International Journal of Biological Macromolecules 101 (2017) 455–463

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Chitin extraction from blue crab (Portunus segnis) and shrimp

(Penaeus kerathurus) shells using digestive alkaline proteases from P.
segnis viscera
Marwa Hamdi, Amal Hammami, Sawssen Hajji, Mourad Jridi ∗ , Moncef Nasri, Rim Nasri
Laboratory of Enzyme Engineering and Microbiology, University of Sfax, National Engineering School of Sfax, B.P. 1173, 3038 Sfax, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: Since chitin is closely associated with proteins, deproteinization is a crucial step in the process of extract-
Received 19 October 2016 ing chitin. Thus, this research aimed to extract chitin from Portunus segnis and Penaeus kerathurus shells
Received in revised form 2 February 2017 by means of crude digestive alkaline proteases from the viscera of P. segnis, regarding deproteinization
Accepted 9 February 2017
step, as an alternative to chemical treatment. Casein zymography revealed that five caseinolytic pro-
Available online 20 March 2017
teases bands exist, suggesting the presence of at least five different major proteases. The optimum pH
and temperature for protease activity were pH 8.0 and 60 ◦ C, respectively, using casein as a substrate.
Keywords:
The crude enzymes extract was highly stable at low temperatures and over a wide range of pH from 6.0
Blue crab and shrimp shells chitins
Enzymatic deproteinization
to 12.0. The crude alkaline protease extract was found to be effective in the deproteinization of blue crab
Digestive alkaline proteases and shrimp shells, to produce chitin. The best efficiency in deproteinization (84.69 ± 0.65% for blue crab
shells and 91.06 ± 1.40% for shrimp shells) was achieved with an E/S ratio of 5 U/mg of proteins after 3 h
incubation at 50 ◦ C. These results suggest that enzymatic deproteinization of crab and shrimp wastes by
fish endogenous alkaline proteases could be a potential alternative in the chitin production process.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction total enzyme production [11]. As crude preparations or purified,

Nowadays, with the development of the concept of environmen-
tally friendly technologies, by-products from aquatic organisms
may have broad possible valorization options in various industrial
disciplines and the production of commercial products [1]. Indeed,
after industrial processing, as much as 70% of fish and shellfish are
consisting of by-product (viscera, heads, cut-offs, bone, skin, etc.),
traditionally converted to low value materials, as feed for farmed
animals, fertilizers, or as a problem and discarded [2]. Recently,
abundant scientific researches reported the versatile potentials of
by-products, obtained after processing fish and crustaceans, to be
converted into highly valuable bio-compounds like collagen and
gelatin [3], bioactive peptides [4], enzymes and specific proteins
[5,6], chitin, chitosan and pigments [7,8]. This will be beneficial for
economics and environment.
Fish viscera, one of the most important by-products of fish-
ing industry, is known to be a rich source of proteases [9,10].
Proteases, particularly alkaline proteases, constitute the class of
enzymes most used worldwide, accounting for 60% of the world’s
∗ Corresponding author.
E-mail address: jridimourad@gmail.com (M. Jridi).
proteases have diverse applications in a wide variety of industries
[12,13]. Aspartic protease and pepsin (high activity between pH
2.0 and 4.0), as well as serine proteases, such as trypsin and chy-
motrypsin (highly active between pH 8.0 and 10.0) are the most
important digestive enzymes from fish and aquatic invertebrates
viscera [14–16]. In another hand, various researches investigated
the application of digestive proteases in the process of depro-
teinization of crustacean shells, considered as a severe problem
for the environment and a rich source of valuable by-products
[17], such as protein (20–40%), carotenoproteins [18,19] and chitin
(20–30% on dry weight basis) [11,20].
The main sources of raw material for the production of chitin
are cuticles of various crustaceans, principally crabs and shrimps.
Chitin, a homopolymer of N-acetyl-d-glucosamine residues linked
by ␤-(1,4) bonds, is the most abundant renewable natural resource
after cellulose [5]. Due to their numerous biological activities
(biodegradability, biocompatibility, non-toxicity, etc.), chitin and
mainly its deacetylated form (chitosan) are considered as inter-
esting bio-compounds, with a great economic importance (food
industry, pharmacy, cosmetics, textiles, chemical industries, etc.)
[17]. Nevertheless, chitin in the cuticles of crustaceans is closely
associated with proteins, minerals, lipids and pigments that have
to be eliminated. Conventionally, chitin was extracted from crus-

http://dx.doi.org/10.1016/j.ijbiomac.2017.02.103
0141-8130/© 2017 Elsevier B.V. All rights reserved.
456 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463
tacean shells through chemicals processing for demineralization
and deproteinization, by treatment with strong acids and bases
[21]. However, the use of these chemicals may cause a partial
deacetylation and depolymerization of the chitin [22], affecting its
final physiological and structural properties [11,23], and therefore
limiting its use in various applications. Hence, the deproteinization,
using bacterial fermentation or proteolytic enzymes, is an alterna-
tive approach to overcome the effect of strong acids or bases on
the extracted chitin [24–27]. This procedure can be considered as
a contribution in the biotechnological valorization [28–31].
Blue crab (Portunus segnis) and shrimp (Penaeus kerathurus)
belong to the order of Decapoda and are classified in family Por-
tunidae and Penaeidae, respectively. Among the crustaceans, the
body of blue crabs and shrimps is covered with a pigmented shell.
P. segnis species is widespread in the west of the Atlantic Ocean.
It proliferates easily and it was introduced, recently, in the east-
ern Atlantic, in the north and east of the Mediterranean and Japan.
Nowadays, it is found, extensively, in the Mediterranean, especially,
in Tunisian coats causing various damages regarding fishing. As
well, blue crabs are being captured and commercialized for human
consumption, considering its excellent organoleptic characteris-
tics.
The present work describes biochemical characterization of the
alkaline crude enzyme preparation from P. segnis, as well as its
application in chitin extraction from different indigenous Tunisian
marine sources (P. segnis and P. kerathurus shells). Physicochemical
characteristics of commercial and extracted chitins were compared
using FTIR spectroscopy.
2. Materials and methods
2.1. Reagents
Acrylamide, N,N -methylene-bis-acrylamide, casein sodium salt
from bovine milk, trichloroacetic acid (TCA), Tween 20, ethylene
diamine tetra-acetic acid (EDTA), phenyl-methylsulfonyl fluoride
(PMSF) and 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) were pur-
chased from Sigma Company Co. (St. Louis, MO, USA). Sodium
dodecyl sulphate (SDS), N,N,N,N -tetramethyl ethylenediamine
(TEMED), and Coomassie Brilliant Blue R-250 were purchased from
Bio-Rad Laboratories (Mexico City, Mexico). All other reagents were
of analytical grade.
2.2. Materials
The shrimp (P. kerathurus) shells and blue crabs (P. segnis) were
obtained in fresh conditions from the shrimp processing plant and
fishery market, located at Sfax, Tunisia, respectively. After the crabs
were washed with tap water, their viscera and shells were sep-
arated, washed thoroughly with cold distilled water. Viscera were
used immediately for enzyme extraction. Commercial chitin (1398-
61-4; MW = 770.8 g/mol) was provided by MP Biomedicals France.
2.3. Preparation of crude alkaline proteases
P. segnis viscera (150 g) were homogenized for 5 min with 150 ml
of extraction buffer (10 mM Tris–HCl, pH 8.0), by means of Moulinex
(Kenwood BL 440) homogenizer. The resulting preparation was
centrifuged at 8000 × g for 30 min at 4 ◦ C (MPW-350 R centrifuge).
The pellet was discarded and the supernatant, referred to as P. seg-
nis protease extract, was collected and then frozen at −20 ◦ C. All
enzymatic essays were conducted within a week after extraction.
2.4. Caseinolytic activity assay
Protease activity in the crude enzyme extract was measured by
the method described by Kembhavi et al. [32] using casein as a sub-
strate. Thus, 500 ␮l of 0.1 M Tris–HCl (pH 8.0), containing 1% (w/v)
casein was mixed with 500 ␮l of the alkaline protease extract, suit-
ably diluted, and incubated for 15 min at 50 ◦ C. The reaction was
stopped by the addition of 500 ␮l of TCA 20% (w/v). After being
allowed to stand at room temperature for 15 min, the mixture
was centrifuged at 10,000 × g for 15 min to remove the precipitate
proteins. The estimation of trichloroacetic acid-soluble hydrolysis
products was realized spectrophotometrically at 280 nm. A stan-
dard curve was generated using solutions of 0–50 mg/l tyrosine.
One unit of protease activity was defined as the amount of enzyme
required to liberate 1 ␮g of tyrosine per minute under the experi-
mental conditions used.
2.5. Polyacrylamide gel electrophoresis and detection of protease
activity by zymography
The samples were separated by sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE) as described by
Laemmli [33], using 5% (w/v) stacking and 12% (w/v) separating
gels. Samples were prepared by mixing the crude enzyme extract
with the SDS-PAGE sample buffer (10 mM Tris–HCl, pH 8.0; 2.5%
SDS; 10% glycerol; 5% ␤-mercaptoethanol and 0.002% bromophenol
blue). The sample (50 U) was heated at 100 ◦ C for 5 min before load-
ing in the gel. After electrophoresis, the gel was stained with 0.25%
(w/v) Coomassie Brilliant Blue R-250 in 45% (v/v) ethanol–10%
acetic acid (v/v) and destained with 5% (v/v) ethanol and 7.5% (v/v)
acetic acid.
Casein zymography was performed as described by Garcia-
Carreno et al. [34]. The sample was not heated before loading in
the gel. After electrophoresis, the gel was submerged in buffer A
(100 mM Tris–HCl (pH 8.0)) containing 2.5% (v/v) Triton X-100, with
shaking for 1 h to remove SDS and allow enzyme renaturation. Tri-
ton X-100 was removed by washing the gel with buffer A for 1 h.
The gel was then immersed in 100 ml of 1.5% (w/v) casein in buffer
A for 30 min at 4 ◦ C, then further incubated for 25 min at 37 ◦ C to
allow for the digestion of the protein substrate (casein) by the active
enzymes. After washing, the gel was stained with Coomassie Bril-
liant Blue R-250 for zymography analysis. The development of clear
bands on the blue background of the gel indicated the presence of
protease activity.
2.6. Biochemical properties of the crude enzyme
2.6.1. pH profile and stability
The optimum pH of P. segnis alkaline proteases was studied over
a pH range of 6.0–13.0, by assaying caseinolytic activity at 50 ◦ C. For
the measurement of pH stability, the crude enzyme extract was
incubated for 60 min at 4 and 25 ◦ C in different buffers (pH 6.0, 7.0,
8.0, 9.0, 10.0, 11.0, 12.0 and 13.0). After incubation, the residual
activity was determined at pH 8.0 and 60 ◦ C, and reported compared
to the non-incubated enzyme carried out in the same condition
(control = 100%). The following buffer systems were used: 100 mM
sodium acetate buffer for pH 6.0, Tris–HCl buffer for pH 7.0–8.0,
glycine–NaOH buffer for pH 9.0–11.0, Na2 HPO4 –NaOH buffer for
pH 12.0 and KCl–NaOH buffer for pH 13.0.
2.6.2. Thermal profile and stability
To investigate the effect of temperature, the proteolytic activity
of P. segnis crude extract was tested at temperature range from 20
to 90 ◦ C, at pH 8.0 using casein as a substrate. Thermal stability was
examined by incubating crude enzyme extract for (15, 30, 45 and
60 min) at different temperatures from 20 to 70 ◦ C. After cooling
 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463 457

the proteases in an ice bath for 5 min, the remaining activity was
assayed at optimal conditions (pH 8.0 and 60 ◦ C). The non-heated
crude enzyme extract was considered as control (100% activity).

2.6.3. Effects of metal ions and enzymes inhibitors on proteolytic

activity
The influence of various metal ions, at a concentration of 5 mM,
on enzyme activity was investigated by adding the monovalent
(Na+ or K+ ) or divalent (Mg2+ , Ca2+ , Zn2+ , Cu2+ , Fe2+ , Ba2+ , or Mn2+ )
metal ions to the reaction mixture. The activity of the crude enzyme
extract without any metallic ions was considered as 100%.
The effects of some enzyme inhibitors such as PMSF, DTNB,
EDTA and ␤-mercaptoethanol on enzyme stability were studied.
The crude enzyme was pre-incubated with each inhibitor for 30 min
at 30 ◦ C. The remaining enzyme activity was tested using casein as
a substrate at 60 ◦ C and pH 8.0. Control was assayed under the same
conditions without inhibitors.

2.7. Chitin extraction

2.7.1. Preparation and chemical analysis of P. segnis and P.
kerathurus shells powders
Blue crab and shrimp shells were cooked in distilled water (1:2
ratio (w/v)) for 20 min at 90 ◦ C. The cooked samples were dessi-
® Demineralization of shells deproteinized shells by P. segnis crude
cated at room temperature, and milled to powder in a Moulinex
blender. Then, after drying, they were kept in glass bottles at room
temperature until used. This crabs/shrimps waste homogenate was
then characterized [35].
The moisture and ash contents of crab and shrimp shells were
determined according to the AOAC standard methods 930.15 [36]
and 942.05 [37], respectively. Total nitrogen content was measured
by Kjeldahl method. Corrected protein concentration was obtained
by subtracting chitin nitrogen from total nitrogen and multiplying
by the factor 6.25 [38,39]. Lipids were determined gravimetrically
after Soxhlet extraction of dried shells using hexane according to
the AOAC (1984) analytical methods [39].
2.7.2. Deproteinization of P. segnis and P. kerathurus shells by P.
segnis crude protease extract
P. segnis alkaline proteases were used for the deproteinization
of crab and shrimp shells. Two commercial enzymes, Purafect (R
2000E) and Neutrase P1236 (Bacillus amyloliquofaciens proteases)
were chosen as a control for deproteinization experiments. Depro-
teinization reactions were carried out in a thermostated stirred
Pyrex reactor (300 ml).
Crab/Shrimp shell powders (20 g) were mixed with 100 ml dis-
tilled water. The pH and temperature of the reaction mixtures were
adjusted to pH 8.0, 50 ◦ C for P. segnis proteases, pH 10.0, 50 ◦ C for
Purafect and pH 7.0, 50 ◦ C for Neutrase. Then, the crab/shrimp shell
proteins were subjected to deproteinization with each protease
during 3 h. Reactions were stopped by heating the mixtures at 90 ◦ C
during 20 min to inactivate enzymes. The solid phases were pressed
manually through four layers of gauze. Deproteinized products in
the solid phase were washed thoroughly until a neutral pH, and
then, dried overnight at 50 ◦ C. Degree of deproteinization (DDP),
expressed as a percentage, was calculated by the following equation
[40]:
[(PO × O) − (PR × R)]
DDP (%) = × 100
PO × O
where PO and PR are the protein concentrations (%) before and after
deproteinization; while, O and R represent the mass (g) of original
sample and hydrolyzed residue in dry weight basis, respectively.
Fig. 1. SDS-PAGE (L1) and activity staining zymogram (L2) of alkaline proteases
from the viscera of Portunus segnis.
2.7.3. Chemical demineralization
extract was performed using 0.55 M hydrochloric acid baths, in 1:10
(w/v) ratio, as described by Tolaimate et al. [21]. The degree of dem-
ineralization (DDM), expressed as a percentage, was evaluated by
the following equation [40]:
[(MO × O) − (MR × R)]
DDM (%) = × 100
MO × O
where MO and MR are the ash contents (%) before and after deminer-
alization; while, O and R represent the mass (g) of original sample
and demineralized residue in dry weight basis, respectively.
2.7.4. Fourier transform infrared spectroscopy (FTIR)
Infrared spectra of prepared and commercial chitins were car-
ried out by means of a Perkin Elmer type FTIR1000 spectrometer
at room temperature and KBr pellets. The sample pellets were pre-
pared at a pressure of 5 tons for 2 min. Pellets were scanned at room
temperature (25 ◦ C) in the 500–4000 cm−1 spectral range [41]. The
incertitude of this measurement was 2 cm−1 . The spectra obtained
were analyzed by an OPUS 3.0 software.
2.8. Statistical analysis
All experiments were carried out in triplicate, and average val-
ues with standard deviation errors are reported. Mean separation
and significance were analyzed using the SPSS software package
ver. 17.0 professional edition (SPSS, Inc., Chicago, IL, USA) using
ANOVA analysis. Differences were considered significant at p < 0.05.
3. Results and discussion
3.1. SDS-PAGE and Zymography of the blue crab alkaline
proteases
Prior to analysis, the estimated number of proteases in the
endogenous enzymatic extract from blue crabs viscera was
revealed by zymography, largely considered as rapid and sensitive
method of assay (detecting nanogram of proteins), using casein as
a substrate (Fig. 1 L2).
As shown in Fig. 1 L1, SDS-PAGE profile revealed the separation
of various proteins bands. Likewise, five clear bands of protease
458 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463

activity with different molecular weights can be observed in the Table 1

Effects of various metal ions and inhibitors on the activity of digestive alkaline
crude enzyme extract (Fig. 1 L2), suggesting the existence of at
proteases of P. segnis.
least five major proteases in P. segnis viscera. This result was sim-
ilar to those reported by Lassoued et al. [9], Younes et al. [11] and Metal ions Concentration Relative activity (%)
Nasri et al. [27] for proteases extracted from the viscera of thorn- Control – 100.00 ± 0.00
back ray (Raja clavata), red scorpionfish (Scorpaena scrofa) and goby Mg2+ 5 mM 109.42 ± 0.89
(Zosterisessor ophiocephalus) viscera, respectively. Cu2+ 5 mM 84.62 ± 1.03
Ca2+ 5 mM 98.22 ± 0.28
Na+ 5 mM 101.54 ± 0.53
3.2. Digestive alkaline proteases extraction and characterization Zn2+ 5 mM 47.33 ± 1.06
Ba2+ 5 mM 109.1 ± 1.41
3.2.1. pH profile and stability K+ 5 mM 102.56 ± 0.84
Fe2+ 5 mM 15.66 ± 0.48
The effect of pH on the activity and stability of P. segnis alka-
Mn2+ 5 mM 76.88 ± 0.80
line proteases was determined over a pH range of 6.0–13.0. The
alkaline enzyme extract was active at different pH with an opti- Inhibitors Concentration Target proteases Residual activity (%)
mum at pH 8.0 (Fig. 2a). The relative activities, at pH 6.0, 7.0, 11.0
None – 100. 00 ± 0.00
and 12.0, compared to that obtained at pH 8.0, were about 66%, PMSF 5 mM Serine proteases 60.51 ± 1.15
75%, 48% and 28%, respectively. As described in various studies, 10 mM 34.78 ± 1.46
the optimum pH of digestive alkaline proteases from some fish DTNB 5 mM Cysteine protease 48.16 ± 1.24
species was at pH 8.0, such as thornback ray [9], golden gray mullet 10 mM 41.41 ± 2.32
EDTA 5 mM Metallo proteases 78.10 ± 1.03
[14], silver mojarra [42] and hybrid catfish [43]. However, the opti-
10 mM 41.39 ± 1.27
mum pH of blue crab crude protease extract was lower than optima ␤-Mercaptoethanol 5 mM 100.00 ± 0.00
pH obtained with proteases extracted from red scorpionfish [11], 10 mM 92.49 ± 1.16
painted comber [12], giant Amazonian fish pirarucu [13], farmed The relative activity of the digestive alkaline proteases was evaluated by incubating
giant catfish [16] and vermiculated sailfin catfish [44]. The decrease the enzyme in the presence of several metal ions in the reaction mixture. Crude
in enzyme activity at pH values outside optimum pH is possibly due extract was pre-incubated with various enzyme inhibitors for 30 min at 30 ◦ C and
to protein conformational changes (surface charge distributions the residual activity was determined at pH 8.0 and 60 ◦ C. Enzyme activity with-
out addition of any inhibitor was considered as 100%. The results are presented by
and irreversible protein denaturation) [16].
mean ± standard deviation.
The pH stability profiles at 4 and 25 ◦ C of the crude alkaline pro-
tease extract are reported in Fig. 2b. P. segnis crude extract is highly
stable in a wide pH range from 6.0 to 12.0, retaining about 95% extracts from scorpionfish [11], farmed giant catfish [16], goby,
of its original activity between 7.0 and 12.0, and more than 85% thornback ray [27] and silver mojarra [42]. These findings suggest
at pH 6.0, after pre-incubation for 1 h at 4 ◦ C. Whereas, more than the suitability for biotechnological applications of the crude alka-
75% of the original activity was obtained after pre-incubation for line protease extract, blue crab viscera, which may be desirable
1 h at 25 ◦ C, in the same pH range. However, at pH 13.0, the crude for many biotechnological, food processing and ecological applica-
enzyme extract maintained about 60% of its initial proteolytic activ- tions, because of saving energy (high stability at low temperatures)
ity. This result was similar to those reported by Younes et al. [11], [13,16].
Silva et al. [42] and Klomklao et al. [43]. This high stability of blue
crabs alkaline proteases suggests that it can be a potential alterna- 3.2.3. Effects of metal ions and inhibitors on protease activity
tive in various industrial applications, which require high alkaline The effects of several metal ions, on the activity of the crude
conditions. alkaline proteases, at a concentration of 5 mM, were studied at pH
8.0 and 60 ◦ C (Table 1). The findings revealed that several metal ions
3.2.2. Influence of temperature on enzyme activity and stability (Ca2+ , K+ , Cu2+ , Na+, and Mn2+ ) did not affect the protease activity
The investigation of thermal profile and stability of blue crab of P. segnis crude protease extract, while Mg2+ and Ba2+ increased
endogenous enzymes was determined at pH 8.0 using casein as a slightly the protease activity to around 109%. In spite of various
substrate and as a function of temperature (20–90 ◦ C). Fig. 2c shows researches [5,6,11], that described the improvement of protease
that digestive proteases are active at temperatures from 20 to 90 ◦ C, activity in the presence of calcium (classic activator by preserving
and displayed maximum activity at 60 ◦ C. The relative activities at specific stable structure of the enzyme), in our study, blue crabs
55 and 70 ◦ C, were about 77% and 81%, respectively, of that at 60 ◦ C. digestive alkaline proteases are not calcium dependent. However,
Nevertheless, a significant decline in enzyme activity was observed Zn2+ and Fe2+ , affect significantly the enzyme activity with 52.7%
beyond 70 ◦ C, due to thermal denaturation. and 84.4%, respectively.
The optimal temperature of P. segnis proteases was similar On another hand, the nature of proteases in the crude enzyme
to crude enzymes extracted from Macrobrachium rosenbergii [6], extract was studied using specific class of enzyme inhibitors.
golden gray mullet [14], farmed giant catfish [16], Scomber japoni- Table 1 displays the effects of various inhibitors, such as chelat-
cus [30], zebra blenny [31] and skipjack tuna [45] viscera. However, ing agent and a specific group reagent, at a concentration of
it was higher than optimal temperatures of goby and scorpionfish 5 mM on the activity of the crude enzyme extract from P. segnis.
digestive alkaline proteases, which showed optimal tempera- Results revealed that proteases from blue crab viscera were rel-
tures at 50 and 55 ◦ C, respectively [27], as well as silver mojarra atively inhibited, when incubated with PMSF, a serine protease
(Diapterus rhombeus) purified protease (55 ◦ C) [42]. inhibitor. In fact, the residual activity was around 60.51 ± 1.15%
Thermal stability profiles of P. segnis crude proteases extract are and 34.78 ± 1.46% in the presence of 5 mM and 10 mM PMSF,
depicted in Fig. 2d. P. segnis alkaline proteases were highly sta- respectively, suggesting the presence of serine proteases, espe-
ble at temperatures below 40 ◦ C, retaining around 100% of their cially trypsin and chymotrypsin. Moreover, the activity of P. segnis
initial activities. Furthermore, digestives proteases retained more alkaline proteases was affected, after incubation with EDTA (5 mM)
than 90% of their initial activity at 50 and 60 ◦ C after pre-incubation (metallo-proteinase inhibitor) and DTNB (5 mM) (cysteine protease
for 15 min. However, only 50% of this activity was retained after inhibitor), retaining about 78.10 ± 1.03% and 48.16 ± 1.24%, respec-
pre-incubation for 60 min at 60 ◦ C. The thermal stability of P. segnis tively. Nevertheless, ␤-mercaptoethanol showed no impact on the
digestive alkaline proteases was higher than those of crude enzyme protease activity of P. segnis digestive alkaline proteases, which
 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463 459

Fig. 2. Effects of pH and temperature on activity and stability of P. segnis proteases. (a) The protease activity was assayed in the pH range 6.0–13.0, at 50 ◦ C. Buffer solutions
used for pH activity and stability were presented in Section 2. The optimum activity was taken as 100%. (b) The pH stability was determined by incubating the crude enzyme
extract in different buffers for 1 h at 4 and 25 ◦ C. (c) The temperature profile was determined by assaying protease activity at temperatures between 20 and 90 ◦ C, at pH 8.0.
(d) Temperature stability was determined by incubating the crude enzyme at temperatures from 30 to 70 ◦ C for 60 min. The residual activities were measured at the optimum
conditions. The activity of the enzyme before incubation was taken as 100%. Values are means of three independent experiments.

exhibited that there is no disulfide bonds in the active site. These son and other factors, within a broad range of 13–50% for protein,
findings revealed the existence of several types of proteases in the 10–25% for chitin and 15–-70% for mineral materials [35].
crude extract (serine, cysteine and metallo proteases).

3.3.2. Enzymatic deproteinization of P. segnis and P. kerathurus

3.3. Chitin extraction
Chitin is closely associated with proteins. Therefore, depro-
teinization in chitin extraction process is crucial. In the present
study, proteolytic enzymes from P. segnis viscera were used to
remove proteins.
3.3.1. Chemical characterization of blue crab and shrimp shells
Two different marine sources, blue crab (P. segnis) and shrimp
(P. kerathurus) shells, were used for the extraction of chitin. The
chemical compositions of shells are reported in Table 2. Blue crabs
and shrimp shells used in this study had a moisture content of
10% and 12%, respectively. The differences observed between the
two sources essentially concern the protein and chitin contents.
Indeed, blue crab shells contained low protein content (11.25%)
than shrimp shells (21.87%). However, regarding chitin content,
shrimp shells had lower amount (23.57%) than blue crab shells
(27.53%). Both materials were characterized by a high ash content
(59.11 ± 0.45%, and 52.64 ± 0.5%, of dry weight, for blue crab and
shrimp shells, respectively). In fact, the calcium carbonate, a main
mineral of crab and shrimp shells, as well as other crustaceans,
allowing calcification of their exoskeleton, can explain high levels of
minerals. The amount of fat in both shells is very low (1.07 ± 0.11%
and 1.09 ± 0.2%, respectively). The overall percentages of protein,
chitin and minerals in the crustacean shell varied by species, sea-
shells by P. segnis alkaline proteases
Deproteinization of blue crab and shrimp shells using P. segnis
proteases was first carried out at pH 8.0 and 50 ◦ C, using different
E/S ratios (1–10 U/mg protein) (Fig. 3a). Control experiments, per-
formed without addition of the enzyme, lead to remove about 37%
and 47% of proteins from the chitinous matrix of blue crabs and
shrimp, respectively. In fact, despite proteins probably related to
chitin by covalent bonds, which require a chemical or enzymatic
treatment for their recovery, some proteins may be associated to
the chitinous matrix by electrostatic forces or hydrogen bonds that
can be dissociated by thermal treatment [46–48]. The addition of
1 U of P. segnis proteases/mg proteins for blue crab and shrimp
shells, improve the efficiency of proteins removal and eliminate
around 75% and 79% of proteins, respectively. However, beyond
a ratio of E/S = 5, which allowed high protein removal of 85% and
91% for blue crabs and shrimp shells, respectively, no significant
improvement in the deproteinization rate was observed. In fact, the
chitinous matrix in crustacean shells acts as a protein-associated
protector against the actions of digestive proteases and no addi-
tional hydrolysis reaction can take place [11,17,24].
Besides E/S ratio, enzymatic deproteinization efficiency was also
found to be dependent on reaction conditions (temperature and
incubation time). In fact, it is essential to consider not only the opti-
mal temperature of the crude digestive alkaline proteases, but also
its stability during proteolysis progression. To this end, the effect
460 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463
Table 2
Characterization of the dried raw materials and purified chitins.
Composition (%)a Blue crab shells
Dry matter 89.87 ± 0.06
Ash 59.11 ± 0.46
Proteins 11.25 ± 0.72
Lipids 1.07 ± 0.03
Chitin 27.53 ± 1.04
Yield – 19.06 ± 1.65
a
Physico-chemical composition was calculated based on the dry matter.
of temperature (40, 50 and 60 ◦ C), as well as the time of enzymatic
deproteinization process (3 and 6 h), were studied. Deproteiniza-
tion reactions were performed using an E/S ratio of 5 U/mg of
proteins at pH 8.0.
As shown in Fig. 3b, the maximum proteins removal was
obtained with deproteinization process carried out at 50 ◦ C
(84.69 ± 0.64% and 91.06 ± 1.40%, for blue crab and shrimp shells,
respectively). However, reactions conducted at 40 and 60 ◦ C
revealed lower deproteinization degrees, although crude protein
extract exhibited higher stability at 40 ◦ C and higher activity at
60 ◦ C, than 50 ◦ C, as reported in Fig. 2d. In fact, at 40 ◦ C, around
75.96 ± 1.97% and 70.25 ± 1.94% of associated proteins were elimi-
nated from P. segnis and P. kerathurus shells, respectively. Likewise,
regarding reactions accomplished at 60 ◦ C, protein removal rates
were 77.01 ± 1.0% for blue crab shells, and 71.33 ± 2.03% for shrimp
shells.
Regarding the influence of incubation time, and as shown in
Fig. 3b, the percentage of protein removal increased with increasing
Fig. 3. Optimization of proteins removal rate: (a) Deproteinization degree at pH 8.0
and 50 ◦ C for 3 h using various E/S ratios (0, 1, 3, 5 and 10). (b) Effect of temperature
and proteolysis reactions duration at 40, 50 and 60 ◦ C during 3 and 6 h. The results
  
are presented by mean ± standard deviation. a,b,c and a ,b ,c Different letters with dif-
ferent E/S ratios indicate significant differences (p ≤ 0.05). A,B Different letters with
different temperatures and reaction times indicate significant differences (p ≤ 0.05).
Blue crab chitin Shrimp shells Shrimp chitin
81.95 ± 0.44 87.18 ± 0.05 91.20 ± 0.52
0.00 52.64 ± 0.50 0.00
1.17 ± 0.11 21.87 ± 1.13 4.02 ± 0.63
0.00 1.09 ± 0.20 0.00
– 23.57 ± 0.10 –
– 22.23 ± 0.94
incubation time from 3 to 6 h when deproteinization reactions were
conducted at 40 ◦ C. Indeed, it was improved from 75.96 ± 1.97%
to 85.10 ± 1.28%, for blue crab shells and from 70.25 ± 1.94% to
91.14 ± 0.67%, for shrimp shells. Further, regardless the source
of the chitinous matrix (blue crab or shrimp shells), almost the
same deproteinization rate was reached for reactions performed
at 40 ◦ C during 6 h and 50 ◦ C during 3 h (p > 0.05). Equally, 85.21%
and 92.93% of associated proteins were removed from blue crabs
and shrimp shells, respectively, when deproteinization was accom-
plished at 50 ◦ C during 6 h, which is not significant (p > 0.05)
(Fig. 3b).
On the other hand, deproteinization efficiency of P. segnis alka-
line proteases was similar to those reported by Maruthiah et al.
[24], Ghorbel-Bellaaj et al. [49] and Maruthiah and Palavesam [50],
for Bacillus sp APCMST-RS3 proteases, elastase from Pseudomonas
aeruginosa A2, and Paracoccus saliphilus APCMST-CS5 protease,
respectively. Nevertheless, the protein removal rate obtained using
P. segnis alkaline proteases was higher than those obtained with
other fish proteases reported in literature, such as crude proteases
extracted from red scorpionfish (S. scrofa) [11] and golden gray
mullet (Liza aurata) [14]. In addition, P. segnis alkaline proteases
were more able to remove associated proteins than proteases
from Aspergillus clavatus ES1, Bacillus licheniformis NH1 and Vibrio
metschnikovii J1 [51], as well as Bacillus sp. TKU004 [48] and Bacillus
invictae [47]. Zhang et al. [52] revealed, in their work, that fermenta-
tion of shrimp shells powder with Serratia marcescens B742 leaded
to a deproteinization efficacy of 83.56% after four days of incuba-
tion. The alimentation composition of P. segnis (mainly consisting
of other crustaceans and mollusks), that could define the nature
and specificity of its enzymes, may explain the high deproteiniza-
tion rates of extracted proteolytic enzymes from blue crab viscera
[11].
The fact that deproteinization cannot reach 100% could be
explained by the non-accessibility of enzymes to some proteins
protected by chitin, since they were associated by covalent bonds,
and it was proposed that small number of the C-2 amino groups of
chitin and some peptides are covalently linked [17,47]. Further-
more, enzymes could be inhibited by peptides produced during
deproteinization. In fact, enzymatic process generates, simulta-
neously, chitin, as well as a supernatant enriched with bioactive
molecules including low-molecular-weight biopeptides and free
amino acids [26]. When present in the reaction mixture, during
the deproteinization process, these biopeptides could hinder the
enzymes activities and thereby, no additional proteolysis could
take place. In this report, to determine whether liberated peptides
may hinder proteases activity during enzymatic process, the depro-
teinized residue was re-suspended in distilled water, and then,
subjected to a further deproteinization during 3 h, at 50 ◦ C and pH
8.0. Results revealed no enhancement in the deproteinization rate
(85.89 ± 1.29% in two successive steps vs 84.69 ± 0.64% in one step
deproteinization; p > 0.05). These results disprove our hypothesis
regarding inhibition of enzymes by peptides produced during the
proteolysis reaction.
On the other hand, commercial enzymes, Purafect and Neu-
trase, were used as control group for deproteinization experiments
 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463
Fig. 4. Comparison of P. segnis and P. kerathurus shells deproteinization efficiency by
means of P. segnis proteases and two commercial enzymes (Purafect and Neutrase).
The digestion reactions were realized, during 3 h, at pH 8.0, 50 ◦ C; pH 10.0, 50 ◦ C and
pH 7.0, 50 ◦ C, for P. segnis alkaline proteases, Purafect and Neutrase, respectively. The
  
results are presented by mean ± standard deviation. a,b,c and a ,b ,c Different letters
with different enzymes indicate significant differences (p ≤ 0.05).
at an E/S ratio equal to 5 U/mg of proteins. The deproteinization
degree obtained with Purafect was higher than that achieved with
blue crab crude proteases extract, reaching 90% and 92%, for blue
crab and shrimp shells, respectively (Fig. 4). Nevertheless, protein
removal rate accomplished with Neutrase was lower, around 70%
and 68%, for blue crabs and shrimp shells, respectively.
3.3.3. Chemical demineralization
To prepare pure chitin from blue crab and shrimp shells, the
obtained residues (85% and 91% for blue crab and shrimp shells,
respectively) using 5 U/mg of proteins, were treated with 0.55 HCl
solution in order to eliminate associated minerals as a second stage.
In this work, the adopted process was inspired from that reported
by Younes et al. [51], in which they demonstrated that three succes-
sive baths of HCl (0.55 M), during 30 min each at 4 ◦ C, allowed a full
shrimp waste demineralization. The blue crab and shrimp shells
demineralization degree obtained in this study was 100%, validat-
ing the effectiveness of this treatment in the minerals removal,
which was similar to that reported by Younes et al. [51].
The yields of chitin extraction obtained after deproteiniza-
tion and demineralization processes were about 19.06 ± 1.65% and
22.23 ± 0.94% for P. segnis and P. kerathurus, respectively (Table 2),
which were higher than that reached by Hajji et al. [20] for
crab (Carcinus mediterraneus) shells (10%). Nevertheless, they were
nearly similar to those obtained by Lopes et al. [17] for enzymatic
deproteinization of lobster Munida spp. shells via Alcalase (22%)
and Lassoued et al. [9] using Raja clavata crude digestive alkaline
proteases (21%) for shrimp (M. monoceros) wastes.
3.3.4. FTIR analysis
The structure of chitins was investigated by FTIR spectroscopy,
and compared with the commercial one (Fig. 5). Considering FTIR
spectrum, blue crab and shrimp chitins exhibited typical ␣-chitin
structure, with absorbance bands around 3445, 2935, 2875, 1654,
1560, 1392, 1021, 896 and 674 cm−1 [53]. Concentrated bands, cor-
responding to the intramolecular hydrogen bond OH (3)–O (5),
were detected at approximately 3445 cm−1 . The bands due to NH of
the amide group at 3265 and 3106 cm−1 are assigned to the vibra-
tional modes involved in intermolecular hydrogen bonding CO–HN
and the intramolecular bonds -NH groups, respectively [54]. The
peaks at 2935 cm−1 and 2875 cm−1 were assigned for stretch-
ing vibration of –CH3 and –CH2 , respectively. Moreover, splitting
461
Fig. 5. FT-IR spectra of commercial chitin (Com C) and chitins extracted from blue
crab P. segnis (CC) and shrimp P. kerathurus (SC) shells.
of the amide I band in the blue crab and shrimp chitins spectra
was observed, with occurrence of two peaks at 1654 cm−1 that is
attributed to the intermolecular hydrogen bonds CO–HN and at
1628 cm−1 due to the intramolecular hydrogen bond CO–HOCH2 .
This is typical and consistent with the ␣-chitin structure [55,56].
As well, a distinct band at 1429 cm−1 (–CH2 groups) occurred in
the FTIR spectra of extracted chitins [51]. Otherwise, no bands at
1540 cm−1 in the FTIR spectrum of blue crab (P. segnis) and shrimp
(P. kerathurus) chitin, proving the efficacy of deproteinization step,
and thereby, the purity of the extracted chitin [9]. FTIR spectra of
blue crabs and shrimp chitins were well matched with the com-
mercial one. High proportion of individual functional groups was
clearly seen in all chitins samples tested.
4. Conclusion
P. segnis viscera is found to be a source of alkaline proteases
active and highly stable in a broad range of pH 6.0–12.0. Alka-
line proteases showed an optimal temperature at 60 ◦ C and higher
stability was observed at low temperatures (<40 ◦ C). Additionally,
the findings established that crude protease extract is highly effec-
tive in the deproteinization of crab and shrimp wastes to produce
chitin. The highest deproteinization degrees (around 85% and 91%
for P. segnis and P. kerathurus shells, respectively) were obtained
with reaction carried out using an E/S ratio of 5 U/mg of proteins,
at 50 ◦ C and pH 8.0, for 3 h. Spectroscopic technique FTIR showed
that spectra of blue crab and shrimp extracted chitins were charac-
teristic of ␣-chitin. Therefore, the effectively application of P. segnis
proteases in chitin extraction from crab and shrimp shells may be
an interestingly alternative process, which would be helpful for the
seafood industries. Additional and comprehensive research is cru-
cial to purify and characterize proteases from blue crab viscera. As
well, bioconversion of chitin to biologically active chitosan seems
to be promising.
Acknowledgement
The “Ministry of Higher Education and Scientific Research”,
Tunisia, funded this work.
References
[1] R.L. Olsen, J. Toppe, I. Karunasagar, Challenges and realistic opportunities in
the use of by-products from processing of fish and shellfish, Trends Food Sci.
Technol. 36 (2014) 144–151.
462 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463
[2] T. Ordóñez-Del Pazo, L.T. Antelo, A. Franco-Uría, R.I. Pérez-Martín, C.G. Sotelo,
A.A. Alonso, Fish discards management in selected Spanish and Portuguese
métiers: identification and potential valorisation, Trends Food Sci. Technol. 36
(2014) 29–43.
[3] T. Rustad, I. Storro, R. Slizyte, Possibilities for the utilization of marine
by-products, Int. J. Food Sci. Technol. 46 (2011) 2001–2014.
[4] E. Sanmartin, J.C. Arboleya, I. Iloro, K. Escuredo, F. Elortza, F.J. Moreno,
Proteomic analysis of processing by-products from canned and fresh tuna:
identification of potentially functional food proteins, Food Chem. 134 (2012)
1211–1219.
[5] M.A. Abdel-Naby, S.A. Ahmed, H.R. Wehaidy, S.A. El-Mahdy, Catalytic, kinetic
and thermodynamic properties of stabilized Bacillus stearothermophilus
alkaline protease, Int. J. Biol. Macromol. 96 (2017) 265–271.
[6] A. Mageswari, P. Subramanian, S. Chandrasekaran, S. Karthikeyan, K.M.
Gothandam, Systematic functional analysis and application of a cold-active
serine protease from a novel Chryseobacterium sp, Food Chem. 217 (2017)
18–27.
[7] M. Nouri, F. Khodaiyan, S.H. Razavi, M. Mousavi, Improvement of chitosan
production from Persian Gulf shrimp waste by response surface methodology,
Food Hydrocolloid. 59 (2016) 50–58.
[8] R.C. Chien, M.T. Yen, J.L. Mau, Antimicrobial and antitumor activities of
chitosan from shiitake stipes, compared to commercial chitosan from crab
shells, Carbohydr. Polym. 138 (2016) 259–264.
[9] I. Lassoued, S. Hajji, S. Mhamdi, M. Jridi, A. Bayoudh, A. Barkia, M. Nasri,
Digestive alkaline proteases from thornback ray (Raja clavata): characteristics
and applications, Int. J. Biol. Macromol. 80 (2015) 668–675.
[10] M.F. Mortuza, M.H. Rahman, M.H. Rahman, A. Nahar, M.R.I. Khan, A.K.M.
Mahbub Hasan, M. Rahman, Isolation, biochemical and genetic
characterization of extracellular protease producing cattle hide dehairing
bacterium – a potential alternative to chemical dehairing, Ecol. Genet.
Genomics 2 (2017) 3–12.
[11] I. Younes, R. Nasri, I. Bkhairia, K. Jellouli, M. Nasri, New proteases extracted
from red scorpionfish (Scorpaena scrofa) viscera: characterization and
application as a detergent additive and for shrimp waste deproteinization,
Food Bioprod. Process. 94 (2015) 453–462.
[12] R. Nasri, H. Abed, M. Karra-châabouni, M. Nasri, A. Bougatef, Digestive alkaline
proteases from Serranus scriba viscera: characteristics, application in the
extraction of carotenoproteins from shrimp waste, and evaluation in laundry
commercial detergents, Biocatal. Agric. Biotechnol. 4 (2015) 355–361.
[13] A.C.V. Freitas-Junior, M.S. Costa, M.Y. Icimoto, I.Y. Hirata, M. Marcondes, L.B.
Carvalho, V. Oliveira, R.S. Bezerra, Giant Amazonian fish pirarucu (Arapaima
gigas): its viscera as a source of thermostable trypsin, Food Chem. 133 (2012)
1596–1602.
[14] I. Bkhairia, N. Ktari, I. Younes, M. Kammoun, M. Nasri, S. Ghorbel, Golden grey
mullet (Liza aurata) alkaline proteases: biochemical characterization,
application in the shrimp wastes deproteinization, laundry commercial
detergents, and preparation of antioxidant protein hydrolysate, J. Aquat. Food
Product Technol. 24 (2015) 597–613.
[15] A.E. Ghaly, V.V. Ramakrishnan, M.S. Brooks, S.M. Budge, D. Dave, Fish
processing wastes as a potential source of proteins, amino acids and oils: A
critical review, J. Microb. Biochem. Technol. 5 (2013) 107–129.
[16] A. Vannabun, S. Ketnawa, S. Phongthai, S. Benjakul, S. Rawdkuen,
Characterization of acid and alkaline proteases from viscera of farmed giant
catfish, Food Biosci. 6 (2014) 9–16.
[17] C. Lopes, L.T. Antelo, A. Franco-Uría, A.A. Alonso, R. Pérez-Martín, Chitin
production from crustacean biomass: sustainability assessment of chemical
and enzymatic processes, J. Clean. Prod. (2017), http://dx.doi.org/10.1016/j.
jclepro.2017.01.082.
[18] T. Senphan, S. Benjakul, H. Kishimura, Characteristics and antioxidative
activity of carotenoproteins from shells of Pacific white shrimp extracted
using hepatopancreas proteases, Food Biosci. 5 (2014) 54–63.
[19] N. Mezzomo, J. Martínez, M. Maraschin, S.R.S. Ferreira, Pink shrimp (P.
brasiliensis and P. paulensis) residue: supercritical fluid extraction of
carotenoid fraction, J. Supercrit. Fluids 74 (2013) 22–33.
[20] S. Hajji, I. Younes, O. Ghorbel-Bellaaj, R. Hajji, M. Rinaudo, M. Nasri, K. Jellouli,
Structural differences between chitin and chitosan extracted from three
different marine sources, Int. J. Biol. Macromol. 65 (2014) 298–306.
[21] A. Tolaimate, J. Desbrieres, M. Rhazia, A. Alagui, Contribution to the
preparation of chitins and chitosans with controlled physico-chemical
properties, Polymer 44 (2003) 7939–7952.
[22] S. Kumari, P.K. Rath, Extraction and characterization of chitin and chitosan
from (Labeo rohit) fish scales, Proc. Mater. Sci. 6 (2012) 482–489.
[23] M.C. Gortari, R.A. Hours, Biotechnological processes for chitin recovery out of
crustacean waste: a mini-review, Electron. J. Biotechnol. 16 (2013) 14-.
[24] T. Maruthiah, B. Somanath, G. Immanuel, A. Palavesam, Deproteinization
potential and antioxidant property of haloalkalophilic organic solvent
tolerant protease from marine Bacillus sp. APCMST-RS3 using marine shell
wastes, Biotechnol. Rep. 8 (2015) 124–132.
[25] O. Ghorbel-Bellaaj, S. Hajji, I. Younes, M. Chaabouni, M. Nasri, K. Jellouli,
Optimization of chitin extraction from shrimp waste with Bacillus pumilus A1
using response surface methodology, Int. J. Biol. Macromol. 61 (2013)
243–250.
[26] S. Mhamdi, N. Ktari, S. Hajji, M. Nasri, A. Sellami Kamoun, Alkaline proteases
from a newly isolated Micromonospora chaiyaphumensis S103:
characterization and application as a detergent additive and for chitin
extraction from shrimp shell waste, Int. J. Biol. Macromol. 94 (2017) 415–422.
[27] R. Nasri, I. Younes, I. Lassoued, S. Ghorbel, O. Ghorbel-Bellaaj, M. Nasri,
Digestive alkaline proteases from Zosterisessor ophiocephalus, Raja clavata, and
Scorpaena scrofa: characteristics and application in chitin extraction, J. Amino
Acids Special Issue: Proteins Enzymes Mar. Resour. 2011 (2011), 9 pp.
[28] I. Younes, S. Hajji, V. Frachet, M. Rinaudo, K. Jellouli, M. Nasri, Chitin
extraction using enzymatic treatment. Antitumor, antioxidant and
antimicrobial activities of chitosan, Int. J. Biol. Macromol. 69 (2014) 489–498.
[29] I. Younes, S. Sellimi, M. Rinaudo, K. Jellouli, M. Nasri, Influence of acetylation
degree and molecular weight of homogeneous chitosans on antibacterial and
antifungal activities, Int. J. Food Microbiol. 185 (2014) 57–63.
[30] A.K.M. Asaduzzaman, B.S. Chun, Characterization of digestive enzymes from
de-oiled mackerel (Scomber japonicus) muscle obtained by supercritical
carbon dioxide and n-hexane extraction as a comparative study, J. Food Sci
Technol. 52 (2015) 3494–3503.
[31] N. Ktari, H. Ben Khaled, I. Younes, I. Bkhairia, S. Mhamdi, I. Hamza, M. Nasri,
Zebra blenny (Salaria basilisca) viscera as a source of solvent-stable proteases:
characteristics, potential application in the deproteinization of shrimp wastes
and evaluation in liquid laundry commercial detergents, J. Food Sci. Technol.
51 (2014) 3094–3103.
[32] A.A. Kembhavi, A. Kulkarni, A. Pant, Salt-tolerant and thermostable alkaline
protease from Bacillus subtilis NCIM No. 64, Appl. Biochem. Biotechnol. 38
(1993) 83–92.
[33] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
of bacteriophage T4, Nature 227 (1970) 680–685.
[34] F.L. Garcia-Carreno, L.E. Dimes, N.F. Haard, Substrate-gel electrophoresis for
composition and molecular weight of proteases or proteinaceous proteases
inhibitors, Anal. Biochem. 214 (1993) 65–69.
[35] S. Hajji, O. Ghorbel-Bellaaj, I. Younes, K. Jellouli, M. Nasri, Chitin extraction
from crab shells by Bacillus bacteria. Biological activities of fermented crab
supernatants, Int. J. Biol. Macromol. 79 (2015) 167–173.
[36] AOAC, Official Methods of Analysis, 15th ed., Association of Official Analytical
Chemists, Washington, DC, USA, 1990.
[37] AOAC, Official Methods of Analysis, 16th ed., Association of Official Analytical
Chemists, Washington, DC, USA, 1995.
[38] K.K. Tshinyangu, G.L. Hennebert, Protein and chitin nitrogen contents and
protein content in Pleurotus ostreatus var. columbinus, Food Chem. 57 (1996)
223–227.
[39] AOAC, Official Methods of Analysis, 14th ed., Association of Official Analytical
Chemists, Washington, DC, USA, 1984.
[40] M.S. Rao, J. Munoz, W.F. Stevens, Critical factors in chitin production by
fermentation of shrimp biowaste, Appl. Microbiol. Biotechnol. 54 (2000)
808–813.
[41] B.A. Juarez-de La Rosa, P. Quintana, P.L. Ardisson, J.M. Yanez-Limon, J.J.
Alvarado-Gil, Effects of thermal treatments on the structure of two black coral
species chitinous exoskeleton, J. Mater. Sci. 47 (2012) 990–998.
[42] J.F. Silva, T.S. Esposito, M. Marcuschi, K. Ribeir, R.O. Cavalli, V. Oliveira, R.S.
Bezerra, Purification and partial characterization of a trypsin from the
processing waste of the silver mojarra (Diapterus rhombeus), Food Chem. 129
(2011) 777–782.
[43] S. Klomklao, S. Benjakul, H. Kishimura, M. Chaijan, 24 kDa trypsin: a
predominant protease purified from the viscera of hybrid catfish (Clarias
macrocephalus × Clarias gariepinus), Food Chem. 129 (2011) 739–746.
[44] A.G. Villalba-Villalba, J.C. Ramírez-Suárez, E.M. Valenzuela-Soto, G.C. Sánchez,
G.C. Ruiz, R. Pacheco-Aguilar, Trypsin from viscera of vermiculated sailfin
catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and
characterization, Food Chem. 141 (2013) 940–945.
[45] S. Klomklao, H. Kishimura, Y. Nonami, S. Benjakul, Biochemical properties of
two isoforms of trypsin purified from the intestine of skipjack tuna
(Katsuwonus pelamis), Food Chem. 115 (2009) 155–162.
[46] C. Jeuniaux, M.F. Voss-Foucart, Chitin biomass and production in the marine
environment, Biochem. Syst. Ecol. 19 (1991) 347–356.
[47] A. Hammami, M. Hamdi, O. Abdelhedi, M. Jridi, M. Nasri, A. Bayoudh,
Surfactant and oxidant-stable alkaline proteases from Bacillus invictae:
characterization and potential applications in chitin extraction and as
detergent additive, Int. J. Biol. Macromol. 96 (2017) 272–281.
[48] S.L. Wang, T.Y. Kao, C.L. Wang, Y.H. Yen, M.K. Chern, Y.H. Chen, A solvent
stable metalloprotease produced by Bacillus sp. TKU004 and its application in
the deproteinization of squid pen for ␤-chitin preparation, Enzyme Microb.
Technol. 39 (2006) 724–731.
[49] O. Ghorbel-Bellaaj, I. Younes, H. Maâlej, S. Hajji, M. Nasri, Chitin extraction
from shrimp shell waste using Bacillus bacteria, Int. J. Biol. Macromol. 51
(2012) 1196–1201.
[50] T. Maruthiah, A. Palavesam, Characterization of haloalkalophilic organic
solvent tolerant protease for chitin extraction from shrimp shell waste, Int. J.
Biol. Macromol. 97 (2017) 552–560.
[51] I. Younes, S. Hajji, M. Rinaudo, M. Chaabouni, K. Jellouli, M. Nasri,
Optimization of proteins and minerals removal from shrimp shells to produce
highly acetylated chitin, Int. J. Biol. Macromol. 84 (2016) 246–253.
[52] H. Zhang, S. Yun, L. Song, Y. Zhang, Y. Zhao, The preparation and
characterization of chitin and chitosan under large-scale submerged
fermentation level using shrimp by-products as substrate, Int. J. Biol.
Macromol. 96 (2017) 334–339.
[53] F.A. Al Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Extraction and
characterization of chitin and chitosan from marine sources in Arabian Gulf,
Carbohydr. Polym. 77 (2009) 410–419.
 M. Hamdi et al. / International Journal of Biological Macromolecules 101 (2017) 455–463 463

[54] G. He, Z. Wang, H. Zheng, Y. Yin, X. Xiong, R. Lin, Preparation, characterization [56] S. Liu, J. Sun, L. Yu, C. Zhang, J. Bi, F. Zhu, M. Qu, C. Jiang, Q. Yang, Extraction
and properties of amino ethyl chitin hydrogels, Carbohydr. Polym. 90 (2012) and characterization of chitin from the beetle Holotrichia parallela
1614–1619. Motschulsky, Molecules 17 (2012) 4604–4611.
[55] M.H. Mohammed, P.A. Williams, O. Tverezovskaya, Extraction of chitin from
prawn shells and conversion to low molecular mass chitosan, Food
Hydrocolloid. 31 (2013) 166–171.