Single-Step Purification of Chitosanases from Bacillus Cereus

International Journal of Biological Macromolecules 82 (2016) 291–298

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ournal homepage: www.elsevier.com/locate/ijbiomac

Single-step puriﬁcation of chitosanases from Bacillus cereus using expanded bed chromatography

a,∗ b

Nathália Kelly de Araújo , Maria Giovana Binder Pagnoncelli ,

a a

Vanessa Carvalho Pimentel , Maria Luiza Oliveira Xavier ,

a a a

Carlos Eduardo Araújo Padilha , Gorete Ribeiro de Macedo , Everaldo Silvino dos Santos

Laboratório de Engenharia Bioquímica, Departamento de Engenharia Química, Universidade Federal do Rio Grande do Norte – UFRN, Brazil

Laboratório de Bromatologia, Departamento de Farmácia, Universidade Federal do Rio Grande do Norte – UFRN, Brazil

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

Article history: A chitosanase-producing strain was isolated and identiﬁed as Bacillus cereus C-01. The puriﬁcation and

Received 22 June 2015

characterization of two chitosanases were studied. The puriﬁcation assay was accomplished by ion

Received in revised form

exchange expanded-bed chromatography. Experiments were carried out in the presence and in the

25 September 2015

absence of cells through different expansion degree to evaluate the process performance. The adsorption

Accepted 28 September 2015

experiments demonstrated that the biomass does not affect substantially the adsorption capacity of the

Available online 1 October 2015

matrix. The enzyme bound to the resin with the same extent using clariﬁed and unclariﬁed broth (0.32

and 0.30 U/g adsorbent, respectively). The fraction recovered exhibited 31% of the yield with a 1.26-fold

Keywords:

increase on the speciﬁc activity concerned to the initial broth. Two chitosanases from different elution

Chitosan ◦

Chitosanase steps were recovery. Chit A and Chit B were stable at 30–60 C, pH 5.5–8.0 and 5.5–7.5, respectively.

◦ ◦ 2+ 2+

B. cereus The highest activity was found at 55 C, pH 5.5 to Chit A and 50 C, pH 6.5 to Chit B. The ions Cu , Fe

2+ 2+

Bioseparation and Zn indicated inhibitory effect on chitosanases activities that were signiﬁcantly activated by Mn .

Ion exchange The methodology applied in this study enables the partial puriﬁcation of a stable chitosanase using a

Expanded bed adsorption

feedstock without any pre-treatment using a single-step puriﬁcation.

1. Introduction temperature and reaction time [9]. Thus, the enzymatic hydrolysis

method has been proposed to be preferable for producing bioactive

Chitosan, a natural cationic polysaccharide, is produced by the COS [10].

deacetylation of chitin. The chitosan molecule has shown various Chitosanases have previously been produced by a number of

properties such as functional, renewable, nontoxic and biodegrad- organisms, including fungi [2,11], plants [12], and bacteria [13,14].

able biopolymer. In recent years, chitosan has been used as artiﬁcial The chitosanases produced by Bacillus cereus are currently inves-

skin, absorbable surgical suture, wound healing accelerator and tigated by several studies [15–20]. The process of puriﬁcation of

also as chitoligosaccharides (COS) source [1]. The oligosaccharides these enzymes normally involves ammonium sulfate precipitation,

from chitosan have many biological properties such as: antitu- gel ﬁltration and packed ion exchange chromatography [16,21–23].

mor [2,3], immunoenhancing [1], antibacterial [4,5], antidiabetic These traditional methods imply in a large number of steps, exten-

[6] and hypocholesterolemic [7]. These functions are dependent on sive work time and scale up difﬁculties. Alternatively, the expanded

oligosaccharide chemical structure and molecular size [1,8]. bed adsorption (EBA) combines separation, concentration and cap-

The COS is obtained mainly using chemical and/or enzymatic ture of the target protein. This competitive biotechnological process

hydrolysis [8]. Enzymes are highly speciﬁc and act under mild has been proposed as a single-unit operation [24]. EBA allows

conditions. Moreover, the use of enzymes allows the reaction con- the capture of target proteins from feedstock without any pre-

trol mainly product formation by adjusting concentration, pH, clariﬁcation steps, which is helpful to industrial processes since it

contributes to reduction of time and costs [25].

The aim of this present study is to evaluate chitosanase puriﬁca-

tion using an economic as well as a fast chromatographic method.

∗ Initially, the adsorption behavior of chitosanase onto Streamline-

Corresponding author. Tel.: +55 84 3215 3769x214; fax: +55 84 3215 3770.

E-mail address: nakar [email protected] (N.K.d. Araújo). DEAE as well as biomass inﬂuence were either determined. After, a

http://dx.doi.org/10.1016/j.ijbiomac.2015.09.063

292 N.K.d. Araújo et al. / International Journal of Biological Macromolecules 82 (2016) 291–298

puriﬁcation protocol was carried out in order to recover and purify carried out using culture broth with cells and without cells. The

◦

the chitosanase. cells were removed by centrifugation for 20 min (4 C, 3000 rpm).

The empirical correlation of Richardson–Zaki [27] was applied

to deﬁne the bed expansion characteristics:

2. Materials and methods

u = uT · ε (1)

2.1. Materials

u is the superﬁcial velocity, ε is the bed voidage, uT is the ter-

Chitosan (85% deacetylated; molecular weight: 90–190 kDa)

minal velocity in an inﬁnite medium and n is the exponent of

acquired from Sigma–Aldrich Co. (Saint Louis, USA) was solubi-

Richardson–Zaki which expresses the ﬂow regime. The settled bed

lized using 0.1 M HCl [14]. Streamline DEAE resin was purchased

voidage was considered to have a value of 0.4 according to [28]. All

from GE healthcare (Uppsala, Sweden). BCA Protein Assay Kit was

experiments were carried out in triplicate.

acquired from Pierce (Rockford, USA). PC33 Siemens Kit was pro-

vided by Maria Alice Hospital, Natal/Brazil. Buffer solutions and

other chemicals were reagent grade and were used as supplied.

2.5. Operation of EBA

2.2. Isolation and identiﬁcation of chitosanolytic bacterium

The puriﬁcation assays were developed using EBA method-

ology. The streamline anionic exchanger resin containing the

The microorganism was isolated from soil samples (Natal/Brazil

ligand Diethylaminoethyl (DEAE) and a homemade EBA column

◦ ◦

S05 52 11 , Wo35 13 08.4 ). Serial dilutions from soil samples

(2.6 cm × 30.0 cm) were used to EBA chromatography. Glass micro-

−1

were inoculated on plates containing peptone (6.0 g L ), chitosan

spheres were added to enhance ﬂuid distribution at the column

−1 −1

(2.0 g L ), magnesium sulfate (0.5 g L ), potassium phosphate

inlet. The ﬂuid was pumped using a peristaltic pump (model

−1 −1 ◦

dibasic (1.0 g L ) and agar (15 g L ) and incubated at 30 C for 2

Perimax 12, Spetec). The vertical alignment of the column was

days. A single colony showing prominent chitosanolytic activity

guaranteed in all assays. All experiments were performed at room

was selected and subjected to morphological analyses, Gram- temperature.

staining and pathogenicity test with PC33 Siemens Kit. Polymerase

The column with 6.0 cm settled-bed height of resin was equil-

chain reaction (PCR) was performed to amplify part of the bacterial

ibrated with 50 mM Tris–HCl pH 8.5 buffer (buffer A) to give a

16S rRNA gene to additional microbial identiﬁcation. The forward

stable expansion degree of H/H0 = 1.5. A volume of 150 mL cul-

and reverse primers were 27F (5 -AGA GTT TGA TCC TGG CTC AG-

ture broth was then loaded (ﬂow-through step) onto column at

−

3 ) and 1525R (5 -AAG GAG GTG ATC CAG CC-3 ), respectively. The 1

150 cm h superﬁcial velocity. After, the bed was washed with

PCR products were analyzed by electrophoresis on agarose gel. Sub-

buffer A (120 mL). The elution was carried out by step-wise gra-

sequently, they were puriﬁed, quantiﬁed and used in sequencing

dient mode with 50 mM Tris–HCl pH 8.5 buffer containing 0.3 M

reaction. Four sequencing reactions were run for each sample using

(buffer B), 0.7 M (buffer C), and 1 M (buffer D) NaCl. The both steps,

the primers 518F (5 -CCA GCA GCC GCG GTA ATA CG-3 ), 800R (5 -

washing and elution, were conducted at the superﬁcial velocity of

−

TAC CAG GGT ATC TAA TCC-3 ) and 1492R (5 -TAC GGY TAC CTT 1

100 cm h . In all puriﬁcation steps, several fractions were collected

GTTA CGA CTT-3 ). The nucleotide sequence of the 16S rRNA gene

for protein quantiﬁcation, enzyme assay and electrophoresis.

of C-1 was determined by an ABI 3730 DNA Sequencer (Applied

Biosystems, USA) and compared to16S rRNA sequences in a NCBI

BLAST search. 2.6. Dynamic binding capacity

The adsorption performance of the expanded bed was studied in

2.3. Chitosanase production

an EBA column loaded with streamline DEAE to a settled bed height

of 6.0 cm. The effect of microbial cells on the breakthrough curves

Cells from the stock cultures were transferred to 50 mL aliquots

behavior was studied. The bed was expanded to an expansion

of medium and dispensed into 250 mL Erlenmeyer ﬂasks for the

−1

degree of 2.0 with buffer A at a superﬁcial velocity of 150 cm h .

investigation of chitosanase production. Subsequently, they were

◦ Subsequently, the unclariﬁed and clariﬁed feedstock was loaded

incubated at 120 rpm for 72 h at 30 C. The cultivation medium

−1 [39]. For clariﬁed feedstock, the centrifugation was carried out for

consisted of (g L ): peptone 6.0, chitosan 2.0, magnesium sulfate ◦

20 min (4 C, 3000 rpm). Then the protein fractions were withdrawn

0.5 and potassium phosphate dibasic 1.0. During cultivation, the

and assayed for protein concentration and chitosanase activity. The

culture broth was collected to measurement of chitosanase activ-

dynamic binding capacity of the bed was checked assuming that

ity and protein content. Cell growth was monitored by extraction

the enzymatic activity at the column exit reached 10% of the initial

and quantiﬁcation of total DNA content. The pellet originated from

activity (U/U0 = 0.1). This methodology was accomplished to avoid

3 mL of culture was used for DNA extraction using sodium dodecyl

the loss of the target product in the ﬂow-through.

sulfate (SDS) and phenol/chloroform/isoamyl alcohol [26]. The cul-

The dynamic binding capacity, QB (U of absorbed enzyme per

ture broth was used for further puriﬁcation by using expanded bed

gram of settled adsorbent) was calculated as follows: adsorption (EBA). V

U f V − V − CdV

0 f m V

2.4. Hydrodynamic of the bed m QB = (2) m

ads

The expansion characteristic of the bed was investigated with

◦ −1

U is the initial enzymatic activity (U mL ), V is the ﬁnal volume

an initial settled bed height of 6.0 cm at 25 C. The expanded bed 0 f

of the feed solution (mL), V is the volume at 10% breakthrough

height was visually checked as a function of incremental velocity m

−

1 (mL) and m is the adsorbent mass (g).

ranging from 30 to 200 cm h . The expansion degree to each veloc- ads

Three additional experiments at superﬁcial velocity of 100, 150

ity was registered and expressed as the ratio of the height of the

−1

and 200 cm h (expansion degree of 1.2, 1.5 and 2.0, respectively)

expanded (H) to the settled (H0) bed adsorbent. The ﬂow rate was

were performed using unclariﬁed broth to estimate the effect of the

kept constant for 10 min in each step before the estimation of the

expansion degree on the chitosanase breakthrough behavior.

bed height to ensure bed stabilization [38]. The experiments were

N.K.d. Araújo et al. / International Journal of Biological Macromolecules 82 (2016) 291–298 293

2.7. Effect of pH and temperature on the enzyme activity electrophoresed in a 12% PAGE containing 0.1% chitosan and then

placed on ice. After electrophoresis, the gel was rinsed with de-

The optimum pH for puriﬁed chitosanases was studied by using ionised water and incubated in 50 mM sodium acetate buffer, pH

◦

soluble chitosan as substrate with different pH values (3.0–10.0). 5.5, at 50 C for 1 h. The chitosanase activity on the gel was visu-

For pH stability proﬁle, the samples were dialyzed against a 50 mM alized by staining the gel with 0.1% Congo Red dye, followed by

◦

buffer solution at various pH values (3.0–10.0) for 24 h at 4 C. The de-staining with 1.0 M NaCl [31].

buffer systems were glycine–HCl (pH 3), acetate (pH 4.0–5.0), phos-

phate (pH 6.0–8.0) and bicarbonate-carbonate (pH 9.0–10.0). After 3. Results and discussion

dialysis, the enzyme activity was determined by the measurement

of the residual activity at pH 6.0. The optimum temperature for the 3.1. Identiﬁcation of chitosanase-producing strain

puriﬁed chitosanases was determined by the enzymatic activity at

◦

different temperatures (ranging from 30 to 80 C). The stability pro- B. cereus C-01, a chitosanase-producing strain, was isolated from

◦ ◦

ﬁle was determined after pre-incubation of the samples for 60 min soil samples in Natal/Brazil (S05 52 11 , Wo35 13 08.4 ). The Bacil-

◦

at various temperatures (30–80 C) and then the residual activity lus C-01 strain showed the highest chitosanase activity from 15

was measured. overall strains isolated. This strain did not show positivity for any

microorganisms identiﬁed by PC33 Siemens Kit. The C-01 was

2.8. Effect of various chemicals and surfactants on the enzyme selected and then maintained on nutrient agar. This material was

activity used throughout the study.

Strain C-01 is a gram-positive and grows in both aerobic

The action of various chemical agents on chitosanolytic activity and anaerobic environments. The culture showed colonies with

of puriﬁed enzymes was investigated. The puriﬁed enzyme samples rounded, irregular border, average size of 4 mm and white color.

2+ 2+ 2+ 2+ 2+ 2+

were pre-incubated with 5 mM of Mg , Cu , Fe , Ca , Zn , Mn According to the results of 16S rDNA nucleotide sequence (approx-

2+ ◦

and Ba ions in phosphate buffer 50 mM (pH 7.0) at 4 C during 2 h, imately 1.5 kbp), the strain C-01 showed identity with two strains

followed by measurement of residual chitosanolytic activity. The exhibiting 100% similarity (B. cereus and Bacillus thuringiensis). Sev-

enzyme sample incubated with buffer was used as control. eral works have showed difﬁculties to distinguish strains of B.

cereus and B. thuringiensis and have suggested that the two strains

2.9. Analytical methods are considered a single species [32–36]. Therefore, the isolate was

identiﬁed as B. cereus.

The chitosanase activity of the crude enzyme and chromato-

␮

graphic samples were measured by using 250 L of enzyme 3.2. Production of chitosanase from B. cereus C-01

sample incubated with 250 ␮L of 1% soluble chitosan (pH 6.0).

The mixture was heated in a water bath for 30 min and the tem- The chitosanase activity was measured in the supernatant of

◦

perature was adjusted to 55 C. The reaction was interrupted by B. cereus C-01 strain during various cultivation times. After 12 h

boiling the samples for 10 min [14]. The reducing sugars were of adaptation phase, an exponential increase of the protein con-

assayed in a spectrophotometer (Thermo Spectronic) using the tent as well as of the enzymatic activity was observed (Fig. 1). The

3,5-dinitrosalicylic acid method, in which -glucosamine was B. cereus C-01 started to secrete signiﬁcant levels of extracellular

taken as standard [29]. One unit (U) of chitosanase was deﬁned chitosanase after 6 h of cultivation and the maximal production

␮ d

as the amount of enzyme capable of generating 1.0 mol of - achieved up to 0.8 U/mL after 36 h (Fig. 1). Thereafter, the chi-

glucosamine per minute under the conditions described above. tosanase activity decreased remarkably. This behavior can possibly

The protein content was determined using the Bicinchoninic be explained by the presence of the protease activity in the medium

Acid method with a BCA Protein Assay Kit in which bovine serum and by the decline in the growth of the microorganism. The pro-

albumin (BSA) as taken as standard. The mass balance for protein duction of enzymes was closely related to the cell growth (noted

and enzyme activity was carried out in order to estimate the recov- by the increase in DNA concentration), i.e., the product is growth-

ery and puriﬁcation factor. In this case, the method used was based associated that is typical of a primary metabolism product. From

on the area under the curve [28] with the area being calculated Fig. 1, it also can be observed that the microorganism is at expo-

using the Software Origin (Microsoft Co., USA, version .6.0). nential growth phase between 8 and 30 h of the cultivation. After

40 h of growth it was observed a decrease of cell growth, which

2.10. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis is expected since cultivation was in a discontinuous mode. There-

(SDS–PAGE) and zymogram analysis fore after this time this decrease in the biomass can be due to

cellular lysis occurring in the cultivation. Also, it was observed an

After the ion-exchange adsorption, the selected samples were increase in the pH maybe due to consumption of amino acids or

subjected to electrophoresis of 12% polyacrylamide gel in the proteins. Taken together, these factors can contribute to culture

presence of SDS in order to estimate the purity of protein. decline. Results shown in Fig. 1 conﬁrm that the production of chi-

◦

These samples were heat treated at 100 C for 5 min according tosanase is cell-growth dependent and B. cereus C-01 is a promising

to the methodology described by Laemmli [30]. Twenty micro- chitosanase producer.

liters of each sample and the protein standards (marker) were Other studies using B. cereus for the production of chitosanase

applied to the gel. Different protein standards were used: phos- and chitinase also reported maximum yield of enzyme after the ﬁrst

phorylase b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic day of cultivation. The relationship between chitosanase activity

anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and and cell growth was veriﬁed as well [16,22,37].

␣

-lactalbumin (14.4 kDa) (Bio-Rad Co., Richmond, EUA). After elec-

trophoresis, the protein bands on the gel were visualized by silver 3.3. Hydrodynamic of the bed

staining. Besides the SDS–PAGE in denaturing conditions, the chi-

tosanase activity in gel was performed without reducing agent Clariﬁed and unclariﬁed culture broths were used in order

(SDS and ␤-Mercaptoethanol) and heating, i.e., a non-denaturing to obtain the characteristics of the bed expansion (Fig. 2). The

electrophoresis (zymogram) was carried out, as shown at right Richard–Zaki equation was applied to determine the hydrodynamic

(columns 6 and 7) of Fig. 6. In this case, the samples were stability of the adsorbent bed during expansion (ﬂuidization). The

294 N.K.d. Araújo et al. / International Journal of Biological Macromolecules 82 (2016) 291–298

◦

Fig. 1. Time-course of cell growth, chitosanase activity and protein content. C-01 was grown aerobically in broth medium at 30 C for 72 h: (᭿) chitosanase activity, () total

•

protein, ( ) pH and (ᮀ) DNA content.

Fig. 2. Behavior of bed height in according with superﬁcial velocity of ﬂuid.

Fig. 3. Effect of the presence of cells on the breakthrough curve behavior of chi-

Unclariﬁed (ᮀ) and clariﬁed culture broth (᭿).

tosanase into the resin Streamline DEAE. Unclariﬁed (ᮀ) and clariﬁed culture broth

(᭿).

expansion indexes were 4.76 and 4.03. The terminal velocities were

−1 −1

893.36 cm h and 807.70 cm h for clarified and unclarified cul- the unclarified broth should be applied directly to expand the bed.

ture broth, respectively. A decrease in the value of terminal settling The use of unclariﬁed culture has an important economic impact

velocity was due to an increase in the viscosity of the mobile phase. because the biological sample can be used without any previous

These results are in agreement with other studies reported in liter- treatment.

ature [38,39].

Some variables have been able to inﬂuence the bed expansion 3.4. Dynamic binding capacity

degree such as: liquid superﬁcial velocity, density of adsorbent par-

ticles and viscosity of feedstock [40]. The bed expansion increases The adsorption experiments were conducted to evaluate the

linearly with the superficial velocity. At a constant superficial veloc- dynamic binding capacity of chitosanase with clarified and unclari-

−1

ity of 200 cm h , the adsorbent was expanded evenly at a degree of ﬁed broth. The breakthrough curve analysis is essential to estimate

2.2 and 2.0 by using the clariﬁed and unclariﬁed broth, respectively. the maximum loading amount of target protein. Usually, a loss

It could be veriﬁed that expanded-bed height was higher in of 10% of target protein in the efﬂuent is acceptable [39].

clariﬁed culture broth. As reported by Anspach (1999) [40], the Fig. 3 presents the inﬂuence of culture broth on the shape of

interaction of adsorbent particles with cell surface, DNA and other breakthrough curves. The curves were similar for both systems,

substances may be the most limiting factor for obtaining a stable displaying little interaction between biomass in the expanded bed.

bed. These feedstock composition leads aggregation and conse- For unclariﬁed broth, the equilibrium was achieved quickly.

quently bed instabilities and channeling [40]. Nevertheless, the The results also showed that the binding capacity of unclari-

small difference between the bed expansion degree indicates that ﬁed culture broth (0.3 U/g adsorbent) was slightly lower compared

N.K.d. Araújo et al. / International Journal of Biological Macromolecules 82 (2016) 291–298 295

Fig. 5. The chromatogram of the chitosanase broth during adsorption onto Stream-

line DEAE using EBA. Content of protein () and enzyme activity (᭿).

Fig. 4. Effect of expansion degree on the breakthrough curve behavior of chitosanase

into the resin Streamline DEAE by using unclariﬁed broth. Expansion degree of 1.2

(᭿), 1.5 (•) and 2.0 ().

to the clariﬁed one (0.32 U/g adsorbent). This slightly difference

could suggest that the adsorption capacity of streamline-DEAE is

not hampered by the presence of the cell, at least until U/U0 = 0.1.

This result supports the behavior of the breakthrough curves. Other

authors have also reported weak inﬂuence of biomass on the

adsorption capacity and on the rupture curve [38,41].

Three experiments at expansion degree of 1.2, 1.5 and 2.0

were performed using unclariﬁed culture broth. Fig. 4 presents the

dynamic behavior of the process. For an expansion degree of 2.0

the adsorption of chitosanase into the resin was very fast. Sixty

millilitres of culture broth was enough to saturated 70% of resin.

The saturation condition (U/U0 = 1.0) was achieved after 80 mL of

culture broth. For an expansion degree of 1.2 and 1.5 the saturation

condition was not achieved. Under the industrial point of view, it

is more interesting to work with a breakthrough up to 10% because

the losses are reduced. In addition, for an expansion degree of 2.0

and 1.5, a breakthrough of 10% was found after 20 and 30 mL of cul- Fig. 6. SDS–PAGE analysis of the chitosanase from B. cereus C-01. Lanes: M – molec-

ture broth, respectively. High yields can be obtained reducing the ular markers; 1 – culture supernatant; 2 – load; 3 – washing; 4 – fraction eluted with

0.3 M NaCl; 5 – fraction eluted with 0.7 M NaCl; 6 – zymogram analysis for fraction

operation time, consequently, increasing the process productivity

[38]. eluted with 0.3 M NaCl; 7 – zymogram analysis for fraction eluted with 0.7 M NaCl.

3.5. Puriﬁcation of chitosanase

factors between 1.2 and 2.3 were obtained during the puriﬁcation

study of antigen of hepatitis B by EBA [43].

An extracellular chitosanase was partially puriﬁed from the cul-

The protein peak eluted with 0.3 M NaCl showed an apparent

ture supernatant of B. cereus C-01. The EBA chromatogram proﬁle

molecular mass of 33 kDa while the other one eluted with 0.7 M

is shown in Fig. 5.

NaCl showed an apparent molar mass of approximately 44 kDa.

From these results, it can be notice a larger peak of enzyme activ-

The molecular mass of these chitosanases were similar to other

ity at elution compared to the protein. As shown in Table 1, the

chitosanases produced by Bacillus strains, such as B. cereus TKU030

elution steps of EBA were combined to give an overall puriﬁcation

(43 kDa) [16], B. cereus TKU022 (44 kDa) [17], B. cereus S1 (45 kDa)

of about 1.26-fold for chitosanase. The overall activity yield of puri-

[44], B. subtilis 168 (30 kDa) [31] and B. subtilis CH (29 kDa) [45].

ﬁed chitosanase was 31%. The ﬁnal amount of protein obtained was 2

Even though the literature reported that the two distinct peaks

about 29 mg.

of chitosanases with different molecular mass can be related to

By the analysis of SDS–PAGE, it was possible to identify two

the production of enzymes with different mechanism of action

protein bands that were concentrated by chromatographic process

(endo and exochitosanases) in the present study we can not specu-

(Fig. 6). The ﬁrst one was eluted with 0.3 M NaCl and the second

late the real mechanism, since the occurrence of two exo-enzymes

with 0.7 M. Chitosanase activities of puriﬁed enzymes were demon-

could also be possible. Other studies have also been reported the

strated by the zymogram (Fig. 6, lanes 6 and 7). A puriﬁcation factor

production of two distinct forms of chitosanases [46,47]. The use

of 1.75 was obtained considering the fraction of 0.3 M NaCl. These

of single-step for enzyme puriﬁcation allowed the puriﬁcation of

analyses represent a good result if one considers that just a single

a chitosanase with good qualitative results as well as enzymatic

puriﬁcation step has been used. A puriﬁcation factor of 1.61 was

activity.

obtained for puriﬁcation of amylase using EBA [42]. Puriﬁcation

296 N.K.d. Araújo et al. / International Journal of Biological Macromolecules 82 (2016) 291–298

Fig. 7. Effects of pH on the activity (᭿) and stability of chitosanase (•) of Chit A (a) and Chit B (b). The pH stability proﬁle to the enzyme was determined by measuring the

◦

residual enzyme activity at pH 6 after 24 h of incubation in different pH values (3.0–10.0) at 4 C.

Fig. 8. Effects of temperature on the activity (᭿) and stability of chitosanase (•) of Chit A (a) and Chit B (b). The enzymatic samples were incubated for 60 min at temperature

◦ ◦

ranging from of 30 C to 80 C.

3.6. Effect of pH and temperature that the enzymes have a larger range of pH stability compared to

others studies. For example, for this same period of incubation, it

The fraction that was eluted with 0.3 M NaCl was named Chit A was found stability in a short range of pH (6.0–7.0) for chitosanase

and that one eluted with 0.7 M NaCl was named Chit B. These frac- puriﬁed from culture broth of Serratia sp. TKU016 [49]. Addition-

tions were used for the characterization experiments. The effect of ally, chitosanase produced by Janthinobacterium showed stability

pH on the catalytic activity was studied by using chitosan as sub- in a pH range from 5.0 to 7.0 [50]. Ultimately, it was veriﬁed that

strate under the standard assay conditions. The Chit A exhibited extreme pH reduces the enzymatic activity over time.

maximum values at pH 5.5 and Chit B at pH 6.5 (Fig. 7). The optimum The effect of temperature on chitosanase activity was also stud-

◦

pH of these chitosanases was similar to the others chitosanases ied. The optimum temperature for Chit A it was 55 C and for Chit

◦

already characterized; most of them reported the optimum pH B it was 50 C (Fig. 8). The enzyme solutions were incubated for

ranging from 4.0 to 6.0 [37,48]. After 24 h of incubation in differ- 60 min at various temperatures and the residual activity was then

ent pH, it was observed a plateau between pH 5.5 and 8.5 for Chit measured to assay the thermal stability. The Chit A maintained its

◦

A and between pH 5.5 and 7.5 for Chit B. These results showed initial activity at a range of temperature between 30 and 60 C. It

Table 1

Puriﬁcation of the chitosanase from B. cereus C-01.

Step Total Total activity (U) Speciﬁc Puriﬁcation factor Yield (%)

protein (mg) activity (U/mg)

Culture broth 121 63 0.52 1.0 100

Load 31 17 0.55 1.0 27

Washing 60 24 0.4 0.77 48

Streamline DEAE (Eluate 0.3 M NaCl) 9.0 8.0 0.9 1.75 13

Streamline DEAE (Eluate 0.7 M NaCl) 12 8.8 0.7 1.35 14

Streamline DEAE (Eluate 1.0 M NaCl) 8.0 2.2 0.27 0.52 4.0

N.K.d. Araújo et al. / International Journal of Biological Macromolecules 82 (2016) 291–298 297

respectively). The fraction recovered after the elution showed 31%

of yield and increased the speciﬁc activity to 1.26-fold compared to

initial one. Two protein fractions with chitosanolytic activity eluted

at different NaCl concentrations were recovered. One protein eluted

with 0.3 M NaCl showed an apparent molecular mass of 33 kDa and

was named Chit A. Other eluted with 0.7 M NaCl showed 44 kDa

◦

(Chit B). Chit A and Chit B were stable at 30–60 C, pH 5.5–8.0 and

◦

5.5–7.5, respectively. The highest activity was found at 55 C, pH 5.5

◦ 2+ 2+ 2+

to Chit A and 50 C, pH 6.5 to Chit B. The ions Cu , Fe and Zn

showed inhibitory effect on chitosanase activities. In contrast, the

enzymes were activated by Mn . The use of Streamline DEAE ionic

exchanger to expanded bed adsorption can be considered a good

system for the recovery of chitosanases from B. cereus C-01 since

it reduces the puriﬁcation steps. Reduction in costs and time are of

great importance for industrial processes, therefore the procedure

Fig. 9. Effect of ions on chitosanase activity of Chit A (a) and Chit B (b). The enzymatic described here has high potential for large scale applications.

◦ 2+ 2+ 2+ 2+ 2+

samples were incubated at 4 C for 2 h in the presence of: Mg , Cu , Fe , Ca , Zn , 2+ 2+

Mn and Ba . Acknowledgments

◦

was possible to keep 91% of its activity at 55 C. The temperature of

◦

The authors thank the Coordenac¸ ão de Aperfeic¸ oamento de

50 C was the best for activity of Chit B, also this enzyme showed

Pessoal de Nível Superior (CAPES) (2011116590) and the Con-

the similar stability from Chit A. The enzyme stability under its

selho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq)

optimum temperature is extremely important for industrial appli-

◦

(407648/2013-1) for the ﬁnancial support.

cations of enzymes. Temperature above 60 C promoted reduction

of both enzymes, i.e., the enzyme denaturation occurs after this

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