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Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation
Journal Pre-proof Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation Liufeng Zheng, Xinying Yu, Changhao Wei, Leyun Qiu, Chenwei Yu, Qian Xing, Yawei Fan, Zeyuan Deng PII:
S0023-6438(19)31332-5
DOI:
https://doi.org/10.1016/j.lwt.2019.108990
Reference:
YFSTL 108990
To appear in:
LWT - Food Science and Technology
Received Date: 2 September 2019 Revised Date:
22 December 2019
Accepted Date: 24 December 2019
Please cite this article as: Zheng, L., Yu, X., Wei, C., Qiu, L., Yu, C., Xing, Q., Fan, Y., Deng, Z., Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation, LWT - Food Science and Technology (2020), doi: https:// doi.org/10.1016/j.lwt.2019.108990. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Author Contributions Liufeng Zheng and Zeyuan Deng designed the experiments, interpreted the results, and wrote the manuscript. Xinying Yu, Changhao Wei, Chenwei Yu, and Leyun Qiu conducted the experiments and prepared the manuscript. Qian Xing and Yawei Fan revised the manuscript.
1
Production and characterization of a novel alkaline protease from a
2
newly isolated Neurospora crassa through solid-state fermentation
3 4
Liufeng Zhenga,†, Xinying Yua,†, Changhao Weia, Leyun Qiua, Chenwei Yua, Qian
5
Xinga, Yawei Fana, Zeyuan Denga,b,*
6
a
7
Nanchang 330047, Jiangxi, China
8
b
9
China
State Key Laboratory of Food Science and Technology, Nanchang University,
Institute for Advanced Study, University of Nanchang, Nanchang 330031, Jiangxi,
10 11
*
Correspondence to: Zeyuan Deng. E-mail address: [email protected]
†
L. Zheng and X. Yu contributed equally to this work.
12 13 14
1
15
ABSTRACT
16
Microbial proteases are widely used to prepare protein hydrolysates with
17
health-promoting biopeptides. Here, a newly isolated strain of Neurospora crassa
18
(named as CGMCC3088) was used to produce proteases through solid-state
19
fermentation of okara as the substrate. The optimal fermentation conditions are: okara,
20
10 g; water, 21 mL; initial pH, 5.0; incubation temperature, 30°C; inoculation amount,
21
2 mL; fermentation time, 72 h, with a corresponding protease activity of 1959.82 U/g.
22
The protease was further purified by ammonium sulphate precipitation, followed by
23
ion-exchange chromatography on DEAE-Sepharose and Sephadex G-75. The
24
molecular weight of the protease was 30 kDa, and further mass spectrometry analysis
25
clearly indicated that it was a novel protease. The protease had the optimal activity at
26
55°C and pH 9. The enzyme activity was partially inhibited by SDS and metal ions,
27
whereas little affected by organic solvents. The protease was completely inactivated
28
by phenylmethylsulfonyl fluoride, indicating its dominant serine protease activity. The
29
enzyme preferably hydrolyzed casein, and kinetic analysis showed that its Km and
30
Vmax were 2.18 mg/mL and 36.36 µg/mL/min, respectively. Therefore, Neurospora
31
crassa CGMCC3088 has the potential to produce a novel organic solvent-stable
32
alkaline protease, which may be applied to the preparation of bioactive ingredients.
33 34
Keywords: Neurospora crassa; Solid-state fermentation; Fermentation conditions;
35
Alkaline protease; Purification.
36 2
37
1. Introduction
38
Proteases are a large category of enzymes and account for approximately 60% of
39
total global enzyme production (Li, Scott, Hemar, Zhang, & Otter, 2018). It has been
40
well documented that proteases can efficiently hydrolyze proteins to biopeptides,
41
which have excellent antioxidant, anti-inflammatory and anti-microbial activities
42
(Cermeno et al., 2019; Sultan, Huma, Butt, Aleem, & Abbas, 2018), and may help to
43
prevent various chronic diseases such as obesity and cardiovascular diseases (Cicero,
44
Fogacci, & Colletti, 2017). Currently, proteases of microbial origin have been widely
45
applied to the production of protein hydrolysates with bioactivities owing to their low
46
cost, high stability and specificity (Chew, Toh, & Ismail, 2019; dos Santos Aguilar &
47
Sato, 2018). However, more novel microbial proteases are still needed from natural
48
resources despite of the currently available commercial proteases.
49
Microorganisms in naturally fermented foods are an attractive source of proteases
50
due to their non-toxic nature and functional properties (Tamang, Shin, Jung, & Chae,
51
2016). Meitauza, a traditional fermented food produced from soybean residue, serves
52
as a health functional food and is widely consumed by Chinese people because of its
53
pleasant flavor and high biopeptide contents (Vong & Liu, 2016). Notably, protease
54
activity, which increases gradually with ripening, is one of the most important factors
55
that determine the quality of Meitauza (Liu, Han, Deng, Sun, & Chen, 2018). Since no
56
protease activity can be detected in the raw material, it can be speculated that
57
microorganisms used in the production of Meitauza can yield proteases. Therefore,
58
Meitauza may be an ideal material for exploring novel microorganisms with a high 3
59
yield of proteases. In our previous work, a kind of fungus, Neurospora crassa
60
CGMCC3088, was firstly isolated from Meitauza. This fungus can produce cellulases
61
to effectively degrade fiber, and has a strong ability to yield carotenoids with potential
62
benefits and nutrition to human health (P. Liu, Li, & Deng, 2016). More recently, our
63
group demonstrated that the nutritional quality and antioxidant activity of soybean
64
meal can be significantly improved through fermentation using this newly isolated
65
fungus (J. Li et al., 2019). Therefore, Neurospora crassa CGMCC3088 can be
66
regarded as a novel functional micoorganism. However, the ability of Neurospora
67
crassa CGMCC3088 to produce proteases has never been reported before.
68
Solid-state fermentation (SSF) is an important way for the production of enzymes.
69
Production of enzymes by SSF with agroindustrial wastes as the substrates has
70
attracted great interests due to its inherent advantages including low cost, high yield
71
and environmental friendliness (Leite, Silva, Salgado, & Belo, 2019; Sadh, Duhan, &
72
Duhan, 2018). Okara, also known as soybean residue and the major insoluble
73
byproduct from the production of soy milk and tofu, is largely under-utilized in food
74
industry. As an agroindustrial waste, okara has high nutritional values with abundant
75
dietary fiber, protein, lipid, vitamin, mineral, and isoflavone (B. Li et al., 2019).
76
Therefore, it is a promising and safe substrate in biotransformation process. Actually,
77
okara has been successfully used as an excellent substrate for SSF to improve its
78
nutritional composition and antioxidant activity (Gupta, Lee, & Chen, 2018).
79
However, there has been no report on the application of SSF to produce proteases
80
using okara as the substrate. 4
81
Therefore, this study aims to optimize the fermentation conditions for achieving the
82
maximum production of proteases from Neurospora crassa CGMCC3088 under SSF
83
of okara. Then, we performed the purification and characterization of a novel
84
extracellular alkaline protease, which can serve as a potential biocatalyst in food
85
processing, particularly in the preparation of biopeptide-enriched hydrolysates.
86 87
2. Materials and methods
88
2.1. Materials
89
Neurospora crassa CGMCC3088 was newly isolated from Meitauza by our lab (P.
90
Liu et al., 2016). The okara was obtained from a local supermarket. Chromatographic
91
columns used for protease purification were purchased from Amersham Biosciences
92
(Freiburg, Germany). All chemicals were of chemical grade.
93 94
2.2. Chemical composition analysis of soybean okara and its solid-state fermentation
95
The moisture, protein, fat, fiber and ash of okara were detected according to the
96
methods described by AOAC (2005). The phenol-sulfuric acid method was used for
97
soluble sugar determination as we previously described (Zheng, Wei, et al., 2018).
98
The preparation of inoculum and SSF of okara were carried out as previously
99
described (P. Liu et al., 2016). Briefly, dried okara (10 g) and distilled water were
100
added to Erlenmeyer flasks, which were sterilized at 121°C for 20 min. Spore
101
suspension was subsequently inoculated after cooling. After fermentation, the matter
102
was mixed with distilled water (1:6, w/v) by shaking on a rotary shaker. The whole 5
103
contents were then centrifuged at 12000 × g for 20 min, and the supernatant was used
104
as crude enzyme extract. For peptide determination, an equal volume of 10% (w/v)
105
trichloroacetic acid was added, followed by mixing and a 10-min standing to
106
precipitate the proteins. After centrifugation at 4000 × g for 15 min, the supernatant
107
was collected and used for peptide measurement by the Biuret method with a Biuret
108
reagent kit (Nanjing Jiancheng Bioengineering Institute, China).
109 110
2.3. Optimization of fermentation conditions for protease and soluble protein
111
production
112
The effects of culture conditions, including water amount, initial pH, incubation
113
temperature, fermentation time, and incubation amount of fungus, were determined by
114
single factor method. Furthermore, the most effective factors were optimized by
115
Box-Behnken design of response surface methodology (J. Li et al., 2019). The
116
response data obtained based on the above design on protease activity or soluble
117
protein content in crude enzyme extract were fitted to the following second-order
118
polynomial equation: =
+
+
+
119
, where Y, b0, bi, bij, and bii are the predicted response, intercept term, linear
120
coefficient, interaction coefficient, and squared coefficient, respectively, and Xi and Xj
121
are coded independent variables. The response surface and contour plots of the
122
predicted responses were used to estimate the optimal value of each parameter. The
123
crude enzyme extract under the optimal culture conditions was used for further 6
124
protease purification by chromatography.
125 126
2.4. Determination of soluble protein content and protease activity
127
Soluble protein content was quantitatively determined by a Bio-Rad Protein Assay
128
Kit with bovine serum albumin (BSA) as the standard. Protease activity was
129
determined using casein as the substrate based on the national professional standard
130
method. Briefly, the crude enzyme or purified protease was incubated with casein at
131
the concentration of 2 g/100 mL for 10 min at 40°C, and the reaction was
132
subsequently terminated by 0.4 mol/L of trichloroacetic acid. The supernatant
133
obtained by centrifugation at 10000 × g for 5 min was incubated with sodium
134
carbonate and Folin reagent for 20 min at 40°C. The absorbance was measured at 660
135
nm. A standard curve was generated by using 0, 10, 20, 30, 40 and 50 µg/mL of
136
tyrosine, and one unit of protease activity was defined as 1 µg of tyrosine equivalent
137
released per mL per min.
138 139
2.5. Protease purification
140
2.5.1. Ammonium sulfate precipitation
141
The crude enzyme extract was fractionated in 20% saturated (NH4)2SO4 solution to
142
remove hybrid proteins. The supernatant was collected by centrifugation at 10000 × g.
143
Subsequently, (NH4)2SO4 at 70% saturation was used to fractionate the supernatant
144
for the second time. The pellets were obtained and re-dissolved by the addition of five
145
volumes of distilled water. After dialysis with distilled water, the enzyme-containing 7
146
solution was lyophilized.
147 148
2.5.2.
Diethylaminoethyl
149
chromatography
(DEAE)-Sepharose
Fast
Flow
anion-exchange
150
After lyophilization, DEAE-sepharose Fast Flow anion exchange column (2.16 ×
151
12.6 cm) was used to further purify the protease. Briefly, the column pre-equilibrated
152
with 50 mmo/L Tris-HCl buffer (pH 7.5) was stepwise eluted with 0–0.7 mol/L of
153
NaCl at a flow rate of 4.0 mL/min. 8 mL of each fraction was collected and assayed
154
for protease activity and protein content.
155 156
2.5.3. Sephadex G-75 gel filtration chromatography
157
After anion-exchange chromatography, the fraction with the highest protease
158
activity was subjected to gel filtration on a Sephadex G-75 column (1.6 × 60 cm),
159
which was equilibrated with 50 mmo/L Tris-HCl buffer (pH 8.5). 4 mL of each
160
fraction was collected at a flow rate of 0.4 mL/min. The purified protein in the
161
fraction with the highest protease activity was freeze-dried and stored at −80°C.
162 163 164
2.6. Molecular weight and zymography determination The molecular weight was determined by sodium dodecyl sulphate-polyacrylamide
165
gel electrophoresis (SDS-PAGE) (Zheng, Yu, et al., 2018). The gel was stained with
166
0.1% Coomassie brilliant blue R-250 and de-stained until clear protein bands were
167
achieved. The molecular weight was estimated using the standard protein marker 8
168
(10–180 kDa, Bio-Rad Laboratories).
169
For zymography analysis, the gel was incubated in 50 mmol/L Tris-HCl buffer (pH
170
8.5) containing 0.2% Triton X-100 after SDS-PAGE. After being washed with
171
Tris-HCl buffer to remove Triton X-100, the gel was incubated with 2% (w/v) casein
172
for 20 min at 50°C (Haddar, Bougatef, Agrebi, Sellami-Kamoun, & Nasri, 2009). The
173
protein in gel was then stained with Coomassie brilliant blue R-250.
174 175
2.7. Protease characterization
176
2.7.1. Effects of temperature and pH on protease activity and stability
177
The optimum temperature for protease was determined as described by the standard
178
enzyme assay at different temperatures (30–70°C). The optimum pH was measured at
179
various pH values ranging from 4.0 to 11.0 under the optimum temperature with the
180
following
181
Na2HPO4–NaH2PO4 buffer (pH 6.0–8.0) and borax-boric acid buffer (pH 9.0–11.0).
buffers:
sodium
acetate-acetic
acid
buffer
(pH
4.0–5.0),
182
To assess the temperature stability, the protease was pre-incubated at different
183
temperatures from 30°C to 65°C for 20–120 min. The pH stability was investigated by
184
incubating the protease with different pH buffers at 25°C for 2–10 h. The remaining
185
protease activity was then determined by the standard enzyme assay and expressed as
186
the percentage to the activity measured before pre-incubation.
187 188 189
2.7.2. Effect of metal ions on protease activity The effects of various metal ions including Ca2+, Cu2+, Co2+, Mg2+, Zn2+, and Fe2+ 9
190
at 5 and 10 mmol/L on protease activity were investigated. The protease was
191
pre-incubated with individual metal ions at room temperature for 1 h, and the
192
remaining protease activity was then determined by the standard enzyme assay. The
193
enzyme without metal ions was taken as 100%.
194 195
2.7.3. Effect of organic solvents on protease activity
196
The effects of different organic solvents at the concentrations of 1% and 10% were
197
determined by adding methanol, ethanol, acetone, acetonitrile, and isopropanol to the
198
reaction mixture. The remaining protease activity was then determined by the
199
standard enzyme assay. The enzyme without any additives was considered as 100%.
200 201
2.7.4. Effect of surfactants on protease activity
202
The stability of the protease in different surfactants (SDS, Tween-80, Span and
203
Triton X-100) was also studied. The protease was pre-incubated with individual
204
surfactants at the concentration of 2 mmol/L for 1 h at room temperature, and the
205
remaining protease activity was then determined under the standard assay conditions.
206
The enzyme without surfactants was taken as 100%.
207 208
2.7.5. Effect of inhibitors on protease activity
209
The protease was premixed with each of the following inhibitors at room
210
temperature for 1 h. The effects of different inhibitors (1 mmol/L) on the enzyme
211
were assayed, including phenylmethylsulfonyl fluoride (PMSF) for serine proteases, 10
212
ethylenediaminetetraacetic acid (EDTA) for metalloproteases, pepstatin A for aspartic
213
proteases, and iodacetamide (IAM) for cysteine proteases. The enzyme without any
214
inhibitors was considered as 100%.
215 216
2.8. Determination of substrate specificity and kinetic parameters
217
To determine the substrate specificity of pure protease, casein, BSA, gelatin, and
218
hemoglobin (1%, w/v) were used as the substrates. The enzyme activity was detected
219
by the standard enzyme assay. The kinetic parameters were calculated by using casein
220
as the substrate at the concentrations of 0–20 mg/mL. The kinetic parameters Km and
221
Vmax were estimated using Lineweaver-Burk plot (Anitha & Palanivelu, 2013).
222 223 224
2.9. Mass spectrometry and protein identification The band of homogeneous protease was excised from the SDS-PAGE gel and
225
digested
with
trypsin.
The
protease
226
chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)
227
as described by Chen et al. (2016). MS data were obtained using Q-Exactive (Thermo
228
Finnigan, San Jose, CA). MS/MS spectra were analyzed using the MASCOT search
229
engine against the fungal subset of NCBI non-redundant protein sequence database
230
(NCBInr) and SwissProt database. Mass tolerances in MS and MS/MS modes were 20
231
ppm and 0.1 Da. For MASCOT search, matches achieving a molecular weight search
232
score ≥20 were considered as significant.
233 11
was
then
identified
using
liquid
234
2.10. Statistical analysis
235
The values were expressed as means ± standard deviation of three independent
236
replicates. Differences between the mean values were analyzed using one-way
237
ANOVA followed by Duncan test, and P < 0.05 was considered as significant.
238 239
3. Results
240
3.1. Chemical composition of okara and its peptide production by solid-state
241
fermentation
242
The chemical composition of soybean okara was as follows (g/100 g dry matter):
243
protein, 14.94±0.30; fat, 2.18±0.29; fiber, 39.29±1.02; soluble sugar, 4.23±0.07; and
244
ash, 3.53±0.04. The overall chemical composition suggested that soybean okara is a
245
suitable substrate for microbial fermentation. Further SSF of okara with Neurospora
246
crassa resulted in the production of peptides, as confirmed by the disappearance of
247
two major proteins β-conglycinin and glycinin (Figure 1A), as well as a remarkable
248
increase of peptide content in okara after 24 h, 48 h and 72 h of fermentation (P <
249
0.05; Figure 1B). Interestingly, novel proteins were yielded after 48 h and 72 h of
250
fermentation (Figure 1A). Taken together, it can be speculated that SSF of okara with
251
Neurospora crassa can produce proteases and thus contribute to the production of
252
biopeptides.
253 254 255
3.2. Optimal fermentation conditions In order to isolate and purify the protease produced from SSF of okara with 12
256
Neurospora crassa, we firstly optimized the fermentation conditions for protease
257
production. As presented in Figure 2, almost similar curve patterns were observed for
258
both protease activity and soluble protein content, which were significantly affected
259
by all the designed culture conditions (P < 0.05). Based on single factor experiment,
260
amount of added water, initial pH, and incubation temperature were the most
261
influential factors. Thus, these three factors were further optimized using
262
Box-Behnken design. By multiple regression analysis on the experimental data, the
263
following second-order polynomial equation was obtained to describe the protease
264
activity (Y1, U/g) and soluble protein content (Y2, mg/g): = 1960.47 + 131.93 − 40.52 = 10.00 + 0.36
− 26.18 − 167.11
− 0.061
− 0.30
+ 133.85
− 1.18
− 478.28
+ 0.41
+ 0.10
+ 72.07
− 104.55
− 1153.56 − 0.37
− 0.17
− 3.69
265
, where X1, X2, and X3 are the amount of added water, initial pH, and incubation
266
temperature, respectively.
267
The response surface and contour plots generated using the above regression
268
equations are presented in Figure 3. The model predicted that the maximal protease
269
production (1988.39 U/g) was achieved at the amount of added water 20.50 mL,
270
initial pH 5.0, and incubation temperature 30°C. The optimal conditions for the
271
maximal soluble protein content (10.11 mg/g) were determined to be: amount of water
272
added 20.80 mL, initial pH 5.0, and incubation temperature 30°C. Thus, for the
273
convenience of experimental operation, the fermentation conditions were optimized as 13
274
follows according to the single factor tests and response surface analysis: okara 10 g,
275
amount of added water 21 mL, initial pH 5.0, incubation temperature 30°C,
276
inoculation amount 2 mL, and fermentation time 72 h. Under these conditions, the
277
protease activity and soluble protein content reached 1959.82 U/g and 9.99 mg/g,
278
respectively, which were very close to the predicted values. Thus, the model for the
279
optimization of both protease activity and soluble protein content was satisfactory and
280
practicable.
281 282
3.3. Extraction and purification of protease
283
Under the above-mentioned optimal conditions, a crude enzyme solution with
284
protease activity of 195.21 U/mg protein was further purified. During the elution from
285
the DEAE-Sepharose column, five protein peaks were observed (Figure 4A). Protease
286
activity was detected in the first peak, which corresponded to fractions 2–15, with the
287
highest protease activity being detected in fraction 5. Fractions 4–6 were collected,
288
pooled and then subjected to Sephadex G-75 gel filtration chromatography. Fractions
289
14–15 exhibited strong protease activities (Figure 4B) and were pooled to constitute
290
the final sample of purified protease. The purification process is summarized in Table
291
1. The enzyme was purified 28.27-fold with a recovery rate of 31.0%, and the specific
292
activity was enhanced to 5518.37 U/mg protein.
293
With the purification steps, the bands of proteins were reduced or disappeared
294
(Figure 5A). The finally obtained enzyme showed a high purity as confirmed by the
295
presence of a single band, with a molecular weight of approximately 30 kDa (Figure 14
296
5B). Furthermore, only one clear zone was found on SDS-PAGE by zymographic
297
analysis of the purified enzyme, indicating the presence of protease.
298 299
3.4. Identification of the purified protease by LC-ESI-MS/MS analysis
300
The 30 kDa band from SDS-PAGE was subjected to LC-ESI-MS/MS analysis. The
301
acquired data were compared with those in NCBInr and SwissProt database. Five
302
identified proteins are presented in Table 2. The purified protein showed significant
303
matches with the three already characterized proteins, including carbohydrate esterase
304
family 1 protein, carbohydrate-binding module family 1 protein, and endo-1,
305
4-beta-xylanase B precursor, which do not belong to proteases. Moreover, an
306
uncharacterized protein (accession No. CAD70564.1) and acetyl xylan esterase in the
307
databases exhibited little similarities to the purified protein, indicating that it is most
308
likely a novel protease.
309 310
3.5. Effects of temperature and pH on protease activity and stability
311
The purified enzyme was the most active at the temperature of 50–60°C, with a
312
significantly higher activity at the temperature of 55°C (P < 0.05; Figure 6A). The
313
protease activity decreased nearly linearly at temperatures below 55°C and above 60°C
314
(P < 0.05). As shown in Figure 6B, the protease activity significantly increased with
315
increasing pH, reached the maximum at pH 9, and then decreased with further
316
increase of the pH to 11 (P < 0.05). Additionally, the protease activity under alkali
317
conditions (pH 8–11) was significantly higher than that under acidic and medium 15
318
conditions (pH 4–7) (P < 0.05), indicating that it is an alkaline protease.
319
Thermostability studies revealed that the enzyme was relatively stable at the
320
temperature of 30–45°C, with a slight decrease in protease activity with increasing
321
pre-incubation time (Figure 6C). Besides, the protease was stable over a broad range
322
of pH from 6 to 10; it retained >90% of its original activity after 2 h of pre-incubation,
323
and >50% of original activity even after 10 h of pre-incubation in this pH range
324
(Figure 6D). These results also confirmed that the purified enzyme belongs to the
325
alkaline protease family.
326 327
3.6. Effects of metal ions, organic solvents, surfactants and inhibitors on protease
328
activity
329
The protease activity was increased by 5 mmol/L of Ca2+, but inhibited by all other
330
metal ions, especially by 5 mmol/L of Cu2+, with the enzyme retaining only 39% of its
331
original activity (P < 0.05; Figure 7A). The organic solvents had little effect on the
332
protease activity (Figure 7B). Methanol at 1% and 10%, ethanol at 10%, and acetone
333
at 10% only marginally inhibited protease activity (1–3%; P > 0.05). Surfactants,
334
namely Tween-80, Span and Triton X-100, showed negligible effects on the protease
335
activity of the purified enzyme (P > 0.05; Figure 7C), indicating that these surfactants
336
are not required for the activation of the enzyme. However, SDS dramatically
337
inhibited the protease activity by 27% (P < 0.05). Further inhibition studies
338
demonstrated that the purified protease was not a metalloprotease, aspartic protease or
339
cysteine protease, because its activity was not suppressed by EDTA, pepstatin A and 16
340
IAM (Figure 7D). However, the typical serine protease inhibitor PMSF completely
341
inhibited the protease activity (P < 0.05), implying that the purified enzyme belongs
342
to serine protease family.
343 344
3.7. Substrate specificity and kinetic parameters
345
As shown in Figure 8A, the purified enzyme exhibited particularly high protease
346
activity towards casein and moderate activity towards gelatin and hemoglobin, but
347
very low activity towards BSA (only ∼5%; P < 0.05). The kinetic parameters were
348
then calculated using casein as the substrate, and the enzyme showed a
349
Michaelis-Menten type of kinetics (Figure 8B). The kinetic parameters including Km
350
and Vmax values were estimated to be respectively 2.18 mg/mL and 36.36 µg/mL/min
351
using linearized Lineweaver-Burk plot.
352 353
4. Discussion
354
The characteristics of high stability, low production cost, and specificity make
355
microbial proteases highly valuable candidates in food processing, particularly in the
356
preparation of protein hydrolysates with biopeptides (Chew et al., 2019; dos Santos
357
Aguilar & Sato, 2018). Currently, there is a high industrial demand for proteases from
358
fungal sources because of their stability, broad diversity, and substrate specificity
359
(Banerjee & Ray, 2017). Among various fungi, Neurospora crassa has an interesting
360
advantage of high yields of carotenoids and cellulases (P. Liu et al., 2016; Zhou et al.,
361
2019), and thus represents a powerful tool in functional food production. In the 17
362
present study, the newly isolated Neurospora crassa CGMCC3088 from Meitauza can
363
contribute to a high yield of proteases using soybean okara as the substrate, and
364
thereby enhance the yield of biopeptides (Figure 1). An enzyme with a very high
365
protease activity was purified by chromatography, and further characterization and
366
mass spectrometry analysis strongly indicated that the enzyme is a novel organic
367
solvent-stable alkaline serine protease, which could be used in food applications.
368
In the late 20th century, an alkaline protease was firstly produced and purified from
369
Neurospora crassa using submerged fermentation (Lindberg, Eirich, Price,
370
Wolfinbarger, & Drucker, 1981). However, the research was mainly focused on the
371
production of cellulases and carotenoids from Neurospora crassa in the following
372
years until now, and there has been no report about its application to protease
373
production. Neurospora crassa CGMCC3088 was newly isolated by our laboratory
374
from Jiangxi Meitauza, and it was shown to be a mutant strain with enhanced
375
production capacity of cellulases and carotenoids (P. Liu et al., 2016). To the best of
376
our knowledge, this is the first report of isolation and purification of a protease from
377
SSF of okara with this mutant strain. The specific activity of the purified protease was
378
significantly increased compared with that reported by Lindberg et al. (1981)
379
(5518.37 vs. 1220 U/mg), and was even higher than that of commercial microbial
380
proteases including alcalase, neutrase and flavourzyme (Bao, Zhao, Wang, & Chi,
381
2017; da Silva & de Castro, 2018). SSF and submerged fermentation are the two main
382
methods for the production of microbial proteases. Although submerged fermentation
383
is often applied for industrial protease production, SSF was found to contribute to a 18
384
higher activity of proteases (Machado de Castro, Fragoso dos Santos, Kachrimanidou,
385
Koutinas, & Freire, 2018), possibly because more carbon sources including sucrose
386
and starch are used for energy transformation during the SSF process, and then more
387
amino acids are biosynthesized for protein production (Zhao et al., 2019). Thus, SSF
388
obviously outperforms submerged fermentation in microbial protease production.
389
Indeed, owing to the high protease production, SSF with microorganisms has been
390
successfully used to efficiently hydrolyze legume proteins to small pepetides and
391
amino acids that are easy to be absorbed, and thereby improve the nutritional
392
composition of the final products (Gupta et al., 2018).
393
Further characterization of enzymatic properties showed that the purified protease
394
belongs to the alkaline protease family (Figure 6). Notably, among different types of
395
proteases, alkaline protease is the most commonly used enzyme in food industry
396
because of its high activity and stability at highly alkaline pH (Guleria, Walia,
397
Chauhan, & Shirkot, 2016; Thakur, Kumar, Sharma, Bhalla, & Kumar, 2018).
398
Commercial alkaline proteases are primarily isolated from the Bacillus species
399
(Contesini, Melo, & Sato, 2018). The fungus in our study was isolated from naturally
400
fermented food, and its safe and non-toxic nature makes it a new ideal microbial
401
strain for producing alkaline proteases. Additionally, most of proteases tend to be
402
inactivated and unstable under the treatment of organic solvents, which severely limits
403
their applications (Doukyu & Ogino, 2010; Si, Jang, Charalampopoulos, & Wee,
404
2018). Recently, various attempts have been made to screen enzymes with natural
405
organic solvent tolerance (de Borba, Machado, Brandelli, & Kalil, 2018; Thakur et al., 19
406
2018). Our purified protease from Neurospora crassa CGMCC3088 remained active
407
in the presence of various organic solvents (Figure 7B). Therefore, Neurospora crassa
408
CGMCC3088 is expected to have promising applications in the production of organic
409
solvent-tolerant proteases and functional foods with high contents of carotenoids and
410
biopeptides.
411 412
5. Conclusions
413
The extracellular alkaline protease produced from the SSF of soybean okara with
414
Neurospora crassa CGMCC3088 was demonstrated to have several superior
415
properties of high industrial values, including alkaline pH, good thermostability and
416
organic solvent tolerance. Importantly, peptide production was observed in soybean
417
okara after SSF with Neurospora crassa, indicating the great application potential of
418
this novel protease in the production of protein hydrolysates with bioactivities.
419
However, further research is needed to apply the purified protease to enzymatic
420
hydrolysis for the production of protein hydrolysates. Besides, it is also necessary to
421
explore the structure-function relationship by using three-dimensional structure
422
modeling and site-directed mutagenesis.
423 424
Acknowledgments
425
This work was supported financially by the Research Program of State Key
426
Laboratory of Food Science and Technology, Nanchang University (Grant Number:
427
SKLF-ZZA-201610). 20
428 429 430
Conflict of interests The authors declare no competing financial interests.
431 432
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433
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Sultan, S., Huma, N., Butt, M. S., Aleem, M., & Abbas, M. (2018). Therapeutic potential of
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Thakur, N., Kumar, A., Sharma, A., Bhalla, T. C., & Kumar, D. (2018). Purification and
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characterization of alkaline, thermostable and organic solvent stable protease from a
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517 518
Vong, W. C., & Liu, S.-Q. (2016). Biovalorisation of okara (soybean residue) for food and nutrition. Trends in Food Science & Technology, 52, 139-147.
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Zhao, G., Ding, L.-L., Pan, Z.-H., Kong, D.-H., Hadiatullah, H., & Fan, Z.-C. (2019).
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Proteinase and glycoside hydrolase production is enhanced in solid-state fermentation
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by manipulating the carbon and nitrogen fluxes in Aspergillus oryzae. Food
522
Chemistry, 271, 606-613.
523
Zheng, L., Wei, H., Yu, H., Xing, Q., Zou, Y., Zhou, Y., & Peng, J. (2018). Fish skin gelatin
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hydrogen peroxide induced intestinal oxidative stress via the pept1-p62-Nrf2 cascade.
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Journal of Agricultural and Food Chemistry, 66, 11601-11611.
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Zheng, L., Yu, H., Wei, H., Xing, Q., Zou, Y., Zhou, Y., & Peng, J. (2018). Antioxidative
528
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antioxidant response element-mediated gene transcription in IPEC-J2 cells. Journal
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of Functional Foods, 51, 104-112.
531
Zhou, R., Ren, Z., Ye, J., Fan, Y., Liu, X., Yang, J., . . . Li, J. (2019). Fermented soybean dregs
532
by Neurospora crassa: a traditional prebiotic food. Applied Biochemistry and
533
Biotechnology, 1-18.
534
23
535
Table 1
536
Purification of protease from the solid-state fermentation of soybean okara with
537
Neurospora crassa. Purification steps
Total activity (U)
Total protein
Specific
Purification
Recovery
(mg)
activity
fold
(%)
(U/mg protein) 74452.11±639.04a
381.39±4.75a
195.21±2.91d
1.00
100.00
51370.03±381.96b
169.78±2.46b
302.56±2.32c
1.55
69.00
DEAE Sepharose
45068.80±483.19c
25.14±0.21c
1792.93±15.86b
9.18
60.53
Sephadex G-75
23081.14±102.86d
4.18±0.05d
5518.37±35.56a
28.27
31.00
Crude extract Ammonium
sulfate
precipitation
538
Means with different letters within the same column differ significantly at P < 0.05.
539
24
540
Table 2
541
Identification of the purified protease from the solid-state fermentation of soybean
542
okara with Neurospora crassa.
Accession number
Protein name
Unique peptides
KHE83333.1
Carbohydrate esterase family 1 protein (Neurospora crassa)
KHE83062.1
Carbohydrate-binding module family 1 protein (Neurospora crassa)
CAD71059.1
Endo-1, 4-beta-xylanase B precursor (Neurospora crassa) Uncharacterized protein (Neurospora crassa) Acetyl xylan esterase (Neurospora crassa)
K.QWSNVLGVEFSR.N K.YNADASR.V R.CAM*EALK.Q R.CAMEALK.Q R.GLQHTPEEWGNFVR.N R.GLQHTPEEWGNFVR.N R.M*YTYVPDK.L R.MYTYVPDK.L R.NSYPGYTGR.R R.NSYPGYTGR.R K.IFEDTWAK.K K.IPSDIPAGDYLLR.A K.IPSDIPAGDYLLR.A K.KPSSSSGDDDFWGVK.D K.KPSSSSGDDDFWGVK.D K.GWM*PGTDR.T K.NHFDAWTR.S R.LGSVTSDGGVYDIYR.T
CAD70564.1
EAA29891.1
Sequence coverage (%)
Theoretical mass (kDa)
19.52
31.08
8.47
10.91
32.86
6.89
10.58
30.78
8.47
R.QGTNAVATAVNSLNAR.C
5.33
30.41
7.83
K.LVGVYAR.G R.GVGHSVPIR.G R.GVGHSVPIR.G
5.13
33.43
6.74
543
25
pI
544
Figure captions
545
Figure 1. Protein electrophoresis profiles (A) and peptide contents (B) of soybean
546
okara before and after the solid-state fermentation with Neurospora crassa. Arrows 1,
547
2 and 3 indicate α’-, α- and β-subunits of β-conglycinin, respectively. Arrows 4 and 5
548
indicate acidic and basic subunits of glycinin, respectively. Peptide contents are
549
expressed as mg of glutathione (GSH) equivalents/g dry weight (DW), and means
550
with different letters differ significantly at P < 0.05.
551 552
Figure 2. Effects of different culture conditions on protease activity and soluble
553
protein content from the solid-state fermentation of soybean okara with Neurospora
554
crassa. Culture conditions including the amount of added water (A), initial pH (B),
555
incubation temperature (C), fermentation time (D), and inoculation amount (E).
556
Means with different letters differ significantly at P < 0.05.
557 558
Figure 3. Surface and contour plot showing the interactive effects of different culture
559
conditions on protease activity and soluble protein content from the solid-state
560
fermentation of soybean okara with Neurospora crassa. (A and D) Amount of added
561
water and initial pH. (B and E) Amount of added water and incubation temperature.
562
(C and F) Initial pH and incubation temperature.
563 564
Figure 4. Chromatographic purification profile of proteases from the solid-state
565
fermentation of soybean okara with Neurospora crassa. (A) Chromatographic 26
566
purification profile of proteases on DEAE-Sepharose fast flow anion exchange
567
column. (B) Chromatographic purification profile of proteases on Sephadex G-75 gel
568
filtration chromatography.
569 570
Figure 5. Protein electrophoresis profile of different fractions of proteases from the
571
solid-state fermentation of soybean okara with Neurospora crassa. (A) SDS-PAGE
572
profile of the purified protease obtained after DEAE-Sepharose fast flow
573
anion-exchange
574
anion-exchange chromatography; line 2, crude supernatant. (B) SDS-PAGE profile
575
(line 1) and zymography (line 2) of purified protease obtained after Sephadex G-75
576
gel filtration chromatography.
chromatography.
Line
1,
purified
protease
obtained
after
577 578
Figure 6. Effects of temperature and pH on enzyme activity (A and B) and stability
579
(C and D) of the purified protease from the solid-state fermentation of soybean okara
580
with Neurospora crassa. Means with different letters differ significantly at P < 0.05.
581 582
Figure 7. Effects of metal ions (A), organic solvents (B), surfactants (C), and
583
inhibitors (D) on the activity of purified protease from the solid-state fermentation of
584
soybean okara with Neurospora crassa. Means with different letters differ
585
significantly at P < 0.05.
586 587
Figure 8. (A) Substrate specificity of the purified protease from the solid-state 27
588
fermentation of soybean okara with Neurospora crassa. Means with different letters
589
differ significantly at P < 0.05. (B) Lineweaver-Burk plot. Enzyme kinetic parameters
590
for hydrolysis of casein by the purified protease were determined using
591
Lineweaver-Burk plot.
592 593
594 595 596
Figure 1
28
597 598
Figure 2
599
29
600 601
Figure 3
602
30
603 604
Figure 4
605
31
606 607 608
Figure 5
32
609 610
Figure 6
611
33
612 613
Figure 7
614
34
615 616
Figure 8
35
Highlights 1. Neurospora crassa CGMCC3088 can be treated as a novel functional fermentation organism. 2. Fermentation conditions were optimized for protease producing of Neurospora crassa CGMCC3088. 3. A novel organic solvent-stable alkaline protease was purified from Neurospora crassa CGMCC3088. 4. The purified protease from Neurospora crassa CGMCC3088 can hydrolyze casein, gelatin and hemoglobin.
Conflict of interest The authors have declared that no competing interests exist.
S0023-6438(19)31332-5
DOI:
https://doi.org/10.1016/j.lwt.2019.108990
Reference:
YFSTL 108990
To appear in:
LWT - Food Science and Technology
Received Date: 2 September 2019 Revised Date:
22 December 2019
Accepted Date: 24 December 2019
Please cite this article as: Zheng, L., Yu, X., Wei, C., Qiu, L., Yu, C., Xing, Q., Fan, Y., Deng, Z., Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation, LWT - Food Science and Technology (2020), doi: https:// doi.org/10.1016/j.lwt.2019.108990. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Author Contributions Liufeng Zheng and Zeyuan Deng designed the experiments, interpreted the results, and wrote the manuscript. Xinying Yu, Changhao Wei, Chenwei Yu, and Leyun Qiu conducted the experiments and prepared the manuscript. Qian Xing and Yawei Fan revised the manuscript.
1
Production and characterization of a novel alkaline protease from a
2
newly isolated Neurospora crassa through solid-state fermentation
3 4
Liufeng Zhenga,†, Xinying Yua,†, Changhao Weia, Leyun Qiua, Chenwei Yua, Qian
5
Xinga, Yawei Fana, Zeyuan Denga,b,*
6
a
7
Nanchang 330047, Jiangxi, China
8
b
9
China
State Key Laboratory of Food Science and Technology, Nanchang University,
Institute for Advanced Study, University of Nanchang, Nanchang 330031, Jiangxi,
10 11
*
Correspondence to: Zeyuan Deng. E-mail address: [email protected]
†
L. Zheng and X. Yu contributed equally to this work.
12 13 14
1
15
ABSTRACT
16
Microbial proteases are widely used to prepare protein hydrolysates with
17
health-promoting biopeptides. Here, a newly isolated strain of Neurospora crassa
18
(named as CGMCC3088) was used to produce proteases through solid-state
19
fermentation of okara as the substrate. The optimal fermentation conditions are: okara,
20
10 g; water, 21 mL; initial pH, 5.0; incubation temperature, 30°C; inoculation amount,
21
2 mL; fermentation time, 72 h, with a corresponding protease activity of 1959.82 U/g.
22
The protease was further purified by ammonium sulphate precipitation, followed by
23
ion-exchange chromatography on DEAE-Sepharose and Sephadex G-75. The
24
molecular weight of the protease was 30 kDa, and further mass spectrometry analysis
25
clearly indicated that it was a novel protease. The protease had the optimal activity at
26
55°C and pH 9. The enzyme activity was partially inhibited by SDS and metal ions,
27
whereas little affected by organic solvents. The protease was completely inactivated
28
by phenylmethylsulfonyl fluoride, indicating its dominant serine protease activity. The
29
enzyme preferably hydrolyzed casein, and kinetic analysis showed that its Km and
30
Vmax were 2.18 mg/mL and 36.36 µg/mL/min, respectively. Therefore, Neurospora
31
crassa CGMCC3088 has the potential to produce a novel organic solvent-stable
32
alkaline protease, which may be applied to the preparation of bioactive ingredients.
33 34
Keywords: Neurospora crassa; Solid-state fermentation; Fermentation conditions;
35
Alkaline protease; Purification.
36 2
37
1. Introduction
38
Proteases are a large category of enzymes and account for approximately 60% of
39
total global enzyme production (Li, Scott, Hemar, Zhang, & Otter, 2018). It has been
40
well documented that proteases can efficiently hydrolyze proteins to biopeptides,
41
which have excellent antioxidant, anti-inflammatory and anti-microbial activities
42
(Cermeno et al., 2019; Sultan, Huma, Butt, Aleem, & Abbas, 2018), and may help to
43
prevent various chronic diseases such as obesity and cardiovascular diseases (Cicero,
44
Fogacci, & Colletti, 2017). Currently, proteases of microbial origin have been widely
45
applied to the production of protein hydrolysates with bioactivities owing to their low
46
cost, high stability and specificity (Chew, Toh, & Ismail, 2019; dos Santos Aguilar &
47
Sato, 2018). However, more novel microbial proteases are still needed from natural
48
resources despite of the currently available commercial proteases.
49
Microorganisms in naturally fermented foods are an attractive source of proteases
50
due to their non-toxic nature and functional properties (Tamang, Shin, Jung, & Chae,
51
2016). Meitauza, a traditional fermented food produced from soybean residue, serves
52
as a health functional food and is widely consumed by Chinese people because of its
53
pleasant flavor and high biopeptide contents (Vong & Liu, 2016). Notably, protease
54
activity, which increases gradually with ripening, is one of the most important factors
55
that determine the quality of Meitauza (Liu, Han, Deng, Sun, & Chen, 2018). Since no
56
protease activity can be detected in the raw material, it can be speculated that
57
microorganisms used in the production of Meitauza can yield proteases. Therefore,
58
Meitauza may be an ideal material for exploring novel microorganisms with a high 3
59
yield of proteases. In our previous work, a kind of fungus, Neurospora crassa
60
CGMCC3088, was firstly isolated from Meitauza. This fungus can produce cellulases
61
to effectively degrade fiber, and has a strong ability to yield carotenoids with potential
62
benefits and nutrition to human health (P. Liu, Li, & Deng, 2016). More recently, our
63
group demonstrated that the nutritional quality and antioxidant activity of soybean
64
meal can be significantly improved through fermentation using this newly isolated
65
fungus (J. Li et al., 2019). Therefore, Neurospora crassa CGMCC3088 can be
66
regarded as a novel functional micoorganism. However, the ability of Neurospora
67
crassa CGMCC3088 to produce proteases has never been reported before.
68
Solid-state fermentation (SSF) is an important way for the production of enzymes.
69
Production of enzymes by SSF with agroindustrial wastes as the substrates has
70
attracted great interests due to its inherent advantages including low cost, high yield
71
and environmental friendliness (Leite, Silva, Salgado, & Belo, 2019; Sadh, Duhan, &
72
Duhan, 2018). Okara, also known as soybean residue and the major insoluble
73
byproduct from the production of soy milk and tofu, is largely under-utilized in food
74
industry. As an agroindustrial waste, okara has high nutritional values with abundant
75
dietary fiber, protein, lipid, vitamin, mineral, and isoflavone (B. Li et al., 2019).
76
Therefore, it is a promising and safe substrate in biotransformation process. Actually,
77
okara has been successfully used as an excellent substrate for SSF to improve its
78
nutritional composition and antioxidant activity (Gupta, Lee, & Chen, 2018).
79
However, there has been no report on the application of SSF to produce proteases
80
using okara as the substrate. 4
81
Therefore, this study aims to optimize the fermentation conditions for achieving the
82
maximum production of proteases from Neurospora crassa CGMCC3088 under SSF
83
of okara. Then, we performed the purification and characterization of a novel
84
extracellular alkaline protease, which can serve as a potential biocatalyst in food
85
processing, particularly in the preparation of biopeptide-enriched hydrolysates.
86 87
2. Materials and methods
88
2.1. Materials
89
Neurospora crassa CGMCC3088 was newly isolated from Meitauza by our lab (P.
90
Liu et al., 2016). The okara was obtained from a local supermarket. Chromatographic
91
columns used for protease purification were purchased from Amersham Biosciences
92
(Freiburg, Germany). All chemicals were of chemical grade.
93 94
2.2. Chemical composition analysis of soybean okara and its solid-state fermentation
95
The moisture, protein, fat, fiber and ash of okara were detected according to the
96
methods described by AOAC (2005). The phenol-sulfuric acid method was used for
97
soluble sugar determination as we previously described (Zheng, Wei, et al., 2018).
98
The preparation of inoculum and SSF of okara were carried out as previously
99
described (P. Liu et al., 2016). Briefly, dried okara (10 g) and distilled water were
100
added to Erlenmeyer flasks, which were sterilized at 121°C for 20 min. Spore
101
suspension was subsequently inoculated after cooling. After fermentation, the matter
102
was mixed with distilled water (1:6, w/v) by shaking on a rotary shaker. The whole 5
103
contents were then centrifuged at 12000 × g for 20 min, and the supernatant was used
104
as crude enzyme extract. For peptide determination, an equal volume of 10% (w/v)
105
trichloroacetic acid was added, followed by mixing and a 10-min standing to
106
precipitate the proteins. After centrifugation at 4000 × g for 15 min, the supernatant
107
was collected and used for peptide measurement by the Biuret method with a Biuret
108
reagent kit (Nanjing Jiancheng Bioengineering Institute, China).
109 110
2.3. Optimization of fermentation conditions for protease and soluble protein
111
production
112
The effects of culture conditions, including water amount, initial pH, incubation
113
temperature, fermentation time, and incubation amount of fungus, were determined by
114
single factor method. Furthermore, the most effective factors were optimized by
115
Box-Behnken design of response surface methodology (J. Li et al., 2019). The
116
response data obtained based on the above design on protease activity or soluble
117
protein content in crude enzyme extract were fitted to the following second-order
118
polynomial equation: =
+
+
+
119
, where Y, b0, bi, bij, and bii are the predicted response, intercept term, linear
120
coefficient, interaction coefficient, and squared coefficient, respectively, and Xi and Xj
121
are coded independent variables. The response surface and contour plots of the
122
predicted responses were used to estimate the optimal value of each parameter. The
123
crude enzyme extract under the optimal culture conditions was used for further 6
124
protease purification by chromatography.
125 126
2.4. Determination of soluble protein content and protease activity
127
Soluble protein content was quantitatively determined by a Bio-Rad Protein Assay
128
Kit with bovine serum albumin (BSA) as the standard. Protease activity was
129
determined using casein as the substrate based on the national professional standard
130
method. Briefly, the crude enzyme or purified protease was incubated with casein at
131
the concentration of 2 g/100 mL for 10 min at 40°C, and the reaction was
132
subsequently terminated by 0.4 mol/L of trichloroacetic acid. The supernatant
133
obtained by centrifugation at 10000 × g for 5 min was incubated with sodium
134
carbonate and Folin reagent for 20 min at 40°C. The absorbance was measured at 660
135
nm. A standard curve was generated by using 0, 10, 20, 30, 40 and 50 µg/mL of
136
tyrosine, and one unit of protease activity was defined as 1 µg of tyrosine equivalent
137
released per mL per min.
138 139
2.5. Protease purification
140
2.5.1. Ammonium sulfate precipitation
141
The crude enzyme extract was fractionated in 20% saturated (NH4)2SO4 solution to
142
remove hybrid proteins. The supernatant was collected by centrifugation at 10000 × g.
143
Subsequently, (NH4)2SO4 at 70% saturation was used to fractionate the supernatant
144
for the second time. The pellets were obtained and re-dissolved by the addition of five
145
volumes of distilled water. After dialysis with distilled water, the enzyme-containing 7
146
solution was lyophilized.
147 148
2.5.2.
Diethylaminoethyl
149
chromatography
(DEAE)-Sepharose
Fast
Flow
anion-exchange
150
After lyophilization, DEAE-sepharose Fast Flow anion exchange column (2.16 ×
151
12.6 cm) was used to further purify the protease. Briefly, the column pre-equilibrated
152
with 50 mmo/L Tris-HCl buffer (pH 7.5) was stepwise eluted with 0–0.7 mol/L of
153
NaCl at a flow rate of 4.0 mL/min. 8 mL of each fraction was collected and assayed
154
for protease activity and protein content.
155 156
2.5.3. Sephadex G-75 gel filtration chromatography
157
After anion-exchange chromatography, the fraction with the highest protease
158
activity was subjected to gel filtration on a Sephadex G-75 column (1.6 × 60 cm),
159
which was equilibrated with 50 mmo/L Tris-HCl buffer (pH 8.5). 4 mL of each
160
fraction was collected at a flow rate of 0.4 mL/min. The purified protein in the
161
fraction with the highest protease activity was freeze-dried and stored at −80°C.
162 163 164
2.6. Molecular weight and zymography determination The molecular weight was determined by sodium dodecyl sulphate-polyacrylamide
165
gel electrophoresis (SDS-PAGE) (Zheng, Yu, et al., 2018). The gel was stained with
166
0.1% Coomassie brilliant blue R-250 and de-stained until clear protein bands were
167
achieved. The molecular weight was estimated using the standard protein marker 8
168
(10–180 kDa, Bio-Rad Laboratories).
169
For zymography analysis, the gel was incubated in 50 mmol/L Tris-HCl buffer (pH
170
8.5) containing 0.2% Triton X-100 after SDS-PAGE. After being washed with
171
Tris-HCl buffer to remove Triton X-100, the gel was incubated with 2% (w/v) casein
172
for 20 min at 50°C (Haddar, Bougatef, Agrebi, Sellami-Kamoun, & Nasri, 2009). The
173
protein in gel was then stained with Coomassie brilliant blue R-250.
174 175
2.7. Protease characterization
176
2.7.1. Effects of temperature and pH on protease activity and stability
177
The optimum temperature for protease was determined as described by the standard
178
enzyme assay at different temperatures (30–70°C). The optimum pH was measured at
179
various pH values ranging from 4.0 to 11.0 under the optimum temperature with the
180
following
181
Na2HPO4–NaH2PO4 buffer (pH 6.0–8.0) and borax-boric acid buffer (pH 9.0–11.0).
buffers:
sodium
acetate-acetic
acid
buffer
(pH
4.0–5.0),
182
To assess the temperature stability, the protease was pre-incubated at different
183
temperatures from 30°C to 65°C for 20–120 min. The pH stability was investigated by
184
incubating the protease with different pH buffers at 25°C for 2–10 h. The remaining
185
protease activity was then determined by the standard enzyme assay and expressed as
186
the percentage to the activity measured before pre-incubation.
187 188 189
2.7.2. Effect of metal ions on protease activity The effects of various metal ions including Ca2+, Cu2+, Co2+, Mg2+, Zn2+, and Fe2+ 9
190
at 5 and 10 mmol/L on protease activity were investigated. The protease was
191
pre-incubated with individual metal ions at room temperature for 1 h, and the
192
remaining protease activity was then determined by the standard enzyme assay. The
193
enzyme without metal ions was taken as 100%.
194 195
2.7.3. Effect of organic solvents on protease activity
196
The effects of different organic solvents at the concentrations of 1% and 10% were
197
determined by adding methanol, ethanol, acetone, acetonitrile, and isopropanol to the
198
reaction mixture. The remaining protease activity was then determined by the
199
standard enzyme assay. The enzyme without any additives was considered as 100%.
200 201
2.7.4. Effect of surfactants on protease activity
202
The stability of the protease in different surfactants (SDS, Tween-80, Span and
203
Triton X-100) was also studied. The protease was pre-incubated with individual
204
surfactants at the concentration of 2 mmol/L for 1 h at room temperature, and the
205
remaining protease activity was then determined under the standard assay conditions.
206
The enzyme without surfactants was taken as 100%.
207 208
2.7.5. Effect of inhibitors on protease activity
209
The protease was premixed with each of the following inhibitors at room
210
temperature for 1 h. The effects of different inhibitors (1 mmol/L) on the enzyme
211
were assayed, including phenylmethylsulfonyl fluoride (PMSF) for serine proteases, 10
212
ethylenediaminetetraacetic acid (EDTA) for metalloproteases, pepstatin A for aspartic
213
proteases, and iodacetamide (IAM) for cysteine proteases. The enzyme without any
214
inhibitors was considered as 100%.
215 216
2.8. Determination of substrate specificity and kinetic parameters
217
To determine the substrate specificity of pure protease, casein, BSA, gelatin, and
218
hemoglobin (1%, w/v) were used as the substrates. The enzyme activity was detected
219
by the standard enzyme assay. The kinetic parameters were calculated by using casein
220
as the substrate at the concentrations of 0–20 mg/mL. The kinetic parameters Km and
221
Vmax were estimated using Lineweaver-Burk plot (Anitha & Palanivelu, 2013).
222 223 224
2.9. Mass spectrometry and protein identification The band of homogeneous protease was excised from the SDS-PAGE gel and
225
digested
with
trypsin.
The
protease
226
chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)
227
as described by Chen et al. (2016). MS data were obtained using Q-Exactive (Thermo
228
Finnigan, San Jose, CA). MS/MS spectra were analyzed using the MASCOT search
229
engine against the fungal subset of NCBI non-redundant protein sequence database
230
(NCBInr) and SwissProt database. Mass tolerances in MS and MS/MS modes were 20
231
ppm and 0.1 Da. For MASCOT search, matches achieving a molecular weight search
232
score ≥20 were considered as significant.
233 11
was
then
identified
using
liquid
234
2.10. Statistical analysis
235
The values were expressed as means ± standard deviation of three independent
236
replicates. Differences between the mean values were analyzed using one-way
237
ANOVA followed by Duncan test, and P < 0.05 was considered as significant.
238 239
3. Results
240
3.1. Chemical composition of okara and its peptide production by solid-state
241
fermentation
242
The chemical composition of soybean okara was as follows (g/100 g dry matter):
243
protein, 14.94±0.30; fat, 2.18±0.29; fiber, 39.29±1.02; soluble sugar, 4.23±0.07; and
244
ash, 3.53±0.04. The overall chemical composition suggested that soybean okara is a
245
suitable substrate for microbial fermentation. Further SSF of okara with Neurospora
246
crassa resulted in the production of peptides, as confirmed by the disappearance of
247
two major proteins β-conglycinin and glycinin (Figure 1A), as well as a remarkable
248
increase of peptide content in okara after 24 h, 48 h and 72 h of fermentation (P <
249
0.05; Figure 1B). Interestingly, novel proteins were yielded after 48 h and 72 h of
250
fermentation (Figure 1A). Taken together, it can be speculated that SSF of okara with
251
Neurospora crassa can produce proteases and thus contribute to the production of
252
biopeptides.
253 254 255
3.2. Optimal fermentation conditions In order to isolate and purify the protease produced from SSF of okara with 12
256
Neurospora crassa, we firstly optimized the fermentation conditions for protease
257
production. As presented in Figure 2, almost similar curve patterns were observed for
258
both protease activity and soluble protein content, which were significantly affected
259
by all the designed culture conditions (P < 0.05). Based on single factor experiment,
260
amount of added water, initial pH, and incubation temperature were the most
261
influential factors. Thus, these three factors were further optimized using
262
Box-Behnken design. By multiple regression analysis on the experimental data, the
263
following second-order polynomial equation was obtained to describe the protease
264
activity (Y1, U/g) and soluble protein content (Y2, mg/g): = 1960.47 + 131.93 − 40.52 = 10.00 + 0.36
− 26.18 − 167.11
− 0.061
− 0.30
+ 133.85
− 1.18
− 478.28
+ 0.41
+ 0.10
+ 72.07
− 104.55
− 1153.56 − 0.37
− 0.17
− 3.69
265
, where X1, X2, and X3 are the amount of added water, initial pH, and incubation
266
temperature, respectively.
267
The response surface and contour plots generated using the above regression
268
equations are presented in Figure 3. The model predicted that the maximal protease
269
production (1988.39 U/g) was achieved at the amount of added water 20.50 mL,
270
initial pH 5.0, and incubation temperature 30°C. The optimal conditions for the
271
maximal soluble protein content (10.11 mg/g) were determined to be: amount of water
272
added 20.80 mL, initial pH 5.0, and incubation temperature 30°C. Thus, for the
273
convenience of experimental operation, the fermentation conditions were optimized as 13
274
follows according to the single factor tests and response surface analysis: okara 10 g,
275
amount of added water 21 mL, initial pH 5.0, incubation temperature 30°C,
276
inoculation amount 2 mL, and fermentation time 72 h. Under these conditions, the
277
protease activity and soluble protein content reached 1959.82 U/g and 9.99 mg/g,
278
respectively, which were very close to the predicted values. Thus, the model for the
279
optimization of both protease activity and soluble protein content was satisfactory and
280
practicable.
281 282
3.3. Extraction and purification of protease
283
Under the above-mentioned optimal conditions, a crude enzyme solution with
284
protease activity of 195.21 U/mg protein was further purified. During the elution from
285
the DEAE-Sepharose column, five protein peaks were observed (Figure 4A). Protease
286
activity was detected in the first peak, which corresponded to fractions 2–15, with the
287
highest protease activity being detected in fraction 5. Fractions 4–6 were collected,
288
pooled and then subjected to Sephadex G-75 gel filtration chromatography. Fractions
289
14–15 exhibited strong protease activities (Figure 4B) and were pooled to constitute
290
the final sample of purified protease. The purification process is summarized in Table
291
1. The enzyme was purified 28.27-fold with a recovery rate of 31.0%, and the specific
292
activity was enhanced to 5518.37 U/mg protein.
293
With the purification steps, the bands of proteins were reduced or disappeared
294
(Figure 5A). The finally obtained enzyme showed a high purity as confirmed by the
295
presence of a single band, with a molecular weight of approximately 30 kDa (Figure 14
296
5B). Furthermore, only one clear zone was found on SDS-PAGE by zymographic
297
analysis of the purified enzyme, indicating the presence of protease.
298 299
3.4. Identification of the purified protease by LC-ESI-MS/MS analysis
300
The 30 kDa band from SDS-PAGE was subjected to LC-ESI-MS/MS analysis. The
301
acquired data were compared with those in NCBInr and SwissProt database. Five
302
identified proteins are presented in Table 2. The purified protein showed significant
303
matches with the three already characterized proteins, including carbohydrate esterase
304
family 1 protein, carbohydrate-binding module family 1 protein, and endo-1,
305
4-beta-xylanase B precursor, which do not belong to proteases. Moreover, an
306
uncharacterized protein (accession No. CAD70564.1) and acetyl xylan esterase in the
307
databases exhibited little similarities to the purified protein, indicating that it is most
308
likely a novel protease.
309 310
3.5. Effects of temperature and pH on protease activity and stability
311
The purified enzyme was the most active at the temperature of 50–60°C, with a
312
significantly higher activity at the temperature of 55°C (P < 0.05; Figure 6A). The
313
protease activity decreased nearly linearly at temperatures below 55°C and above 60°C
314
(P < 0.05). As shown in Figure 6B, the protease activity significantly increased with
315
increasing pH, reached the maximum at pH 9, and then decreased with further
316
increase of the pH to 11 (P < 0.05). Additionally, the protease activity under alkali
317
conditions (pH 8–11) was significantly higher than that under acidic and medium 15
318
conditions (pH 4–7) (P < 0.05), indicating that it is an alkaline protease.
319
Thermostability studies revealed that the enzyme was relatively stable at the
320
temperature of 30–45°C, with a slight decrease in protease activity with increasing
321
pre-incubation time (Figure 6C). Besides, the protease was stable over a broad range
322
of pH from 6 to 10; it retained >90% of its original activity after 2 h of pre-incubation,
323
and >50% of original activity even after 10 h of pre-incubation in this pH range
324
(Figure 6D). These results also confirmed that the purified enzyme belongs to the
325
alkaline protease family.
326 327
3.6. Effects of metal ions, organic solvents, surfactants and inhibitors on protease
328
activity
329
The protease activity was increased by 5 mmol/L of Ca2+, but inhibited by all other
330
metal ions, especially by 5 mmol/L of Cu2+, with the enzyme retaining only 39% of its
331
original activity (P < 0.05; Figure 7A). The organic solvents had little effect on the
332
protease activity (Figure 7B). Methanol at 1% and 10%, ethanol at 10%, and acetone
333
at 10% only marginally inhibited protease activity (1–3%; P > 0.05). Surfactants,
334
namely Tween-80, Span and Triton X-100, showed negligible effects on the protease
335
activity of the purified enzyme (P > 0.05; Figure 7C), indicating that these surfactants
336
are not required for the activation of the enzyme. However, SDS dramatically
337
inhibited the protease activity by 27% (P < 0.05). Further inhibition studies
338
demonstrated that the purified protease was not a metalloprotease, aspartic protease or
339
cysteine protease, because its activity was not suppressed by EDTA, pepstatin A and 16
340
IAM (Figure 7D). However, the typical serine protease inhibitor PMSF completely
341
inhibited the protease activity (P < 0.05), implying that the purified enzyme belongs
342
to serine protease family.
343 344
3.7. Substrate specificity and kinetic parameters
345
As shown in Figure 8A, the purified enzyme exhibited particularly high protease
346
activity towards casein and moderate activity towards gelatin and hemoglobin, but
347
very low activity towards BSA (only ∼5%; P < 0.05). The kinetic parameters were
348
then calculated using casein as the substrate, and the enzyme showed a
349
Michaelis-Menten type of kinetics (Figure 8B). The kinetic parameters including Km
350
and Vmax values were estimated to be respectively 2.18 mg/mL and 36.36 µg/mL/min
351
using linearized Lineweaver-Burk plot.
352 353
4. Discussion
354
The characteristics of high stability, low production cost, and specificity make
355
microbial proteases highly valuable candidates in food processing, particularly in the
356
preparation of protein hydrolysates with biopeptides (Chew et al., 2019; dos Santos
357
Aguilar & Sato, 2018). Currently, there is a high industrial demand for proteases from
358
fungal sources because of their stability, broad diversity, and substrate specificity
359
(Banerjee & Ray, 2017). Among various fungi, Neurospora crassa has an interesting
360
advantage of high yields of carotenoids and cellulases (P. Liu et al., 2016; Zhou et al.,
361
2019), and thus represents a powerful tool in functional food production. In the 17
362
present study, the newly isolated Neurospora crassa CGMCC3088 from Meitauza can
363
contribute to a high yield of proteases using soybean okara as the substrate, and
364
thereby enhance the yield of biopeptides (Figure 1). An enzyme with a very high
365
protease activity was purified by chromatography, and further characterization and
366
mass spectrometry analysis strongly indicated that the enzyme is a novel organic
367
solvent-stable alkaline serine protease, which could be used in food applications.
368
In the late 20th century, an alkaline protease was firstly produced and purified from
369
Neurospora crassa using submerged fermentation (Lindberg, Eirich, Price,
370
Wolfinbarger, & Drucker, 1981). However, the research was mainly focused on the
371
production of cellulases and carotenoids from Neurospora crassa in the following
372
years until now, and there has been no report about its application to protease
373
production. Neurospora crassa CGMCC3088 was newly isolated by our laboratory
374
from Jiangxi Meitauza, and it was shown to be a mutant strain with enhanced
375
production capacity of cellulases and carotenoids (P. Liu et al., 2016). To the best of
376
our knowledge, this is the first report of isolation and purification of a protease from
377
SSF of okara with this mutant strain. The specific activity of the purified protease was
378
significantly increased compared with that reported by Lindberg et al. (1981)
379
(5518.37 vs. 1220 U/mg), and was even higher than that of commercial microbial
380
proteases including alcalase, neutrase and flavourzyme (Bao, Zhao, Wang, & Chi,
381
2017; da Silva & de Castro, 2018). SSF and submerged fermentation are the two main
382
methods for the production of microbial proteases. Although submerged fermentation
383
is often applied for industrial protease production, SSF was found to contribute to a 18
384
higher activity of proteases (Machado de Castro, Fragoso dos Santos, Kachrimanidou,
385
Koutinas, & Freire, 2018), possibly because more carbon sources including sucrose
386
and starch are used for energy transformation during the SSF process, and then more
387
amino acids are biosynthesized for protein production (Zhao et al., 2019). Thus, SSF
388
obviously outperforms submerged fermentation in microbial protease production.
389
Indeed, owing to the high protease production, SSF with microorganisms has been
390
successfully used to efficiently hydrolyze legume proteins to small pepetides and
391
amino acids that are easy to be absorbed, and thereby improve the nutritional
392
composition of the final products (Gupta et al., 2018).
393
Further characterization of enzymatic properties showed that the purified protease
394
belongs to the alkaline protease family (Figure 6). Notably, among different types of
395
proteases, alkaline protease is the most commonly used enzyme in food industry
396
because of its high activity and stability at highly alkaline pH (Guleria, Walia,
397
Chauhan, & Shirkot, 2016; Thakur, Kumar, Sharma, Bhalla, & Kumar, 2018).
398
Commercial alkaline proteases are primarily isolated from the Bacillus species
399
(Contesini, Melo, & Sato, 2018). The fungus in our study was isolated from naturally
400
fermented food, and its safe and non-toxic nature makes it a new ideal microbial
401
strain for producing alkaline proteases. Additionally, most of proteases tend to be
402
inactivated and unstable under the treatment of organic solvents, which severely limits
403
their applications (Doukyu & Ogino, 2010; Si, Jang, Charalampopoulos, & Wee,
404
2018). Recently, various attempts have been made to screen enzymes with natural
405
organic solvent tolerance (de Borba, Machado, Brandelli, & Kalil, 2018; Thakur et al., 19
406
2018). Our purified protease from Neurospora crassa CGMCC3088 remained active
407
in the presence of various organic solvents (Figure 7B). Therefore, Neurospora crassa
408
CGMCC3088 is expected to have promising applications in the production of organic
409
solvent-tolerant proteases and functional foods with high contents of carotenoids and
410
biopeptides.
411 412
5. Conclusions
413
The extracellular alkaline protease produced from the SSF of soybean okara with
414
Neurospora crassa CGMCC3088 was demonstrated to have several superior
415
properties of high industrial values, including alkaline pH, good thermostability and
416
organic solvent tolerance. Importantly, peptide production was observed in soybean
417
okara after SSF with Neurospora crassa, indicating the great application potential of
418
this novel protease in the production of protein hydrolysates with bioactivities.
419
However, further research is needed to apply the purified protease to enzymatic
420
hydrolysis for the production of protein hydrolysates. Besides, it is also necessary to
421
explore the structure-function relationship by using three-dimensional structure
422
modeling and site-directed mutagenesis.
423 424
Acknowledgments
425
This work was supported financially by the Research Program of State Key
426
Laboratory of Food Science and Technology, Nanchang University (Grant Number:
427
SKLF-ZZA-201610). 20
428 429 430
Conflict of interests The authors declare no competing financial interests.
431 432
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Thakur, N., Kumar, A., Sharma, A., Bhalla, T. C., & Kumar, D. (2018). Purification and
515
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516
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517 518
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519
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520
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521
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522
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523
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524
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525
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527
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530
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533
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534
23
535
Table 1
536
Purification of protease from the solid-state fermentation of soybean okara with
537
Neurospora crassa. Purification steps
Total activity (U)
Total protein
Specific
Purification
Recovery
(mg)
activity
fold
(%)
(U/mg protein) 74452.11±639.04a
381.39±4.75a
195.21±2.91d
1.00
100.00
51370.03±381.96b
169.78±2.46b
302.56±2.32c
1.55
69.00
DEAE Sepharose
45068.80±483.19c
25.14±0.21c
1792.93±15.86b
9.18
60.53
Sephadex G-75
23081.14±102.86d
4.18±0.05d
5518.37±35.56a
28.27
31.00
Crude extract Ammonium
sulfate
precipitation
538
Means with different letters within the same column differ significantly at P < 0.05.
539
24
540
Table 2
541
Identification of the purified protease from the solid-state fermentation of soybean
542
okara with Neurospora crassa.
Accession number
Protein name
Unique peptides
KHE83333.1
Carbohydrate esterase family 1 protein (Neurospora crassa)
KHE83062.1
Carbohydrate-binding module family 1 protein (Neurospora crassa)
CAD71059.1
Endo-1, 4-beta-xylanase B precursor (Neurospora crassa) Uncharacterized protein (Neurospora crassa) Acetyl xylan esterase (Neurospora crassa)
K.QWSNVLGVEFSR.N K.YNADASR.V R.CAM*EALK.Q R.CAMEALK.Q R.GLQHTPEEWGNFVR.N R.GLQHTPEEWGNFVR.N R.M*YTYVPDK.L R.MYTYVPDK.L R.NSYPGYTGR.R R.NSYPGYTGR.R K.IFEDTWAK.K K.IPSDIPAGDYLLR.A K.IPSDIPAGDYLLR.A K.KPSSSSGDDDFWGVK.D K.KPSSSSGDDDFWGVK.D K.GWM*PGTDR.T K.NHFDAWTR.S R.LGSVTSDGGVYDIYR.T
CAD70564.1
EAA29891.1
Sequence coverage (%)
Theoretical mass (kDa)
19.52
31.08
8.47
10.91
32.86
6.89
10.58
30.78
8.47
R.QGTNAVATAVNSLNAR.C
5.33
30.41
7.83
K.LVGVYAR.G R.GVGHSVPIR.G R.GVGHSVPIR.G
5.13
33.43
6.74
543
25
pI
544
Figure captions
545
Figure 1. Protein electrophoresis profiles (A) and peptide contents (B) of soybean
546
okara before and after the solid-state fermentation with Neurospora crassa. Arrows 1,
547
2 and 3 indicate α’-, α- and β-subunits of β-conglycinin, respectively. Arrows 4 and 5
548
indicate acidic and basic subunits of glycinin, respectively. Peptide contents are
549
expressed as mg of glutathione (GSH) equivalents/g dry weight (DW), and means
550
with different letters differ significantly at P < 0.05.
551 552
Figure 2. Effects of different culture conditions on protease activity and soluble
553
protein content from the solid-state fermentation of soybean okara with Neurospora
554
crassa. Culture conditions including the amount of added water (A), initial pH (B),
555
incubation temperature (C), fermentation time (D), and inoculation amount (E).
556
Means with different letters differ significantly at P < 0.05.
557 558
Figure 3. Surface and contour plot showing the interactive effects of different culture
559
conditions on protease activity and soluble protein content from the solid-state
560
fermentation of soybean okara with Neurospora crassa. (A and D) Amount of added
561
water and initial pH. (B and E) Amount of added water and incubation temperature.
562
(C and F) Initial pH and incubation temperature.
563 564
Figure 4. Chromatographic purification profile of proteases from the solid-state
565
fermentation of soybean okara with Neurospora crassa. (A) Chromatographic 26
566
purification profile of proteases on DEAE-Sepharose fast flow anion exchange
567
column. (B) Chromatographic purification profile of proteases on Sephadex G-75 gel
568
filtration chromatography.
569 570
Figure 5. Protein electrophoresis profile of different fractions of proteases from the
571
solid-state fermentation of soybean okara with Neurospora crassa. (A) SDS-PAGE
572
profile of the purified protease obtained after DEAE-Sepharose fast flow
573
anion-exchange
574
anion-exchange chromatography; line 2, crude supernatant. (B) SDS-PAGE profile
575
(line 1) and zymography (line 2) of purified protease obtained after Sephadex G-75
576
gel filtration chromatography.
chromatography.
Line
1,
purified
protease
obtained
after
577 578
Figure 6. Effects of temperature and pH on enzyme activity (A and B) and stability
579
(C and D) of the purified protease from the solid-state fermentation of soybean okara
580
with Neurospora crassa. Means with different letters differ significantly at P < 0.05.
581 582
Figure 7. Effects of metal ions (A), organic solvents (B), surfactants (C), and
583
inhibitors (D) on the activity of purified protease from the solid-state fermentation of
584
soybean okara with Neurospora crassa. Means with different letters differ
585
significantly at P < 0.05.
586 587
Figure 8. (A) Substrate specificity of the purified protease from the solid-state 27
588
fermentation of soybean okara with Neurospora crassa. Means with different letters
589
differ significantly at P < 0.05. (B) Lineweaver-Burk plot. Enzyme kinetic parameters
590
for hydrolysis of casein by the purified protease were determined using
591
Lineweaver-Burk plot.
592 593
594 595 596
Figure 1
28
597 598
Figure 2
599
29
600 601
Figure 3
602
30
603 604
Figure 4
605
31
606 607 608
Figure 5
32
609 610
Figure 6
611
33
612 613
Figure 7
614
34
615 616
Figure 8
35
Highlights 1. Neurospora crassa CGMCC3088 can be treated as a novel functional fermentation organism. 2. Fermentation conditions were optimized for protease producing of Neurospora crassa CGMCC3088. 3. A novel organic solvent-stable alkaline protease was purified from Neurospora crassa CGMCC3088. 4. The purified protease from Neurospora crassa CGMCC3088 can hydrolyze casein, gelatin and hemoglobin.
Conflict of interest The authors have declared that no competing interests exist.