTGCTCAAAAAAATCTCGGTC AG


Page 27 of 34


Table 2. The effects of different metal ions and chemical reagents on the enzyme activity of recombinant KerK. Chemicals Control

Concentrations

Relative activity (%) 100 ± 2

−

Ca2+

5 mM

129 ± 3

Mg2+

5 mM

75 ± 1

Mn2+

5 mM

11 ± 2

Fe3+

1 mM

211 ± 7

Cr3+

1 mM

92 ± 2

Zn2+

1 mM

36 ± 1

Co2+

1 mM

15 ± 1

Ni2+

1 mM

9±1

EDTA

5 mM

20 ± 2

PMSF

5 mM

2±1

β-Mercaptoethanol

1%

220 ± 7

SDS

1%

2±1

Triton X-100

1%

40 ± 1

Tween-80

1%

67 ± 3

Tween-20

1%

65 ± 1



Page 28 of 34 28

Table 3. Proteolytic and keratinolytic activities of keratinase KerK from native and recombinant B. amyloliquefaciens K11.

Substrate

Enzymatic activity (U/ml) Recombinant Native B. amyloliquefaciens K11 B. amyloliquefaciens K11

Feather keratin powder

238.4 ± 6.1


1490 ± 24

Page 29 of 34


Figure 1. 119x180mm (300 x 300 DPI)



Figure 2. 39x59mm (300 x 300 DPI)


Page 30 of 34

Page 31 of 34


Figure 3. 65x50mm (300 x 300 DPI)



Figure 4. 129x104mm (300 x 300 DPI)


Page 32 of 34

Page 33 of 34


Figure 5. 83x50mm (300 x 300 DPI)



Graphic for table of contents 85x47mm (300 x 300 DPI)


Page 34 of 34

Construction of a Rapid Feather-Degrading Bacterium by

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