Biocatalysis and Agricultural Biotechnology 31 (2021) 101918

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Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Statistical optimization of halophilic chitosanase and protease production

by Bacillus cereus HMRSC30 isolated from Terasi simultaneous with chitin
extraction from shrimp shell waste
Hilmi Amanah Aditya Cahyaningtyas a, Wasana Suyotha a, *, Benjamas Cheirsilp a,
Shigekazu Yano b
a
Biotechnology for Bioresource Utilization Laboratory, Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, 90112,
Thailand
b
Department of Biochemical Engineering, Graduate School of Sciences and Engineering, Yamagata University, Jonan, Yonezawa, Yamagata, 992-8510, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Chitosanase is an industrially important enzyme involved in the production of bioactive chitooligosaccharides. In
Bacillus cereus HMRSC30 the present study, Bacillus cereus HMRSC30 isolated from the Indonesian shrimp paste was found to have high
Shrimp paste ability to assimilate shrimp shell powder (SSP), and selected as the microbial tool to produce low-cost chito­
Shrimp shell
sanase under statistical optimization. The highest chitosanase production (4.85 U/mL) was achieved under the
Chitosanase
optimal conditions: 20 g/L SSP, 3 g/L (NH4)2SO4, 0.3 g/L Mg2SO4, 1.5 g/L KH2PO4, 1.5 g/L K2HPO4, pH 5.5 with
Statistical optimization
a 15% (v/v) inoculum size at 35 ◦ C for 3 days incubation. Besides chitosanase, a high amount of protease
(33.47–72.19 U/mL) was also synthesized during 7 days fermentation. The optimal fermentation conditions also
simultaneously deproteinized (76.36–97.86%) and demineralized (24.07–67.22%) the SSP. These observations
indicated the potential of the strain HMRSC30 in a biological process for the enzyme production simultaneous
with chitin extraction. The purified chitosanase with an apparent MW of 41 kDa exhibited the highest specificity
to 75–85% deacetylated chitosan in soluble form (100%), followed by colloidal form (73.6%) and soluble chitin
(73.4%) in an acidic pH of 5.5 at 55 ◦ C. Additionally, it was discovered that the chitosanase obtained was a
halophilic enzyme which was fully activated by 2.5% (w/v) NaCl and displayed activity of 81.73% even in the
presence of 7.5% (w/v) NaCl. The enzyme yielded a putative dimer, and trimer of glucosamine as the pre­
dominant hydrolysis products. This study revealed that the properties of chitosanase from the strain HMRSC30
would be useful in industrial applications.

1. Introduction actinomycetes (Boucher et al., 1992). Nevertheless, bacteria have been

Chitosanase is an enzyme which specifically hydrolyzes the β-1,4-
glycosidic linkage in the chitosan molecule to generate monomer and
oligomers of glucosamine. The hydrolysis products by chitosanase are
well-known as bioactive compounds which have a wide potential
application in the food processing (Vela Gurovic et al., 2015), agricul­
tural (Ismail, 2019) and pharmaceutical industries (Cao et al., 2018).
This potential has nowadays made chitosanase gain a lot of interest from
the industrial sector. However, the industrial application of chitosanase
has the major impediment as its high production cost.
Chitosanase can be produced using a variety of microorganisms, such
as bacteria (Gao et al., 2008), fungi (Abdel-Aziz et al., 2014) and
most widely found to be preferable source for industrial chitosanase
production as they produce relatively high activity within a shorter time
of fermentation than fungi or actinomycetes. However, at present, the
high-cost production of chitosanase comes up due to an expensive me­
dium for microbial fermentation. Because bacterial chitosanase is an
inducible enzyme, the chitosan or its derivatives, which have high
market values, are required as inducers as well as a carbon source to
ensure a high yield of chitosanase synthesis (Rodríguez-Herrera et al.,
2008).
In recent years, crustacean by-products have been used as a nutrient
source for the production of enzymes (Liang et al., 2010; Doan et al.,
2018, 2019) and shrimp shell waste is a potential source with a high

* Corresponding author.
E-mail address: wasana.suy@psu.ac.th (W. Suyotha).

https://doi.org/10.1016/j.bcab.2021.101918
Received 23 June 2020; Received in revised form 23 November 2020; Accepted 12 January 2021
Available online 20 January 2021
1878-8181/© 2021 Elsevier Ltd. All rights reserved.
H.A.A. Cahyaningtyas et al.
chitin content of 25–30%. Therefore shrimp-shell waste could play an
important role in producing chitosanase, as well as minerals (30–50%),
proteins (26–40%) and lipids (1.2–2.4%) for microbial growth. This
would be beneficial not only for reducing the cost of production but also
for waste management.
Although production using an inexpensive medium may be possible,
it would provide only a low level of enzyme synthesis. Previous studies
have reported different chitosanase activity using shrimp shell powder
(SSP) by Paenibacillus marcerans TKU029 (0.022 U/mL) (Doan et al.,
2018), Paenibacillus mucilaginosus (0.53 U/ml) (Doan et al., 2019), Ser­
ratia marcescens TKU019 (0.04 U/mL) (Liang et al., 2010) and Bacillus
cereus TKU030 (0.04 U/mL) (Liang et al., 2014). These reported values
were lower than the production using chitosan as an inducer, for
example, chitosanase production using Acinetobacter sp. C-17 (2.8
U/mL) (Zhu et al., 2003), Bacillus sp. L1 (0.8 U/mL) (Zakaria et al.,
2012). Such low productivity can be ascribed to the complexity of the
chitinous substrate, which consists of a lipid-protein matrix surrounded
by a high quantity of minerals (Xu et al., 2013). For this reason, the
fermentation requires a potent bacterium, which is able to degrade the
complex structure and assimilate the nutrients inside of the substrate to
produce the desired high amount of chitosanase.
Chitosanase-producing bacteria with promising properties have been
isolated from different sources with most strains having been isolated
from soil, including Bacillus cereus TP12.24 (0.638 U/mL) (Wangtueai
et al., 2006) and Pseudomonas geniculata (0.722 U/mL) (Kassem et al.,
2013), while Janthinobacterium was isolated from a fresh water lake
(0.035 U/mL) (Johnsen et al., 2010), isolate B1211 from a hot spring
(0.406 U/mL) (Razak et al., 2019), as well as from beaches (Kaur et al.,
2012). However, there have been few reports on chitosanase production
using bacteria isolated from fermented shrimp products although there
is a high potential to discover excellent strains capable of degrading
shrimp shell.
Besides the variety of microorganism, fermentation conditions have
an important impact on enzyme synthesis and the influence of many
factors affecting chitosanase production has been reported. In addition
to the carbon source, the previous researches mentioned the importance
of inorganic nitrogen sources, such as (NH4)2SO4 to enhance chitosanase
synthesis (Zhang et al., 2012; Zhang and Zhang, 2013). Other factors
such as trace elements (Zhang and Zhang, 2013; Ismail, 2019), the initial
pH of the medium (Zhang et al., 2012), the cultivation temperature (Zhu
et al., 2003; Nidheesh et al., 2015) and the inoculum size (Zhang et al.,
2012) have also been noted to have an effect on the growth of micro­
organisms and their metabolism, and all these consequently influence
enzyme production. Interestingly, it was found that synergic in­
teractions of these factors also significantly influence enzyme produc­
tion. Therefore, response surface methodology (RSM) has commonly
been employed to investigate the interaction between each of the pa­
rameters, with a view to reducing the expense of analytical models by
proposing fewer experimental trials, and as a result reducing errors in
establishing the optimum conditions (Thadathil and Velappan, 2014).
According to background above, the aim of the present study was to
isolate bacteria which can effectively utilize shrimp shell waste from the
Indonesian fermented shrimp paste (Terasi). The enzyme production
conditions were optimized using RSM. In designing the experiment, a
Plackett-Burman design (PBD) was firstly used to determine the most
significant variables in chitosanase production using the selected strain,
Afterwards, a central composite design (CCD) was used to optimize the
level of the variables selected. Moreover, enzyme purification was
conducted to evaluate biochemical properties that were necessary in
relation to later applications.
2. Materials and methods
2.1. Materials
Shrimp shells (Penaeus monodon) were kindly supplied by Chotiwat
2
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
Manufacturing Co., Ltd. (Songkhla, Thailand). Indonesian shrimp paste
(Terasi) was acquired from Malang, East Java, Indonesia. Chitosan
powder (75–85% deacetylated), chitin and casein were purchased from
Sigma-Aldrich. Other chemicals and reagents used in this study were of
analytical grade. The chitooligosaccharide was kindly provided by Yaizu
Suisankagaku Industry, Japan.
2.2. Preparation of shrimp shell powder
The head, tail, and legs of the shrimps were removed and the shell
washed three times with tap water then dried at 60 ◦ C for 2 days. The
dried shrimp shell was then ground using a blender and sieved through
the given size of mesh sieve. The shrimp shell powder (SSP) used in this
study was able to pass through a 25-mesh sieve but could not pass
through a 40-mesh sieve. At this size, the SSP was easily able to be
separated from the inoculum after the fermentation process.
2.3. Preparation of chitosan substrate
Colloidal chitosan (1% w/v) was prepared by dissolving 1 g of
powder in 70 mL of 0.1 M acetic acid under magnetic stirring at room
temperature. The pH of the solution was adjusted to 7.0 using NaOH (6
M) and made up to 100 mL by adding distilled water. At this condition,
chitosan was form of soluble-insoluble particles and dispersed in
aqueous. This substrate was used for the enzyme assay.
2.4. Isolation and screening of chitosanase-producing microorganism
One gram of Terasi was mixed into 100 mL of mineral medium
containing 1 g/L (NH4)2SO4, 1 g/L K2HPO4, 1 g/L KH2PO4, 0.7 g/L NaCl
and 0.3 g/L MgSO4 with the addition of 1.5% (w/v) SSP, then incubated
at 37 ◦ C for 7 days in order to enrich the chitosan-assimilating micro­
organisms. The enriched culture was serially diluted in sterilized phys­
iological salt water (0.85% w/v NaCl) and 0.1 mL of the diluted solution
was spread on each of nutrient agar (NA), de Man, Rogosa and Sharpe
(MRS) agar and yeast malt (YM) agar to identify prospective microor­
ganisms. After incubation at 37 ◦ C for 24 h, colonies with different
appearance in shape, size, color and opacity grown on each of the me­
dium were selected and sub-cultured until pure colonies were obtained.
Each pure colony was streaked on a selective agar medium consisting of
mineral medium supplemented with 0.25% (w/v) colloidal chitosan and
15 g/L agar (pH 5.0 and 7.0). The isolates grown on this selective me­
dium were further individually incubated in 5 mL selective medium
broth containing 0.25% (w/v) colloidal chitosan at 30 ◦ C for 5 days.
2.5. Selection of SSP assimilated chitosanase-producing microorganism
The strains that produce supernatants with the highest chitosanase
activity were selected to test their ability to assimilate to SSP. Inoculums
were prepared by transferring a loopful of each selected isolate to a
nutrient broth (NB) medium. After incubation until the optical density of
the cells at 600 nm (OD600) was around 1.0, 5% (v/v) of inoculum was
transferred into a mineral medium supplemented with 15 g/L SSP and
incubated at 30 ◦ C for 5 days. The cell supernatant was sampled at 24 h
intervals for quantitative analysis of the chitosanase activity. In addi­
tion, the level of protease, which is useful for SSP degradation, was also
determined. The most potent isolate with the highest activity was cho­
sen for the next experiment.
2.6. Bacterial identification
The identification of the selected strain was performed based on
morphological characterization, including gram staining (Kaplan and
Kaplan, 1933), spore staining, and molecular classification according to
the similarity of its 16s rDNA. Genomic DNA was extracted and the se­
quences obtained from the polymerase chain reaction (PCR) were
H.A.A. Cahyaningtyas et al. Biocatalysis and Agricultural Biotechnology 31 (2021) 101918

compared to the Gene Bank database using the Basic Local Alignment Table 2
Search Tool (BLAST). The primer pairs used for the PCR were 27F Central composite experimental design and predicted results for chitosanase
(5′ -AGAGTTTGATCMTGGCTCAG-3′ ) and 1492R (5′ -TACGGTTACCTT activity.
GTTACGACTT-3′ ). The DNA fragments were sequenced and a homology Run Factor A: Factor B: Factor 3 C: Actual Predicted
search for the 16S rDNA sequences was conducted with BLAST (Klijn SSP (g/ Inoculum Temperature Value Value (U/
et al., 1994). Alignment similarities and a phylogenetic tree were L) size (%) (◦ C) (U/mL) mL)

created using Mega-X software. 1 25.946 20.946 37.973 3.03 2.40

2 20 5 35 3.33 2.85
3 14.054 20.946 37.973 3.10 2.85
2.7. Experimental design and statistical analysis 4 20 15 35 2.59 2.62
5 20 15 30 1.49 1.40
In the present study a PBD was initially employed to select significant 6 10 15 35 1.17 1.18
variables, with nine independent variables being identified comprising 7 20 25 35 1.57 2.30
8 20 15 35 2.29 2.62
(NH4)2SO4, yeast extract, KH2PO4, K2HPO4, SSP, glucose, incubation 9 14.054 20.946 32.027 0.84 0.49
temperature, inoculum size, and initial pH. All of which have previously 10 25.946 9.05396 32.027 2.35 2.42
been reported to be significant parameters influencing bacterial chito­ 11 25.946 9.05396 37.973 2.05 2.21
sanase. They were examined at the low level (− 1) and high level (+1). 12 14.054 9.05396 37.973 3.01 3.00
13 25.946 20.946 32.027 2.08 1.91
Table 1 shows the levels of each factor used in the experimental design.
14 20 15 40 2.86 3.21
In total, 12 experimental runs were generated using the nine variables. 15 20 15 35 2.52 2.62
The response in each was measured in terms of chitosanase activity (U/ 16 20 15 35 2.20 2.62
mL). The effect of all the variables on the enzyme activity was analyzed 17 14.054 9.05396 32.027 0.88 1.33
by analysis of variance (ANOVA). 18 30 15 35 1.46 1.71
19 20 15 35 3.74 2.62
Thereafter, the three most significant variables were selected and 20 20 15 35 2.45 2.62
their optimal level determined using a CCD to maximize the chitosanase
activity. Each variable was examined at five different levels; the axial
point (-α; +α), low level (− 1), middle level (0) and high level (+1). purity of the enzyme was confirmed by 12.5% Sodium dodecyl sulfate
Twenty experiments each conducted in three replications were carried polyacrylamide gel electrophoresis (SDS-PAGE) analysis as described by
out (Table 2) to determine the chitosanase activity. Laemmli (1970). The proteins were stained with 0.05% Coomassie
brilliant blue R-250.
2.8. Purification of chitosanase of strain HMRSC30 The zymogram analysis was performed in order to determined ac­
tivity of chitosanase in purified enzyme. The polyacrylamide gel was
The inoculum of the selected strain, Bacillus cereus HMRS30, was prepared using 0.1% (w/v) SDS and 0.01% (v/v) glycol chitosan. Sam­
prepared in NB medium and incubated at 30 ◦ C in an orbital incubator ple buffer was prepared without the addition of β-mercaptoethanol.
shaker at 150 rpm until the OD600 reached 1.00. The inoculum (15% v/ After electrophoresis, the gel was soaked in 50 mM acetate buffer
v) was then transferred into a chitosanase-producing medium contain­ contain 1% (v/v) Triton X-100, shaken gently for 6 h and maintained at
ing 20 g/L SSP, 3 g/L (NH4)2SO4, 0.3 g/L Mg2SO4, 1.5 g/L KH2PO4, 1.5 20 ◦ C to renature the enzyme. The enzyme was activated to promote the
g/L K2HPO4, pH 5.5 and incubated at 35 ◦ C for 3 days. The crude su­ reaction by socking the gel in 50 mM acetate buffer pH 5.5 at 37 ◦ C for
pernatant of the chitosanase HMRS30 was collected by centrifugation at 12 h. Afterwards, the gel was strained by 0.1% (w/v) congo red for 15
10,000 g at 4 ◦ C for 15 min. The activity of chitosanase from the strain min and de-stain by washing with 1 N NaCl for several times until the
HMRS30 was recovered by ammonium sulfate precipitation, with the clear mark appeared.
saturation in a range of 30–75%. The precipitant was resuspended in 10
mM acetate buffer, pH 5.0 (Buffer A) and desalted by dialysis against the 2.9. Characterization of purification of chitosanase HMRS30
same buffer at 4 ◦ C for 12 h. The dialysate was continuously loaded onto
a CM-Sepharose CL-6B 500 column (5 cm × 30 cm) equilibrated with The effect of pH on the activity of chitosanase HMRS30 was deter­
Buffer A at a flow rate of 1.0 mL/min. The column was washed by Buffer mined by incubating the reaction mixture containing 50 mM of various
A, followed by elution using Buffer A containing 50–200 mM NaCl. The buffers with pH ranging from 4 to 10. The enzyme was pre-incubated at
enzyme activity in all the fractions was determined and the fractions 4 ◦ C for 12 h in 100 mM of the same range of buffers before the residual
showing the highest chitosanase activity were collected and dialyzed activity was determined. The buffers used were acetate buffer (pH
against Buffer A for 12 h at 4 ◦ C. In this experiment, the protein con­ 4.0–6.0), potassium phosphate buffer (pH 6.0–7.5), Tris-HCl buffer (pH
centration was determined using the absorbance at 280 nm and the 7.5–9.5) and sodium carbonate buffer (pH 9.5–10.0). In order to

Table 1
Placket–Burman design matrix for evaluating factors influencing the activities of chitosanase.
Run Factor A: Factor B: Factor C: Factor D: Factor E: Factor F: Factor G: FactorH: Factor I: Actual Predicted
(NH4)2SO4 SSP (g/ MgSO4 KH2PO4 K2HPO4 Initial Temperature Glucose Inoculum value (U/ value (U/
(g/L) L) (g/L) (g/L) (g/L) pH (◦ C) (g/L) size (%) mL) mL)

1 3.0 15 0.3 0.5 1.5 7.0 37 0.0 20 2.57 2.78

2 1.0 15 0.3 0.5 0.5 5.5 30 0.0 5.0 1.12 0.63
3 3.0 15 0.6 0.5 0.5 5.5 37 10 20 2.90 2.69
4 3.0 15 0.6 1.5 0.5 7.0 30 0.0 5.0 1.08 1.14
5 1.0 15 0.3 1.5 1.5 7.0 30 10 20 1.64 1.66
6 1.0 30 0.6 0.5 1.5 5.5 30 0.0 20 2.18 2.46
7 3.0 30 0.3 1.5 1.5 5.5 37 0.0 5.0 3.99 3.80
8 1.0 30 0.3 0.5 0.5 7.0 37 10 5.0 2.17 2.44
9 1.0 30 0.6 1.5 0.5 7.0 37 0.0 20 3.88 3.39
10 3.0 30 0.3 1.5 0.5 5.5 30 10 20 3.72 3.91
11 1.0 15 0.6 1.5 1.5 5.5 37 10 5.0 1.05 1.47
12 3.0 30 0.6 0.5 1.5 7.0 30 10 5.0 2.08 2.02

3
H.A.A. Cahyaningtyas et al.
measure the optimum temperature, the reaction mixture in 50 mM ac­
etate buffer, pH 5.5 was incubated at temperatures ranging from 25 to
80 ◦ C. The thermal stability was determined by measuring the residual
activity of the chitosanase after incubation at those temperatures for 10
min.
Several substrates comprising 0.5% (w/v) of soluble and insoluble
forms of chitosan, chitin, and carboxymethyl cellulose (CMC) were used
to determine the substrate specificity of the purified chitosanase
HMSR30.
The effect of metal ions on the activity was studied at 55 ◦ C. The
enzyme was incubated in solution of CaCl2, ZnCl2, CuCl2, MgCl2•6H2O,
FeSO4•7H2O, Fe(NO3)3•9H2O, CoCl2•6H2O, and MnSO4•H2O at final
concentration being 1, 5 and 10 mM. The relative activities were
determined under standard assay protocol and compared with a control
without metal ions.
In order to recognize the effect of salt on the activity of chitosanase
HMSR30, the enzymatic reaction was measured in the presence of
0–10% (w/v) NaCl.
2.10. Analysis of hydrolysis product from chitosan by chitosanase
HMRSC30
Hydrolysis was performed using 1% (w/v) soluble chitosan in 0.1 M
acetate buffer (pH 4.5) at 55 ◦ C for 0.5–72 h. The hydrolysis products
collected after different times were put on thin-layer chromatography
(TLC) plates (silica gel 60 F254; Merck, Darmstadt, Germany). The hy­
drolysis products were separated in a developing solvent consisting of n-
butanol: 25% ammonium solution: acetic acid: water (10:1:5:5 v/v), and
detected by spraying with 0.2% ninhydrin dissolved in butanol, and then
heating at 100 ◦ C until the components could be visualized (Huynh
et al., 2019).
2.11. Enzyme assay
2.11.1. Chitosanase assay
The chitosanase activity was determined according to the Miler
method (1959). The reaction mixture containing 250 μl of 1% (w/v)
colloidal chitosan and 25 μl of 1 M acetate buffer (pH 5.5), was pre-
incubated at 37 ◦ C in a water bath for 5 min. Then 225 μl crude
enzyme was added to the mixture and incubated for 1 h. The enzymatic
reaction was terminated by heating at 100 ◦ C for 10 min. The super­
natant was collected by centrifugation at 10,000 g for 10 min, and 300 μl
of supernatant was then added to 400 μl of DNS solution. After incu­
bation at 100 ◦ C for 15 min, the absorbance at 540 nm was measured.
Heat-inactivated enzyme was used to conduct a blank assay. One unit of
chitosanase was defined as the amount of enzyme which liberated 1
μmol of reducing sugar as glucosamine per min (Nanjo, 1990).
2.11.2. Protease assay
A quantity of 130 μl of 0.65% (w/v) casein solution in 50 mM
phosphate buffer was preincubated at 37 ◦ C for 5 min before 25 μl
enzyme was added. The reaction mixture was continuously incubated at
37 ◦ C for 10 min. Then, 130 μl of 10% (w/v) trichloroacetic acid was
added and the mixture incubated again for 30 min at the same tem­
perature. 250 μl of supernatant was then added to 625 μl of 500 mM
Na2CO3 and 125 μl Folin-Ciocalteu reagent, which was then incubated at
37 ◦ C for 30 min before the absorbance at 660 nm was measured. One
unit of chitosanase was defined as the amount of enzyme which released
1 μmol of tyrosine per min (Cupp-Enyard, 2008).
2.12. Characterization of crude chitin
The residual SSP was recovered to determine protein and ash content
in order to indicate the biological chitin extraction occurred during
cultivation of B. cereus HMRSC30. The recovered SSP was considered as
crude chitin in this study.
4
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
The degree of demineralization of crude chitin is generally deter­
mined through ash content analysis. The dried samples were placed in a
muffle at 550 ◦ C for 12 h and the ash was then collected and weighed.
The degree of demineralization (DM) was calculated using the following
equation (Younes et al., 2012):
(Ao × So) − (AR ​ × SR )
DM (%) = × 100
(Ao x So)
where AO and AR are the ash content of the raw SSP and fermented SSP
(crude chitin), respectively, and SO and SR are the dry weight of the raw
SSP used in fermentation and the residual fermented SSP (g) collected
after fermentation, respectively.
The degree of deproteinization (DP) was determined through the
percentage of protein content removal. Chitin extraction from simples
was done according to the procedure described by Tshinyangu and
Hennebert (1996) and the obtained product was used for determination
of chitinous nitrogen. The nitrogen content in samples was measured
according to the Kjeldahl method (AOAC, 1990). The actual protein
content was calculated by the subtraction of chitinous nitrogen from
total nitrogen content, then multiplied by 6.25 (Aranday-García et al.,
2017). Afterwards, DP was calculated using the following equation
(Younes et al., 2012):
(Po × So) − (PR ​ × SR )
DP (%) = × 100
(Po x So)
where PO and PR are the protein concentrations detected in the raw SSP
and fermented SSP (crude chitin), respectively, and SO and SR are the dry
weight of the raw SSP and residual fermented SSP (g), respectively.
The spectral characteristics of the crude chitin were analyzed by
Fourier transform infrared spectroscopy (FTIR) according to the method
described by Gharieb et al. (2015). The sample analyzed was mixed with
KBr powder and the analysis was conducted at wavelengths between
400 and 4000 cm− 1.
3. Results and discussion
3.1. Selection and identification of SSP-assimilating and chitosanase
producing bacteria
Terasi can be considered to be a promising source of bacteria capable
to effectively degrade shrimp shell. In the primary state of isolation, the
sample was mixed with mineral medium supplemented with 1.5% (w/v)
SSP for 1 week to enrich the target bacteria. The enriched culture was
then spread on NA, MRS and YM agar medium to isolate all the pro­
spective bacteria, bacilli and lactobacilli as well as actinomycetes.
Noteworthy, although the mineral medium supplemented with 0.25%
(w/v) soluble and colloidal chitosan were used to isolate the target
bacteria as described previously (Huynh et al., 2019), no colony was
able to grow. As a result, 114 isolates with different appearance mor­
phologies were transferred to a mineral medium contained 0.25% (w/v)
colloidal chitosan in order to screen the chitosan-assimilating bacteria.
Of those, 27 isolates were able to grow and were selected to test their
ability to produce extracellular chitosanase in a broth medium con­
taining 0.25% (w/v) colloidal chitosan. At this stage, 7 isolates with
chitosanase activity exceeding 0.5 U/mL (Supplementary Fig. 1) were
chosen for tertiary screening to ensure their capacity to produce chito­
sanase from SSP. Fig. 1 shows that the isolate HMRSC30 exhibited the
highest chitosanase activity of 2.51 ± 0.21 U/mL after 4 days incuba­
tion. Moreover, the hydrolases involved in degrading the SSP were
determined. Protease activity of 87.34 ± 0.05 U/mL was also detected in
the crude supernatant collected after 4 days incubation.
Morphological observation under a microscope showed that isolate
HMRSC30 was a gram positive (Fig. 2A) and spore-forming (Fig. 2B)
short-rod shaped bacteria. The homology search for 16s rDNA sequences
indicated that isolate HMRSC30 was 100% similar to bacteria belonging
H.A.A. Cahyaningtyas et al.
Fig. 1. Chitosanase activity (A) and protease activity (B) of selected chitosanase-producing bacteria on mineral medium supplemented with 1.5% (w/v) SSP.
to the group of Bacillus cereus as shown in the phylogenetic tree
(Fig. 2C). Therefore, the isolate HMRSC30 was identified as Bacillus
cereus HMRSC30 (Gene accession number MT505694) and was selected
as the optimum microorganism for chitosanase production. A number of
known chitosanase-producing bacteria have been classified within the
Bacillus group. B. cereus is one of the indigenous bacteria found in fer­
mented shrimp products (Chukeatirote, 2016). Following the several
stages of screening employed in this study, it can be considered that
B. cereus HMRSC30 might be useful in degrading shrimp shell in order to
produce chitosanase.
3.2. Screening of significant variables using placket-burman design
Many chemical and physical parameters, including the composition
of the medium, its initial pH, the culture temperature and the inoculum
size have been reported to be important factors influencing the pro­
duction of microbial chitosanase (Zhu et al., 2003; Zhang et al., 2012;
Zhang and Zhang, 2013; Nidheesh et al., 2015). In order to identify the
most influential variables for chitosanase production by B. cereus
HMRSC30, a PBD was used in the selection of the variables tested.
Although the aim of this study was to produce chitosanase using SSP as
the carbon source, the effect of glucose was explored since it is capable
of supporting bacterial growth and consequently may enhance enzyme
production (Reischke et al., 2014). The enzyme activity was determined
at 24 h intervals for 5 days with the highest activity being detected after
5
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
3 days incubation. Table 1 shows the value of the chitosanase activity
detected during 12 experimental runs. The activity was in a range of
1.05 U/mL to 3.99 U/mL. The maximum activity (3.99 U/mL) was found
on run number 7 using a fermentation medium without a glucose sup­
plement, but containing 30 g/L SSP, 3 g/L (NH4)2SO4, 0.3 g/L Mg2SO4,
1.5 g/L KH2PO4, 1.5 g/L K2HPO4, pH 5.5 with a 5% (v/v) inoculum size,
at 37 ◦ C.
The ANOVA of the chitosanase activity is shown in Supplementary
Table 1. The p-value of 0.0416 indicated the significance of the model at
the p ≤ 0.05 level. The regression equation derived was as follows:
Chitosanase ​ = + 2.36586 ​ + ​ 0.35807 ​ * ​ A ​ + ​ 0.63974 ​ * ​ B ​
− ​ 0.17033 ​ * ​ C ​ + ​ 0.19580 ​ * ​ D ​ − ​ 0.12811 ​ * F
+ ​ 0.39539 ​ * ​ G ​ + 0.44916 ​ * ​ J ​ (1)
where A is (NH4)2SO4, B is the concentration of SSP, C is MgSO4. D is
KH2PO4, F is the initial pH, G is the temperature, and J is the inoculum
size.
Among the nine variables, SSP, inoculum size and incubation tem­
perature were found to be three most significant factors affecting chi­
tosanase production by B. cereus HMRSC30. The result was different
from previous reports using PBD to screen main variable. Chitosanase
production by Aspergillus fumigatus YT-1 was significantly affected by
(NH4)2SO4, SSP and MgSO4 (Zhang and Zhang, 2013) while Hashem
(2018) reported the most foremost effects of agitation speed, chitosan
H.A.A. Cahyaningtyas et al.
Fig. 2. Morphological and molecular identification of isolate HMRSC30 by gram staining (A), spore staining (B) test, and analysis of phylogenetic tree (C).
and incubation time on chitosanase production by Dothideomycetes sp.
NRC-SSW. Ismail (2019) found that time period of microwave pre­
treatment of SPB, FeSO4⋅7H2O and KCl were positive factors for chito­
sanase production of Bacillus cereus strain SSW1. These observations
indicated that the effect of factors varied among microorganisms
examined. However, carbon source might be identical factor provides
the remarkable effect towards microbial chitosanases production since
its effect was proved by the reports mentioned above.
Microbial chitosanase is well known as inducible enzyme. The sup­
plement of inducer, especially chitosan, in cultivation medium could
induce the highest level of chitosanase production (Pagnoncelli et al.,
2010; Huynh et al., 2019). In contrast, some microorganism could
produce chitosanase constitutively (Kim et al., 2004). According to re­
sults obtained, the chitosanase production by B. cereus HMRSC30 was
also observed in presence of both glucose and SSP (Table 1). However,
the level of enzyme production was relative lower as compared with that
from the cultivation without glucose. The finding speculated that SSP
could be used as carbon source as well as inducer for chitosanase
HMRSC30 synthesis. The amount of the inoculum inserted into the
medium has also been noted to influence biomass production and tem­
perature in a given range might contribute to microorganism growth and
consequently facilitate the microbial utilization of the SSP (Nidheesh
et al., 2015).
3.3. Determination of optimal level of significant variables
The three independent variables (SSP, inoculum size, and tempera­
ture) which most significantly affected the chitosanase production were
selected to determine their optimal level using a CCD with each variable
being tested at axial point (-α; +α), low level (− 1), middle level (0) and
high level (+1) with 20 experiments being formulated.
6
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
While the ANOVA shown in Supplementary Table 2 for the model
was significant at the 95% confidence level, with the F value of the
model being 3.06, the model was barely significant, with a 4.79% pos­
sibility of the result being the result of coincidence or measurement
noise. A lack of fit coefficient of 0.4429 was obtained, showing that the
model was sufficient to predict the enzyme production (Zhang et al.,
2012). The final three-variable model including interactions between
the variables representing chitosanase activity was as follows:
Chitosanase ​ = − ​ 36.97853 ​ + ​ 13.92966 ​ * ​ A ​ − ​ 0.40248 ​ * ​ B ​
+ ​ 1.45506 ​ * ​ C ​ + ​ 0.023384 ​ * ​ AB ​ 0.26525 ​ * ​ AC ​ + ​ 9.78995E
− 003 ​ * ​ BC ​ − ​ 1.18250 ​ * ​ A2 ​ − ​ 4.75685E − 004 ​ * ​ B2
− ​ 0.012721 ​ * ​ C2 ​
(2)
where A is the concentration of SSP, B is the inoculum size and C is the
temperature.
The model estimated that temperature was a significant independent
variable (p = 0.0065) and its interaction with SSP as shown in supple­
mentary Fig. 2B also significantly influenced the chitosanase production
(p-value of AC = 0.0456). Among the 20 experiments, run number 19
produced the highest chitosanase activity of 3.74 U/mL with the addi­
tion of a 15% inoculum size, 20 g/L SSP and a temperature of 35 ◦ C,
while the predicted result suggested that the maximum activity would
be 2.62 U/mL (Table 2). The actual result therefore yielded a 1.5-fold
increase in chitosanase activity compared with the activity obtained
before optimization (2.51 U/mL), indicating that RSM is a useful way of
determining the optimum cultivation conditions for enhancing the ac­
tivity of chitosanase. The results of this study therefore agree with those
of previous studies reported for Asperigillus fumigatus YT-1 (Zhang and
H.A.A. Cahyaningtyas et al.
Table 3
Comparison of chitosanase production by B. cereus HMRSC30 with other studies.
Microorganism Substrate Chitosanase activity (U/mL)
P. macerans TKU029 1% SSP 0.022
P. mucilaginosus 1% SSP 0.53
B. cereus TKU022 1.5% SSP 0.8
S. marcescens 0.5% SSP 0.16
B. cereus TKU031 1% SSP 0.039
Bacillus sp. KCTC 0377BP 2.5% Chitosan 1.2
P. ehimensis 1% Chitosan 0.5
B. cereus 1% Chitosan oligomer 0.22
Aeromonas 0.501
Zhang, 2013), Bacillus sp. RKY3 (Wee et al., 2009), and Bacillus moja­
vensis (Liaqat et al., 2018).
In comparison with previous researches on chitosanase production
using different strains of B. cereus (Table 3), the activity obtained in this
study was higher than those previously obtained from SSP as carbon
source using B. cereus TKU031 (0.039 U/mL) (Wang et al., 2014),
B. cereus TKU022 (0.6 U/mL) (Liang et al., 2012), B. cereus C-01 (0.8
U/mL) (Araújo et al., 2016) and B. cereus 739 (1.17 U/mL) (Aktuganov,
2002). Moreover, the result was found to be higher than the value ob­
tained by B. cereus (0.22 U/mL; Rodríguez-Herrera et al., 2008), Paeni­
bacillus ehimensis (0.5 U/mL) (Pagnoncelli et al., 2010) and Aeromonas
(0.501 U/mL) (Rodríguez-Herrera et al., 2008) which used chitosan and
chitooligomer as the carbon source. On the other hand, it was lower than
the results reported by Gao et al. (2009) who achieved chitosanase ac­
tivity of 4.85 U/mL using B. cereus strain D-11, which might be due to
the existence of protease activity (Aktuganov, 2002), which was highly
evident with the strain used in the present study.
3.4. Time course bioconversion of SSP to chitosanase, protease and crude
chitin by B. cereus HMRSC30
The chitosanase production was explored again under optimal con­
ditions for 7 days. The highest activity of 4.85 ± 0.09 U/mL was ach­
ieved after 3 days incubation (Fig. 3A), which was shorter than the
period observed before optimization (Fig. 1A) indicating that the culture
conditions affected the production period of chitosanase by B. cereus
HMRSC30. Thereafter, chitosanase activity declined because of the in­
crease in protease production. The level of protease production was also
investigated since it is well-known as an important enzyme assisting the
degradation of SSP. The production of this enzyme gradually increased
during the cultivation period (Fig. 3B) and after 3 days the activity was
46.24 ± 0.37 U/mL, and reached 72.19 ± 0.20 U/mL at 7 days.
Simultaneous production of chitosanase and protease in one-time
fermentation has also been reported for B. cereus TKU022, producing
0.6 U/mL of chitosanase and 0.5 U/mL of protease (Liang et al., 2012)
and Serratia marcescens, which produced 0.04 U/mL and 0.18 U/mL of
chitosanase and protease, respectively (Liang et al., 2010). The result
was also in good agreement with other studies which have reported on
the production of chitosanase using SSP and other crustacean wastes
(Liang et al., 2012; Doan et al., 2018, 2019).
In recent years, several studies have noted the potential of protease-
producing bacteria in biological processes used to extract chitin from
crustacean substrates (Sorokulova et al., 2009; Haddar et al., 2011;
Hongkulsup, 2016). Therefore, in this experiment, an attempt was also
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
Assay condition Reference
0.1 mL Chitosan (1% in 50 mM phosphate buffer), Doan et al. (2018)
0.1 mL enzyme, 30 min, 37 ◦ C
0.1 mL of 1% water soluble chitosan in 20 mM Doan et al. (2019)
Tris-HCl buffer,
0.1 mL enzyme,
30 min 37 ◦ C
1 mL of 0.3% water soluble chitosan in 50 mM Liang et al. (2012)
phosphate buffer, 0.2 mL enzyme,
30 min, 37 ◦ C
1 mL of 0.3% water soluble chitosan in 50 mM Liang et al. (2010)
phosphate buffer, 0.2 mL enzyme, 30 min, 37 ◦ C
1 mL of 0.3% water soluble chitosan in 50 mM Wang et al. (2014)
phosphate buffer, 0.2 mL enzyme,
30 min, 37 ◦ C
0.3 mL chitosan, 0.3 mL enzyme, 10 min, 50 ◦ C Choi et al.(2004)
0.5 mL enzyme, 0.5 mL soluble chitosan (pH 6.0), 30 min, Pagnoncelli et al.(2010)
55 ◦ C
9.5 mL of 1.32% chitosan in 50 mM sodium acetate buffer, Rodríguez-Herrera et al.(2008)
0.5 mL enzyme,
40 ◦ C, 180 min
made to investigate the level of chitin extraction from SSP which
occurred during fermentation. The fermented SSP was recovered for the
determination of its protein and ash content over the course of time
along with enzyme production. Noteworthy, the chitin content in raw
SSP used in our study was 27.33% of its dry weight and contained 6.92%
nitrogen which was the comparable to theoretical value of pure chitin
being 6.9% (Liu et al., 2012). As shown in Fig. 3C, 83.00 ± 0.07%
deproteinization (DP) occurred after 3 days fermentation, which was the
peak time of chitosanase production. The degree of DP increased to
97.86% after 7 days fermentation with high protease production. The
degree of demineralization (DM) was below 40% at an early stage of
fermentation but it reached 67.21 ± 0.04% at 7 days (Fig. 3D). The
results obtained were comparable with those for crude chitin obtained
by bioconversion using B. cereus 8–1 of 78.6% DP and 73% DM (Sor­
okulova et al., 2009), S. marcescens B742 of 48.32% DM (Zhang et al.,
2017), Lactobacillus sp. B2 of 56% DP (Flores-Albino et al., 2012) and
Lactobacillus helveticus of 70% DP and 60% DM (Arbia et al., 2017).
The FTIR analysis shown in Fig. 4 indicated that the spectral char­
acteristics of the fermented SSP sampled after 3 and 7 days of fermen­
tation were similar to those of commercial chitin. The spectrum showed
characteristic bands of an amino group (NH2) at 3447 cm− 1, the trans-
configuration of the NH–CO group of chitin at 3110 cm− 1 and an acetyl
group (CH3CO) at 1655 cm− 1. Additionally, the peak at 2923 cm− 1
indicated CH stretching and that at 1558 cm− 1 indicated the bending
and stretching of N–H (Kumirska et al., 2010; Demir et al., 2016).
In terms of the correlation between the outcomes obtained, it is
interesting to highlight the probability of gaining chitosanase, protease
and crude chitin from one fermentation process of SSP by B. cereus
HMRSC30. However, the level of protease obtained would be increased
by prolonging the fermentation time, which is necessary to assure the
good quality of the chitin with high DP and DM. Unfortunately, the trend
of the chitosanase activity obtained under the cultivation conditions in
this study was to decline with an increasing level of protease production.
Therefore, the optimization of protease for the production of chitin will
be dealt with in a future study.
3.5. Purification of chitosanase HMRSC30
Initially, ammonium sulfate precipitation at 30–75% saturation was
employed to recover the chitosanase HMRSC30 from the crude super­
natant obtained at the optimal cultivation conditions. Thereafter, the
enzyme was purified using a CM-Sepharose CL-6B 500 column (5 cm ×
30 cm) equilibrated with 10 mM acetate buffer (pH 5.0). The activity of
chitosanase with high specific activity was detected in the fraction
7
H.A.A. Cahyaningtyas et al.
eluted by 200 mM NaCl. The specific activity was approximately double
that of the crude supernatant and after purification the recovery
increased 1.84-fold with a 13.18% yield of chitosanase (Table 4).
SDS-PAGE was conducted and the result showed that contaminated
protein was separated from the chitosanase fraction (Fig. 5). Further,
zymogram analysis was conducted to confirm the presence of chitosa­
nase as proposed by Haddar et al. (2011). As shown in lane 4 of Fig. 5,
the result confirmed the chitosanase activity band with a molecular
weight (MW) of 41 kDa, where a clear zone was observed, indicating
chitosan’s hydrolysis activity towards the glycol chitosan contained in
the gel. The result showed that the purified enzyme contained only one
protein capable of degrading chitosan, which can be reliably taken to
indicate that no further purification was possible.
The apparent MW of the chitosanase from B. cereus HMRSC30 esti­
mated by this method was the same as that of purified recombinant
chitosanase of B. cereus D-11 (41 kDa) produced in the study of Gao et al.
(2008). Moreover, it was similar to that in previous reports from the
same strain, i.e., Bacillus TKU022 (44 kDa) (Liang et al., 2012), B. cereus
S1 (45 kDa) (Kurakake et al., 2000) and a Bacillus sp. with a high identity
(99%) to B. cereus (47 kDa) (Zhou et al., 2015).
3.6. Characterization of chitosanase HMRSC30
The effect of pH and temperature on chitosanase HMRSC30 was
examined. The maximum activity was detected in the acidic condition
with acetate buffer pH 5.5 and decreased as the pH level increased
(Fig. 6). The highest chitosanase activity has also been previously re­
ported at an acidic pH in a range of 4–6. For example, chitosanase from
Bacillus sp. (Zhou et al., 2015), Pseudomonas geniculata (Kassem, 2013),
P. mucilaginosus TKU032 (Doan et al., 2019), and B. cereus TKU030
Fig. 3. Time course of chitosanase activity (A), protease activity (B), degree of deproteinization (% DP) (C) and degree of demineralization (% DM) (D) of crude
chitin under optimal conditions for the chitosanase production.
8
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
(Liang et al., 2014). Whereas there have been reports of chitosanase
exhibiting optimum specific activity at pH values of 7–9, including
chitosanase from B. cereus TKU022 (Liang et al., 2012), and B. cereus
GU-02 (Goo and Park, 2014). Meanwhile, Liang et al. (2018) reported
chitosanase activity in both acidic and alkaline conditions. In practical
use, acidic chitosanase is preferable for industrial production because it
can act in conditions where the pH must be lower than 6.5 to dissolve
chitosan. In relation to temperature, the enzyme exhibited high activity
at 55 ◦ C and maintained its stability at over 80% from 30 to 50 ◦ C.
Interestingly, approximately 50% of its original activity was retained at
75–80 ◦ C (Fig. 6B), indicating that this enzyme is thermally stable. Of
note, high temperature (50–80 ◦ C) is one factor enhances the solubility
of chitosan as well as lowering its viscosity (Desbrieres, 2002), allowing
the hydrolysis of chitosan occurs easily. Therefore, chitosanase stable at
such high temperature was particularly useful for application in COS
production.
Fig. 6C shows the substrate specificity of partially purified chitosa­
nase HMRSC30 towards chitosan, chitin and CMC. The enzyme showed
its highest specificity towards soluble chitosan (100%), followed by
colloidal chitosan (73.69%). Similar results were reported by Choi et al.
(2004), Lee et al. (2006) and Yi et al. (2004). On the other hand, its
specificity to CMC was only 1.17%. Although chitosanase HMRSC30
effectively hydrolyzed soluble chitin (73.42%), its hydrolysis ability
reduced remarkably against colloidal chitin (26.67%) and chitin powder
(1.71%). Specificity to chitin is a characteristic often found among
chitosanases, for instance, in the chitosanase from the Bacillus sp. strain
KCTC 0377BP (Choi et al., 2004). This might be because chitosanase has
been theorized to cleave the hetero-linkage of glucosamine (GlcN) and
N-acetylglucosamine (GlcNAc); GlcN-GlcNAc, and GlcNac-GlcN in
polymers of chitin with deacetylation lower than 50% (Zhu et al., 2007).
H.A.A. Cahyaningtyas et al. Biocatalysis and Agricultural Biotechnology 31 (2021) 101918

Fig. 4. FTIR spectrum of commercial chitin, crude chitin recovered after 3 days and 7 days of fermentation.

Table 4
Purification results of chitosanase produced from SSP by B. cereus HMRSC30.
Purification Total Total Specific Purification Yield
step activity protein activity (fold) (%)
(U) (mg) (U/mg)

Crude 885.83 20.13 44.00 1.00 100.0

supernatant
30–75% 180.43 6.75 26.71 0.83 20.36
(NH4)2SO4
CM-Sepharose 116.76 1.44 81.09 1.84 13.18
CL-6B 500

Fig. 6D indicates that chitosanase HMRSC30 was negatively affected

by most of the metal ions tested, with the exception of MnSO4, which
slightly enhanced its activity at a concentration of 1 mM. However, the
activity decreased at higher concentrations of MnSO4. The role of
MnSO4 as an activator has been reported for chitosanase from Bacillus
sp. TS (Zhou et al., 2015) and B. cereus GU-02 (Goo and Park, 2014).
The effect of NaCl is also worth noting here since B. cereus HMRSC30
was isolated from Terasi containing a high salt concentration (2–20%)
(Aristyan et al., 2014). It was thus expected that the enzyme would be
either halotolerant or halophilic. Interestingly, the activity of chitosa­
nase HMRSC30 was positively enhanced by the addition of NaCl. The
activity was obviously increased with 0.5–2.5% NaCl (Fig. 6E). At
concentrations beyond 5.0%, the chitosanase activity gradually
decreased. However, 45% of the activity remained at 10% NaCl. To the
best of our knowledge, halophilic chitosanase has not been reported,
although there are few reports on halophilic chitinase from Aspergillus
flavus (Beltagy et al., 2018), and halotolerant chitinase from Talaromyces
stipitacus (Paranetharan et al., 2018). This observation offers important
opportunities in practical applications of chitosanase. For instance, it
could allow the enzyme to be used in the scaled up production of chi­
tooligosaccharide in a saline environment to prevent microbial Fig. 5. SDS-PAGE (12.5%) analysis of purified chitosanase. M, Markers; Lane 1,
contamination, as well as enhancing the hydrolysis (Miller, 1959) electrophoresis of sample from crude supernatant; 2, 30–75% (NH4)2SO4 pre­
(Reischle et al., 2014). cipitation; 3, cation exchange chromatography; 4, zymogram analysis of the
purified chitosanase with white band indicating chitosanase activity.

9
H.A.A. Cahyaningtyas et al.
Fig. 6. Characterization of purified chitosanase HMRSC30. The results show effect of pH (A), and temperature (B) on the activity and stability of the enzyme,
substrate specificity (C), effect of metal ions (D) and salt concentrations (E) on the activity, and hydrolysis products analyzed by TLC (F). Relative activity for effect of
metal ions and salt was the percentage of activity with addition of each metal ion compared to the control without any chemicals. TLC analysis used GlcN and
chitooligosaccharide (YS–COS) as standards.
3.7. Analysis of hydrolytic product
In order to investigate the mode of action of chitosanase HMRSC30,
the hydrolysis products were analyzed over the course of time by TLC
(Fig. 6F). In the initial stage of hydrolysis at 0.5 h, chitosanase
HMRSC30 cleaved chitosan to oligomers with a degree of polymeriza­
tion larger than 3 (DP3). With extensive digestion up to 72 h, it released
shorter oligomers with a degree of polymerization of 2 (DP2) and 3
(DP3) as the predominant product and a minor oligomer of DP 5, while
monomeric product was undetectable. This result indicated the endo-
splitting of chitosanase HMRSC30. Similar results have been reported
for chitosanases from B. cereus GU-02 (Goo and Park, 2014), B. cereus
D-11 (Gao et al., 2009), and Bacillus sp. MET1299 (Kim et al., 2004), all
of which yielded a final chitosan degradation composed mainly of
oligomers ranging from DP2 to DP5.
4. Conclusion
The B. cereus HMRSC30 isolated from Indonesian Terasi exhibited the
ability to produce chitosanase from shrimp shell waste. Through the
statistical approach adopted in this study, the variables influencing
chitosanase production and their optimum levels were determined to
achieve the highest chitosanase activity of 4.85 U/mL. In addition,
shrimp shell-degrading protease was also produced at a noticeable level
(72.19 U/mL) which positively impacted the chitin extraction. The
10
Biocatalysis and Agricultural Biotechnology 31 (2021) 101918
results exhibited considerable deproteinization (83%) and demineral­
ization (40%) after the optimal cultivation time (3 days) for chitosanase
production. This finding shows the potential of B. cereus HMRSC30 for
the production of chitosanase simultaneous with chitin production in a
single fermentation. Moreover, the purified chitosanase obtained is a
valuable enzyme for practical use in industrial applications. Moreover,
its halophilic property and its ability to maintain its stability under high
thermal and acidic conditions are advantageous in a hydrolytic process
to produce chitooligosaccharides.
CRediT authorship contribution statement
Hilmi Amanah Aditya Cahyaningtyas: Data curation, Visualiza­
tion, Investigation, Software, Writing - original draft. Wasana Suyotha:
Conceptualization, Methodology, Data curation, Supervision, Writing -
review & editing. Benjamas Cheirsilp: Supervision, Writing - review &
editing. Shigekazu Yano: Supervision, Writing - review & editing.
Acknowledgments
This work was supported by the Higher Education Research Pro­
motion and the Thailand’s Education Hub for Southern Region of
ASEAN Countries (TEH-AC) Scholarship from Prince of Songkla Uni­
versity. The authors gratefully acknowledge the Thailand Research Fund
under Grant No. RTA6280014. Thanks are also due to PSU Research and
H.A.A. Cahyaningtyas et al. Biocatalysis and Agricultural Biotechnology 31 (2021) 101918

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