Optimization of reversed micellar extraction of chitosanases produced by Bacillus Cereus.

Optimization of reversed micellar extraction of

chitosanases produced by Bacillus cereus

Ya-Ling Chen

, Chia-Kai Su

, Been-Huang Chiang

*

a_{Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC}

b_{Department of Leisure, Recreation, and Tourism Management, Southern Taiwan University of Technology, Tainan, Taiwan, ROC}

Received 4 April 2005; received in revised form 25 September 2005; accepted 26 September 2005

Abstract

The fermentation broth of Bacillus cereus NTU-FC-4 was precipitated with 70% acetone to obtain crude enzyme. Chitosanases in the crude

enzyme were then extracted by reversed micelles. It was found that proper amount of crude enzyme should be first dissolved in the 50.0 mM

phosphate buffer containing 96.0 mM sodium chloride to make a 1.0 mg/ml protein solution. After adjusting the pH of the crude enzyme solution to

a value of 4.0, the aqueous solution was mixed with an organic solution, the isooctane containing 102.3 mM of the anionic surfactant AOT (sodium

1,2-bis(-2-ethylhexyl) sulfosuccinate). The mixture was shaken in reciprocating shaker bath at 15 8C for 85 min to solubilize the target enzymes in

the reversed micelles formed in the organic phase, thus completed the forward extraction. Then, the reversed micellar phase was separated from the

aqueous phase, and allowed to mixed with 50 mM phosphate buffer containing 1.0 M potassium chloride at pH 10. After mixing the two solutions

at 40 8C for 40 min, the target enzymes in the reversed micelles transferred back to the aqueous solution. The processes recovered approximately

70% of total activity of chitosanases. The purity of the chitosanases was increased to 30-fold as compared to that of the fermentation broth, and the

specific activity of the final product was 60.3 unit/mg.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Chitosanases; Bacillus cereus; Reversed micelles; Extraction; Optimization; Enzyme

1. Introduction

Chitosan has been recognized as a health promoting food

supplement since it possesses antibacterial activity

[1–5]

,

hypocholesterolemic activity

[6–8]

, and anti-hypertensive

action

[9]

. However, increasing attention has recently been

given to the conversion of chitosan to oligosaccharides.

Chito-oligomers show interesting biological activities, such as

antitumor activity

[10–12]

, immuno-enhancing effects

[13]

,

protective effects against infection with some pathogens

[14,15]

, antifungal activity

[16]

, and antimicrobial activity

[3,4]

.

Chitooligosaccharides can be prepared by chemical or

enzymatic hydrolysis. However, drawbacks, such as acid

corrosion, the need for neutralization after reaction, and low

yield of products with degree of polymerization (DP) equal or

larger than 6 (DP) limit the practical application of acid

hydrolysis. Chitosanases, which represent a class of hydrolytic

enzymes, are found in bacteria, fungi, and plants

[17]

. Among

these, bacterial chitosanases appear to be especially useful for

the production of chito-oligomers. Bacillus cereus NTU-FC-4,

a strain originally isolated from Taiwan soil by Hung

[18]

was

found to be able to produce high amounts of extracellular

chitosanases along with a minor amount of chitinase during

fermentation. However, a practical method for extraction and

purification of these enzymes from the culture broth needs to be

established in order to fully explore the industrial applications

of these enzymes. The chitosanases from various sources have

been purified using the conventional protein purification

techniques including ammonium sulfate fractionation, gel

filtration, ion-exchange chromatography, and isoelectric

focus-ing

[17]

. These methods are often used in laboratory practice,

but scaling-up of them for commercial production might

encounter the problem of limited processing capacity.

Reversed micelles are the aggregates of amphiphilic

molecules in an organic solvent. When the reversed micelles

are formed with an anionic surfactant, such as AOT, they would

display a surface of negative charge surrounding an aqueous

www.elsevier.com/locate/procbio

* Corresponding author. Tel.: +886 2 23632821; fax: +886 2 23620849. E-mail address: bhchiang@ntu.edu.tw (B.-H. Chiang).

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.09.018

polar core. Because of the electrostatic interactions, the

positively charged proteins could transfer from the aqueous

phase to the inner core of the reversed micelles, thus effect a

separation

[19–25]

. Reversed micellar extraction is an

attractive separation method for large-scale operation because

the process could be carried out using the existing liquid–liquid

extraction system in the chemical and biochemical industries.

Factors affecting the performance of the reversed micelle

system are rather complicated, including the nature and

concentration of target protein, pH, and ionic strength of the

aqueous phase, extraction temperature, type and concentration

of the surfactant, and the processing time

[26–28]

. Therefore,

investigation of the effects of these processing parameters on

the performance of reversed micellar extraction often requires

tedious experimental works. In this study, all of the processing

parameters were considered and pre-tested to screen the factors

that had a dominant effect on the process performance. Then,

the response surface methodology was used to develop the

mathematical functions describing the relationships between

these factors and the recovery rate of chitosanases during

extraction. Thus, the optimal processing conditions for the

purification of chitosanases by the reversed micellar extraction

could be established.

2. Materials and methods

2.1. Materials

Chitin, glucosamine, dioctyl sulfosuccinate sodium salt (AOT), potassium chloride, polyacrylamide, and Coomassie brilliant blue R-250 were purchased from Sigma Chemical Co. (St. Louis, MO). Crab chitosan with 66% deacetyla-tion was obtained from Ohka Enterprises Co. (Kaohsiung, Taiwan). Other materials used in this study included Soyton and yeast extract (Difco Lab. Sparks, MD), 2,2,4-trimethylpentane (isooctane) (Mallinckrodt Baker Inc., Phillipsburg, NJ), Bio-Rad Dc protein assay kit (Bio-Rad Lab., Hercules, CA), and various chemicals of reagent grade.

2.2. Crude enzyme preparation

The B. cereus isolated from Taiwan soil and was kindly supplied by Professor Lee of the Department of Agricultural Chemistry of National Taiwan University. The microbe was cultured in a 500-ml glass jar containing 150 ml of the medium composed of 0.3% colloidal chitin, 0.5% yeast extract, 0.5% soyton, 0.1% potassium dihydrogen phosphate, and 0.5% magnesium sulfate at pH 6.24. The jars were incubated in reciprocating shaker at 30 8C for 48 h. The fermentation broth was centrifuged at 6500
g for 40 min at 4 8C, and acetone was added to the supernatant until its concentration reached 70%. The resulting solution was centrifuged at 7000 rpm for 10 min at 4 8C. The precipitate was dried by lyophilization and used as crude enzyme.

2.3. Reversed micellar extraction

The aqueous solutions were prepared by dissolving an appropriate amount of the freeze-dried crude enzyme in 50 mM of sodium phosphate buffers at pH 3, 4, or 5. Sodium chloride was added to the aqueous solution to adjust the ionic strength. The organic solution was prepared by dissolving a designated amount of AOT in isooctane. For the forward extraction (i.e. inclusion of enzyme in the reversed micelles), equal volumes (ca. 5 ml) of the organic solution and aqueous solution were mixed in a centrifugal tube (15 ml) at approximately 200 rpm in a reciprocating shaker bath for various time periods and temperatures. The resulting mixture was then centrifuged at 1000
g for 10 min to separate the two phases. The upper layer (reversed micellar solution, the organic phase)

was further processed by the subsequent backward extraction (i.e. release of the enzyme from the reversed micelles to the aqueous solution). For backward extraction, the organic solution from forward extraction and equal volume of 50 mM phosphate solution at pH 10.0 containing 1 M KCl were mixed. The mixture was held at 40 8C in a water bath for 5 min, shaken at 150 rpm for 40 min, and centrifuged at 1000
g for 5 min to separate the two phases. Samples of aqueous phase were then taken for analysis.

2.4. The experimental design

There were six experimental factors that might have affected the recovery of chitosanase activity during reversed micellar extraction. This include protein concentration, pH, and NaCl concentration in aqueous phase; AOT concentra-tion in the organic phase; and extracconcentra-tion temperature and time. To reduce the number of experimental variables to the level that can be handled practically, initial studies were focused on determining the proper protein concentration (0.5–5.0 mg/ml), extraction temperature (10–30 8C), and time (15–155 min). These factors were determined using the aqueous solution containing 50.0 mM of NaCl at pH 4, and the organic solution was 100.0 mM of AOT in isooctane. For studying the proper initial protein concentration, the extractions were carried out at 15 8C for 85 min. Once these variables were determined, the effects of the other three factors on the recovery of chitosanase activity were further determined experimentally based on a Box–Behnken design[29]. Two sets of experiments were designed and carried out. For the first set of experi-ment, the pH were set at 3, 4, or 5; AOT concentrations were 50, 200, or 350 mM, and sodium chloride concentrations were 50, 200, or 350 mM. The pH for the second set of experiment were 4.0, 4.5, or 5.0; AOT concentrations were 50, 100, or 150 mM; and sodium chloride concentrations were 30, 90, or 150 mM. The mathematical equations giving the activity recovery as functions of these variables were then developed.

2.5. Model building and data analysis

A regression procedure in the SAS package (SAS Institute Inc., Cary, NC) was used to fit the activity recovery data into second-order polynomial equations with interaction terms:

Y¼ B0þ Bi X Xiþ Bii X Xi2þ Bi j X Xi j ði 6¼ jÞ (1)

where Y is the dependent variable, B0, Bi, Bii, and Bijregression coefficients of

the model and Xiare magnitudes of the selected critical variables. An F-test for

lack of fit was used to determine whether the regression models adequately fit the experimental data. Once the regression models were developed, non-linear programming techniques were used to search the maximum recovery of chitosanase activity. A commercial linear and non-linear programming package ‘‘AMPL’’ (The Scientific Press, San Francisco, CA)[30]was used to search for the optimal conditions.

2.6. Analytical methods

The protein concentration was determined by the modified Lowry method using Bio-Rad protein Dc protein assay kit [31]. SDS-polyacrylamide gel electrophoresis using 10% acrylamide was performed and stained by Coomas-sie blue R-250[32]. The sheets were destained with acetic acid/methanol/water solution (1/3/6, v/v/v). A pre-stained protein standard (SeeBlue Plus2, Invitro-gen Co., Carlsbad, CA) was used during SDS-PAGE for determining the molecular weights of the separated proteins. Chitosanase activity was deter-mined by measuring the reducing sugar produced from chitosan. Chitosan was dissolved in the 0.2 M acetate buffer at pH 5 to make a 1% (w/v) chitosan solution. A mixture consisting of 1 ml of 1% chitosan solution, 3.5 ml of 0.2 M acetic acid solutions, and 0.5 ml of enzyme solution was then prepared and incubated at 45 8C for 30 min, then boiled for 15 min to stop the reaction. A portion of the mixture (0.5 ml) was mixed with 1.8 ml of water and 2 ml of alkaline ferri-cyanide solution, and the reducing sugar produced was measured colorimetrically[33]using a standard curve constructed by pure compound of glucosamine. One enzyme unit was defined as the amount of enzyme that hydrolyzed 1% chitosan solution to yield 1 mmol of reducing sugar per minute at 45 8C.

3. Results and discussion

3.1. Preparation of the crude enzyme

After incubating the B. cereus at 30 8C for 2 days, the culture

broth was centrifuged, and the crude enzyme was precipitated

by acetone. The above procedure recovered 86% of the total

chitosanase activity from the culture broth, and raised its

specific activity from 2.0 to 24.7 unit/mg protein, a 12-fold

increase. Then, reversed micellar extraction was used to further

purify the chitosanases.

3.2. Effects of protein concentration, extraction

temperature and time

Fig. 1

shows the effect of initial protein concentration on the

extraction performance. It was found that the maximum amount

of chitosanase activity could be recovered at the initial protein

concentration of 1 mg/ml. When the initial protein

concentra-tion was higher than 1 mg/ml, the recovery of chitosanase

activity decreased. It was suspected that the interactions

between protein molecules might interfere the extraction

performance when the protein concentration was too high.

Therefore, the initial crude enzyme concentration was fixed at

1.0 mg/ml for the subsequent studies.

The temperature and time are two important physical

parameters involved in the reversed micellar extraction. Since

these two physical parameters, theoretically, had less

interac-tions with the other chemical parameters, including pH, NaCl

concentration, and AOT concentration, it was decided to

determine them first in order to minimize the number of

independent variables in this study. The effects of extraction

time and temperature on the extraction performance were

investigated and the results are shown in

Fig. 2

. In general, the

extraction conducted at 15 8C accomplished the highest

chitosanase activity recovery among the different temperatures

tested ranging from 10 to 30 8C. It appeared that temperature

below 15 8C might be too low to facilitate mass transfer. A

higher temperature, on the other hand, might have loosened the

structure of the reversed micelles, thus offering less protection

for the enzyme when it passed through the aqueous/organic

inter-phase to enter the micelles. It was noticed that there was a

dramatic decrease in the recovery of the chitosanase activity for

the extraction conducted at high temperature (i.e. 30 8C) for a

long time (i.e. 160 min). In general, increasing the extraction

time would increase the chance for the enzyme to contact the

organic solvent and being inactivated. Dekker et al.

[34]

found

that the maximum amount of protein that could be solubilized

in the reversed micellar phase would be a function of

temperature. Chou and Chiang

[23]

also found that lowering

extraction temperature facilitated the extraction of lysozyme

into the micellar phase. However, the mass transfer rate of the

protein would decrease with decreasing temperature, and

therefore, the extraction time should be increased to

compensate for the reduced mass transfer rate. From another

viewpoint, too long of an extraction time might have increased

the chance for the proteins to contact with the organic solvent,

leading to protein denaturation and affecting the performance.

Based on the results of this study, the forward extraction time

was fixed at 85 min and the temperature was at 15 8C for the

subsequent experiments.

3.3. Optimization of extraction conditions

There were three parameters that needed to be investigated

to search for the optimum process conditions to recover

chitosanase during reversed micellar extraction. This includes

pH (X

), AOT concentration of the organic phase (X

), and

NaCl concentration of the aqueous phase (X

).

Table 1

shows

the extraction conditions and results of the first experimental set

for studying the effects of these variables on the recovery of

Fig. 1. Effect of initial protein concentration in the aqueous phase on the recovery of chitosanase activity during reversed micellar extraction. The forward extraction was carried out at 15 8C for 85 min. The aqueous phase was 150 mM phosphate buffer at pH 4 and the NaCl concentration was 150 mM. The AOT concentration in the organic phase was 100 mM. The backward extraction was carried out at 40 8C for 40 min using 50 mM phosphate buffer containing 1 M KCl at pH 10 as the aqueous phase.

Fig. 2. Effects of temperature and time on the recovery of chitosanase activity. During forward extraction, the aqueous phase was 150 mM phosphate buffer at pH 4 and contained 50 mM NaCl. The AOT concentration in the organic phase was 100 mM. The backward extraction was carried out at 40 8C for 40 min using 50 mM phosphate buffer containing 1 M KCl at pH 10 as the aqueous phase.

chitosanase activity based on a Box–Behnken design

[29]

. A

second-degree polynomial model based on regression analysis

was then developed showing the recovery of chitosanase

activity (Y

) as the function of the processing variables:

Y

¼ 434:852 þ 221:847X

þ 0:034X

þ 0:262X

 24:612X

1

þ 0:005X

X

 0:0003X

 0:066X

X

þ 0:0003X

X

 0:0005X

(2)

The coefficient of determination (R

) of the model for

activity recovery was 0.95, indicating a generally good fit of the

model. The highest possible recovery of chitosanase activity

resulting from the first experimental set was estimated to be

66.4% using the non-linear programming technique, which was

obtained by carrying out the extraction at pH 4.5, sodium

chloride concentration of 112.4 mM, and AOT concentration of

50.0 mM.

The pH of the aqueous phase is an important parameter

affecting reversed micellar extraction

[35–37]

. For the AOT/

isooctane reversed micellar system, significant transfer of

protein occurs when the pH values are below the isoelectric

point (pI) of protein. The pIs of chitosanases secreted from B.

cereus NTU-FC-4 are about 6.8–7.2

[18]

; therefore, the

enzymes could transfer into reversed micellar phase at pH

below 6.8. Eq.

(2)

indicates that the recovery of chitosanase

activity increases with increasing pH. It is known that the

enzymes are more positively charged at a lower pH, rendering a

stronger interaction between cationic proteins with anionic

AOT head, and thus facilitating the extraction. However, the

size of the protein molecule also influences its uptakes by the

reversed micelles. Larger proteins appear to be more difficult to

transfer into reversed micelles, and a pH much lower than its

isoelectric point is needed for an efficient transfer. The small

proteins, on the other hand, can be transferred at a pH very close

to its isoelectric point

[38,39]

. The molecular weights of the

chitosanases produced by B. cereus NTU-FC-4 are around 47–

66 kDa

[18]

, a medium size protein. Considering both the

electrostatic interaction and the size of the enzymes, the second

set of the experiment was carried out with increasing pH from 4

to 5.

The concentration of surfactant affects the size of reversed

micelles

[39]

. With increasing AOT concentration, the size of

reversed micelles increases. Micelle size in the aqueous phase,

however, remains constant once the concentration of the

surfactant exceeds the critical micelle concentration

[40]

.

Therefore, the amount of extracted protein in the reverse

micellar phase (organic phase) also increased with increasing

AOT concentration, and reached a maximum until certain AOT

concentration

[41]

. Results from the first experimental set

suggested that a proper AOT concentration should be around

100 mM. Therefore, it was decided that the AOT concentrations

for the second set of experiment were in the range of 50–

150 mM.

Sodium chloride in the aqueous phase was to provide the

proper ionic strength for the extraction. Addition of a proper

amount of salt into the aqueous phase is desirable for the

extraction of a large amount of protein into the reverse

micellar phase

[41]

. However, when the ionic strength is too

high, the electrostatic screening effect might reduce the

interaction between protein and surfactant molecules

[42]

.

High ionic strength also reduces the electrostatic repulsion

between the surfactant head groups, resulting in a decrease in

the size of the reverse micelles, thus decreases the extent of

protein being solubilized to the reverse micellar phase

[40,43]

. Results of the first experimental set revealed that

NaCl concentration should be around 50 mM for the

extraction, and Eq.

(2)

suggested that further increasing the

NaCl concentration might be helpful for increasing the

Table 1

Recovery of chitosanase activity from crude enzyme by reversed micellar extraction based on Box–Behnken design

No. pH (X1) [AOT] (X2) (mM) [NaCl] (X3) (mM) Recovery (%) 1 3 50 200 3.98 2 3 200 50 2.14 3 3 200 350 0.68 4 3 350 200 3.62 5 3 350 200 5.19 6 4 50 50 68.75 7 4 50 50 62.53 8 4 50 350 2.30 9 4 200 200 50.15 10 4 200 200 49.57 11 4 200 200 52.45 12 4 350 50 47.61 13 4 350 350 3.10 14 5 50 200 28.82 15 5 200 50 50.13 16 5 200 50 55.84 17 5 200 350 3.60 18 5 350 350 1.66 Table 2

Recovery of chitosanase activity from crude enzyme by reversed micellar extraction based on Box–Behnken design and the specific activity of the extract No. pH (X1) [AOT] (X2) (mM) [NaCl] (X3) (mM) Recovery (%) Specific activity (unit/mg) 1 4 50 90 77.5 46.8 2 4 100 30 67.8 67.4 3 4 100 150 72.7 55.0 4 4 150 90 77.1 54.0 5 4 150 90 84.1 60.7 6 4.5 50 30 58.5 48.9 7 4.5 50 30 59.7 47.1 8 4.5 50 150 49.2 35.4 9 4.5 100 90 63.8 46.9 10 4.5 100 90 64.0 47.2 11 4.5 100 90 65.6 45.6 12 4.5 150 30 58.9 36.1 13 4.5 150 150 44.4 32.6 14 5 50 90 45.6 47.5 15 5 100 30 64.5 48.5 16 5 100 30 62.0 45.7 17 5 100 150 41.9 46.5 18 5 150 150 32.1 29.1

chitosanase recovery during reversed micellar extraction.

Therefore, the salt concentrations ranged from 30 to 150 mM

were investigated for the second set of experiment.

Table 2

shows the effects of the three variables discussed

above on the recovery and specific activity of chitosanase (Y

)

based on a Box–Behnken design. Again, a second-degree

polynomial model based on regression analysis was developed

showing the recovery of chitosanase activity as the function of

the three processing variables:

Y

¼ 422:930  168:097X

þ 0:288X

þ 1:393X

þ 18:160X

1

þ 0:0195X

X

 0:002X

 0:237X

X

 0:0004X

X

 0:002X

(3)

The coefficient of determination (R

) of this equation was

0.95, indicating a generally good fit of the model. The highest

possible rate of chitosanase activity recovery was estimated to

be 81.3% using non-linear programming technique, which was

obtained by operating the reversed micellar extraction at pH

4.0, AOT concentration of 102.4 mM, and sodium chloride

concentration of 96.0 mM. A regression equation for the

specific activity (Y

) as the function of the three processing

variables was also established:

Y

¼ 561:859  243:508X

þ 2:008X

 0:637X

þ 27:924X

 0:320X

X

 0:004X

þ 0:103X

X

þ 0:0008X

X

þ 0:0002X

(4)

The R

of this model was 0.98. It was estimated that the

specific activity of the product extracted at pH 4.0, AOT

concentration of 102.4 mM, and sodium chloride concentration

of 96.0 mM was 60.3 unit/mg protein, a 2.4-fold increase from

the acetone precipitate. Changes of activity recovery and

specific activity of chitosanases during acetone precipitation

Table 3

Specific activity and recovery of chitosanase activity during purification processes

Purification step Activity recovery (%) Specific activity (unit/mg protein) Purification (fold) Centrifugation 100 2.0 1 Precipitation by 70% (v/v) acetone 85.9 24.7 12.3 Reversed micellar extraction 69.8 60.3 30.1

Fig. 3. Effects of process variables on the recovery of chitosanase activity during reversed micellar extraction. (a) pH of the aqueous phase was 4; (b) AOT concentration was 102.4 mM; (c) sodium chloride concentration was 96 mM.

Table 4

Effect of NaCl and AOT concentrations on the activity recovery of chitosanase at pH 3.5

Treatment Activity recovery (%) 30 mM NaCl, 100 mM AOT 1.2

90 mM NaCl, 50 mM AOT 33.8 90 mM NaCl, 150 mM AOT 71.7 150 mM NaCl, 100 mM AOT 54.3

Fig. 4. SDS-PAGE of chitosanases. M, mixture of standard protein with various molecular weights; E1, the fermentation broth; E2, the acetone precipitate; E3, the chitosanases extracted by reversed micelles.

and reversed micellar extraction processes are summarized in

Table 3

.

By fixing one of the three variables of the operation

condition for obtaining the highest recovery, the effect of the

rest of two variables on the recovery of chitosanase are

illustrated by

Fig. 3

. As expected, when the pH was fixed at the

value of 4 the highest recovery of chitosanases was found at an

AOT concentration of 102.4 mM and sodium chloride

concentration of 96 mM (

Fig. 3

(a)). However, when fixing

either the AOT concentration at 102.4 mM or the sodium

chloride concentration at 96.0 mM

Fig. 3

(b) and (c), it appeared

that further decreases of pH might increase the chitosanases

activity recovery. Hence, a separate experiment was conducted

at a lower pH of 3.5 and various AOT and sodium chloride

concentrations (

Table 4

). Results indicated that the

chitosa-nases activity recoveries obtained at all of the tested conditions

were lower than those obtained at pH 4 (compared to the data

shown in

Table 2

). It was suspected that the aqueous phase with

pH lower than 4 might damage the chitosanases activity during

extraction.

3.4. Changes of protein profile during purification process

Electrophoretic patterns of fermentation broth, acetone

precipitate, and the extracts of reversed micelles are shown in

Fig. 4

. After reversed micellar extraction, the extract consisted

of two major proteins with molecular weights around 64 and

50 kDa, respectively. Hung

[18]

used the colloidal chitosan to

adsorb the chitosanases from the acetone precipitate, and

purified the enzymes using preparative electrophoresis, and

found that two kinds of chitosanases existed in the acetone

precipitate, namely Chitosanase I and Chitosanase II. The

Chitosanase I had two subunits with molecular weights 56 and

66 kDa, and Chitosanase II had a molecular weight of 47 kDa.

It appeared that the reversed micelles removed some

non-chitosanase proteins as well as a non-chitosanase subunit, and

yielded a more purified chitosanase product.

4. Conclusion

This research demonstrated that separation of chitosanases

from the fermentation broth of B. cereus NTU-FC-4 could be

carried out by two steps. The first step was to fractionate crude

enzyme by 70% acetone precipitation, and the second step was

to purify the chitosanases from crude enzyme using reversed

micellar extraction. When the reversed micellar extraction was

operated at an optimal condition, the complete procedure,

including acetone precipitation and reversed micellar

extrac-tion, recovered approximately 70% of chitosanase activity

from the fermentation broth. The specific activity increased

30-fold.

Extraction of chitosanase directly from fermentation broth

by reversed micelles without organic solvent precipitation

would be another consideration. However, there will be more

non-target contaminants that might interfere with the partition

behavior of target protein. Besides, the cell debris, being larger

molecules, would possibly be precipitated with surfactant in the

interface layer

[44]

. In spite of these possible shortcomings,

future research is needed to investigate the possibility of

applying the technique of reversed micellar extraction to the

fermentation broth directly. Nevertheless, the present results

give evidence of the potential of AOT reversed micelles for the

extraction and purification of chitosanases. However, in order to

establish commercially viable processes, further work will be

necessary to study the scale-up engineering and the recycling of

the organic solution after completed extractions.

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