Preparative Biochemistry and Biotechnology

ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: https://www.tandfonline.com/loi/lpbb20

Microbial cell disruption methods for efficient

release of enzyme L-asparaginase

Tales A. Costa-Silva, Juan Carlos Flores-Santos, Rominne K. B. Freire, Michele

Vitolo & Adalberto Pessoa-Jr

To cite this article: Tales A. Costa-Silva, Juan Carlos Flores-Santos, Rominne K. B. Freire,
Michele Vitolo & Adalberto Pessoa-Jr (2018) Microbial cell disruption methods for efficient release
of enzyme L-asparaginase, Preparative Biochemistry and Biotechnology, 48:8, 707-717, DOI:
10.1080/10826068.2018.1487850

To link to this article: https://doi.org/10.1080/10826068.2018.1487850

Published online: 11 Jul 2018.

Submit your article to this journal

Article views: 150

View related articles

View Crossmark data

Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=lpbb20
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
2018, VOL. 48, NO. 8, 707–717
https://doi.org/10.1080/10826068.2018.1487850

Microbial cell disruption methods for efficient release of enzyme L-asparaginase

Tales A. Costa-Silva
, Juan Carlos Flores-Santos
, Rominne K. B. Freire, Michele Vitolo and Adalberto Pessoa-Jr
Department of Pharmaceutical and Biochemical Technology, School of Pharmaceutical Sciences, University of S~ao Paulo, S~ao Paulo, Brazil

ABSTRACT KEYWORDS
The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, Anticancer enzyme;
sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed L-asparaginase recovery;
in this study, via measurements of the release of total protein and L-asparaginase activity. Three microbial cell disruption;
periplasmic protein;
different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), protein release
Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified
the best procedure for each kind of microorganism. Sonication and glass bead stirring led to
obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins
and specific activity; however, sonication was the best since it obtained 4.61 ± 0.12 IU mg1 after
3 min of operation time. Mechanical methods were also the most effective for yeast cell disruption,
but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared
to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication
proved to be the best procedure due to getting the highest specific activity (9.01 IU mg1) and
total enzyme activity (61.7 IU). The data presented lead to conclude that the mechanical methods
appeared to be the most effective for the disintegration of the all microbial cells studies. This is
the first report related to the experimental comparison of L-ASNase extraction procedures from
different microorganisms, which can also be used for extracting periplasm located enzymes from
other organisms.

Introduction Improvements in the expression systems, growth condi-

The relevance of microorganisms as a source of valuable
compounds (chemicals, enzymes, pigments, antibiotics, vac-
cines, gums) has been established for many years.[1,2] The
vast majority of these products, including many enzymes
and recombinant proteins, are retained within the cells of
their producers.[3–5] The isolation and recuperation of intra-
cellular material requires that the strong cellular structures
be disintegrated by different techniques including mechan-
ical (bead mills, high-pressure homogenizers and cavitation),
physical–chemical, enzymatic, and combined methods.[6]
The efficiency of disruption methods differs for different
microbial species, age of culture, temperature of cultivation,
and cultivation medium.[7] The higher barrier to disintegra-
tion of microorganisms lies in their resistant cell walls.
In the true bacteria, peptidoglycan (whose macromolecules
covalently cross-linked lead to a web like structure) is
responsible for the mechanical resistance of the wall.
Consequently, covalent cross-links of the cell wall must be
broken in order to disrupt the cells and access the intracel-
lular substances.[8] Yeast has a thick cell wall made from
partially phosphorylated mannans and glucans, while fungi
developed cell walls in multi-layer structures from glucan,
glycoproteins, and chitin.[9]
tions, and scaling-up the culture in bioreactor allowed
achieving reduction on the upstream cost due to the increas-
ing of process yield. Therefore, the influence on the overall
cost of production is due to the downstream protocol
employed. In the case of the desired compound is not
excreted by the cells, so they must be disrupted in order to
free it.[10,11] In spite of the wide range of techniques avail-
able, not all are adequate for large-scale use. Thereby, we
focused on studying as disruption methods for selected
microorganisms (Escherichia coli, Leucosporidium muscorum,
and Aspergillus niger) the sonication, the chemical treatment
(detergent: Triton-X-100, SDS and Tween 80; organic solv-
ent: ethyl acetate and toluene) and the freezing followed by
grinding (all useful in lab scale for microbiological and bio-
chemical experimental approaches), as well as the osmotic
shock and agitation with glass beads (both useful in lab and
large scale). All methods cited not demand sophisticated
apparatus or skilled technical training, at least on lab scale.
Moreover, the osmotic shock and agitation with glass beads
are easily scaled up because they require only well-known
and plentiful available equipments and well-established
engineering unit operations. In addition to this, the available
research knowledge regarding the specific features of cell
disruption of different species, especially for filamentous

CONTACT Tales A. Costa-Silva costa.silva@usp.br Department of Pharmaceutical and Biochemical Technology, School of Pharmaceutical Sciences,
University of S~ao Paulo, Av. Prof. Lineu Prestes, 580, B.16, S~ao Paulo, SP, CEP 05508-900 Brazil.

These authors contributed equally to this work.
ß 2018 Taylor & Francis
708 T. A. COSTA-SILVA ET AL.
fungi, is scarce, and more focused research should
be encouraged.
Finally, the product used as biomolecule model was the
commercially important enzyme L-asparaginase (ASNase)—
L-asparagine amido hydrolase: EC 3.5.1.1. Several microor-
ganisms from fungi, yeast, and bacteria are reported as sour-
ces of L-asparaginase[12,13] and in most cases the enzyme is
intracellular. The requisition for this special enzyme will
growth innumerable fold in the next years, due to its com-
mercial utilization as food processing aid in addition to its
pharmacological use.[14] This enzyme is used in the food
industry to reduce the acrylamide formation during frying
starchy foods. Acrylamide is a toxic compound, being classi-
fied by the International Agency for Research on Cancer as
a potential carcinogenic agent for humans.[15] Furthermore,
ASNase has become an essential component in the treat-
ment of children with acute lymphoblastic leukemia (ALL)
becoming one of the most successful examples of a thera-
peutic enzyme, especially in cancer treatment.[12]
Materials and methods
ASNase production
Bacterium––Escherichia coli
The recombinant strain E. coli BL21 (DE3) harboring the
pET15b expression vector (Novagen, WI, USA) which con-
tains the ASNase gene (GenBank KY305877), ampicillin
resistance gene and a signal sequence for transferring
ASNase to the periplasm was used in this study. Inoculum
was performed in 250 mL Erlenmeyer flasks containing
25 mL Luria-Bertani, LB (BD Difco, Maryland, USA) at pH
7.0, supplemented with ampicillin (100 mg.mL1), and incu-
bated in an ExcellaV E24 shaker (New Brunswick, NJ, USA)
R
at 37  C and 250 rpm for 12 h. The culture was prepared
using 0.5 mL of the inoculum in 25 mL (1/10, medium vol-
ume/flask volume ratio) of modified LB medium (addition
of glucose 5 g.L1 and phosphate buffer 0.1 M pH 7.0) in a
shaker, for 4 h. Subsequently, isopropyl b-D-thiogalactopyra-
noside (IPTG) (inducer of expression), was added at a
concentration of 1 mM for 4 or 16 h. Cells were harvested
by centrifugation (10,000 g, 4  C, 5 min), washed in 0.85%
NaCl solution (w/v), and stored at 4  C.
Yeast––Leucosporidium muscorum
The yeast used in this study was isolated from marine sedi-
ments collected in Admiralty Bay, King George Island,
Antarctica, and previously detected as ASNase producer.
Yeast cell envelope is constituted by 80–90% of polysacchar-
ides, which confer hardness and thickness to the cell
wall.[16,17] For biomass production, the yeast was pre-culti-
vated in Czapek Dox liquid medium (pH 5.5) containing:
(0.2% (w/v) glucose, 1% (w/v) L-proline, 0.15% (w/v)
KH2PO4, 0.05% (w/v) KCl, 0.05% (w/v) MgSO47H2O and
0.001% (w/v) ZnSO47H2O, FeSO47H2O and CuSO45H2O)
incubated at 15  C using 250 rpm for 72 h. Initial cell concen-
tration of 0.2 optical density at 600 nm (OD600) was used for
re-inoculate a new Czapek Dox medium with the already
described composition and cultivated at the same conditions
for more 72 h. Cells were harvested by centrifugation
(10,000 g, 4  C, 10 min), washed in sterilized distillated
water and stored at 4  C.
Filamentous fungus––Aspergillus terreus
The filamentous fungus A. terreus was isolated from soil
and taxonomically identified by the Laboratory of Fungi
Taxonomy and Systematics––Federal Pernambuco
University, and then deposited at the Andre Tosello
Foundation – Campinas/Brazil (Strain: CCT 7693). This fun-
gus was selected in the L-asparaginase screening; the enzyme
produced showed antiproliferative effects against leukemia
cell lines (RS4;11 and HL60) and did not show cytotoxicity
for human normal cells and was maintained by weekly
transfers on slants of PDA medium.[18] ASNase production
was carried out by submerged fermentation using Czapek
Dox modified medium. For biomass production, the fungus
was cultivated (1  107 sporesmL1) in Czapek Dox
medium 1, which contains the following ingredients (g/L of
distilled water): 1.4% (w/v) glucose, 10% (w/v) L-proline,
0.2% (w/v) NH4NO3, 0.15% (w/v) KH2PO4, 0.05% (w/v)
KCl, 0.05% (w/v) MgSO47H2O and 0.001% (w/v)
ZnSO47H2O, FeSO47H2O and CuSO45H2O; the pH was
adjusted to 8.5 with KOH. This fermentation step was real-
ized at 120 rpm for 96 h at 30  C. For enzyme production,
the biomass produced was filtrated on Whatman 2 filter
paper, washed with sterilized distillated water, and re-inocu-
lated in the new fermentative medium, which was similar to
Czapek Dox medium 1, except for the glucose concentration
(0.2% w/v) and absence of inorganic nitrogen. The ASNase
production was carried out for 96 h at 30  C and at 120 rpm.
Evaluation of cell disruption methods for
ASNase release
For effective cell disintegration and ASNase release in bac-
terium, yeast and filamentous fungus, different methods
were tested including: sonication, osmotic shock, glass beads
stirring, freezing following by grinding and chemical treat-
ment. In all experiments, an optimized cooling bath system
prepared from 33% (w/v) sodium chloride solution and acet-
one (as cooling liquid) was used to prevent overheating—
Figure 1. All experiments were carried out <10  C.
Mechanical method for microbial cell disruption
Glass bead stirring
The cell suspension was mechanically disrupted by using
acid-washed glass beads in tubes under stirring by a vortex
stirrer Vixar VM3000 (Bioclassi, Joinville, SC, Brazil),
according to Table 1. In all experiments, a 35% of void
space was maintained in the tubes to facilitate the vortexing.
Protease inhibitors (PMSF and Pepstatin) were added
(10 mg mL1) immediately before cell disruption to inhibit
the action of proteases (aspartyl proteases, serine proteases
and certain cysteine proteases). Different agitation periods

were evaluated followed by cooling intervals in the cooling
bath (Fig. 1) to ensure that temperature remained <10  C
during the process. Finally, optimal time disruption was
determined according the disruption profile by monitoring
the following parameters: (1) OD600 of the cell suspension
after disruption, (2) concentration of total protein, and (3)
and activity of ASNase.
Sonication
Suspended microbial cells were submitted to sonicated dis-
ruption using an ultrasonic cell disruptor, Branson Models
250 & 450 (Branson Ultrasonics SonifierTM, Dunbury, CT,
USA). Sonication was performed at 200 W and 20 kHz. The
distance between the bottom of the bottle and the tip end
was maintained at approximately 10 mm throughout this
experiment. The cells were sonicated and put in a cooling
bath system to prevent overheating (temperature <10  C).
The filamentous fungus cells were sonicated for 60 s and
then allowed to cool for 30 s. Yeast cells (25 mg mL1) were
sonicated using the following parameters: 40% amplitude,
20 s pulse-on and 50 s pulse-off during 7 min and the sam-
ples were collected after every two “pulse-on.” Sonication
conditions for bacterial cells were: 20 mL cell suspension
sample of E. coli BL21 (DE3) (50 mg mL1 cell concentra-
tion) was placed in a conical-bottom tube (30 mm outside
diameter, 35 mL capacity) and kept in a cooling bath during
the cell disruption process; the duty cycle (at 30% ampli-
tude—20 kHz, 200 W) was 7 min with pulse-on for 30 s
intervals and 45 s of pulse-off. The homogenate was then
centrifuged (15,000  g, 4  C, 15 min) and the supernatant
was analyzed.
Figure 1. A cooling bath system was made with sodium chloride solution (33%
w/v) and acetone (cooling liquid) for cooling the tubes after the stirring cycle in
the mechanical cell disruption method.
Table 1. Conditions of cellular disruption using glass beads.
Bead diameter Tube volume
Microorganism (mm) (mL)
Bacterium 0.17 2 3:4
Yeast 0.5 2 3:4
Filamentous fungi 4.0 50
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 709
Freezing and grinding
Aspergillus terreus cells were frozen at –80  C for 5 h and
ground in a pre-chilled mortar and pestle at 5  C for
10 min.[19] The powder produced was suspended in Tris-
HCl buffer (20 mM and pH 8.6) supplemented with protease
inhibitors (PMSF and Pepstatin) at 10 mg mL1, under an
agitation speed of 250 rpm for 30 min at 5  C. The hom-
ogenate was centrifuged at 15,000  g for 20 min at 5  C and
the supernatant was used as a crude enzymatic extract.
Physical method for microbial cell disruption
Osmotic shock
Each type of microbial cell was suspended in their appropri-
ate buffer: 33 mM Tris-HCl (pH 7.2) for bacteria, 50 mM
Sodium phosphate (pH 7.4) for yeast and 20 mM Tris-HCl
(pH 8.6) for filamentous fungus. The suspension was treated
in two stages: (1) the suspension was dispersed by stirring
gently for 10 min in cold solution I (0.5 M sucrose, 0.1 or
1.0 mM EDTA, 33 mM Tris-HCl (pH 7.2). Cells harvested
by centrifugation (10,000 g, 4  C for 5 min) were used in
the next stage. (2) The cells were dispersed under gentle stir-
ring for 10 min in cold solution II (0.5 mM MgCl2), then the
cells were centrifuged (10,000 g, 4  C for 15 min). A ratio
of 80 mL of solution per gram of cells was used.
Chemical method for cell disruption
The microbial biomasses were suspended in appropriate buf-
fers containing different detergents (concentrations: 1.5%)
and organic solvents (suspended in 100 mL of Tris-HCl,
20 mM pH 8.6). Triton X-100, SDS, Tween 80, ethyl acetate
and toluene were mixed with microbial cells separately (10%
w/v) and were kept for 2 h at 250 rpm and at 4  C. After the
stirring period, the supernatant was recovered by centrifuga-
tion (15,000  g, 4  C, 15 min) and analyzed.
Analytical method
Measurement of cell density
Cell density was estimated using optical density by light
scattering at 600 nm using a UV/VIS spectrophotometer,
SpectraMax Plus 384 (Molecular Devices,CA, USA). The
cells were diluted to an optical density <1.0 with NaCl
0.85% w/v. The following equation shows the calculation of
% cell disruption:
 
ODB
% cell disruption ¼ 100 
100 : (1)
ODA
where ODA is the optical density at 600 nm at initial time
and ODB is the optical density at 600 nm after a certain cycle.
Stirring/cooling
Beads/cell g mL1 Lysis buffer time (s/s)
Tris-HCl, 20 mM pH 8.0 60/30
Sodium phosphate, 50 mM pH 7.4 60/30
3:6 Tris-HCl, 20 mM pH 8.6 60/45
710 T. A. COSTA-SILVA ET AL.
ASNase activity
The Nessler method was used and expressed as IU.mL1,
0.1 mL sample was incubated in 1 mL Tris-HCl (50 mM, pH
8.6) with 0.1 mL L-asparagine (189 mM) and 0.9 mL of ultra-
pure water at 37  C for 30 min.[20] The reaction was stopped
by adding 0.1 mL of 1.5 M trichloroacetic acid (TCA).
The tubes were shaken by inversion and centrifuged at
10,000 g and 4  C for 5 min. The liberated ammonia was
determined by adding Nessler’s reagent (0.2 mL supernatant,
4.3 mL of ultrapure water, and 0.5 mL of Nessler’s reagent),
agitated by inversion and measured at 436 nm. The standard
curve was prepared with ammonium sulfate (Sigma-Aldrich,
MO, USA). One International Unit (IU) of ASNase activity
is the amount of enzyme which liberates 1 lmol of ammonia
from L-asparagine per minute.
Quantification of total protein
The total protein concentration from the supernatant of dis-
rupted the cell sample was determined by using a BCA pro-
tein assay kit (Pierce Biotechnology, Rockford, IL, USA),
following the method already described.[21] An aliquot of
the sample (25 lL) was used and added to 200 lL of freshly
prepared reagent. It was then incubated for 25 min at 37  C,
and finally measured at 562 nm. Bovine serum albumin
(BSA) in the range 100–1000 lg mL1 was used as the
standard. The following equation shows the calculation of %
of protein released:
 
ODB
% cell disruption ¼ 100   100 : (2)
ODA
where ODA is the protein concentration after the last cycle
and B is the protein concentration after a certain cycle.
Results and discussion
ASNase released by mechanical cell disruption
Mechanical cell disruption offers simultaneous high-yield of
protein releasing and poor selectivity of enzyme required.
This is due to the indiscriminate destruction of intracellular
structures from which all kinds of macromolecules (proteins,
nucleic acids, lipids, among others) are released to the
medium. Particularly problematic are the presence into the
medium after disruption of proteases and desoxiribonucleic
acid (DNA). Proteases hydrolyze any kind of protein,
including the desired one, whereas DNA increases the vis-
cosity of the medium, which complicates the protein extrac-
tion and purification throughout the downstream unit
operations. The collision between the glass beads and cells
cause temperature rising which cause both protein thermal
denaturation and increasing of proteases activity.[22] In order
to circumvent both handicaps our disruption protocol was
designed for maintaining the temperature <10  C (protease
activity reduction) and the disruption time as low as for
diminishing the disarrangement of cell organelles (such as
the nucleus). Figure 2 shows the effect of stirring/cooling
cycles carried out by using mixtures of water/ethanol,
sodium chloride/ethanol (33% w/v) and sodium
Figure 2. Effect of stirring/cooling cycles on the temperature of the suspension
during disruption (disruption time duration ¼ accumulated stirring cycles) of
Escherichia coli BL21 (DE3) cells: water and ethanol (~), 33% w/v sodium chlor-
ide and ethanol (w) and 33% w/v sodium chloride and acetone ().
chloride/acetone (33% w/v) as cooling bath systems. Stirring
cycles for 30 s followed by cooling cycles of 30 s in a cooling
bath prepared from water/ethanol and sodium chloride/etha-
nol; stirring cycles for 60 s followed by cooling cycles of 30 s
in a cooling bath sodium chloride/acetone. Cell suspension
(50 mg mL1) was stirred at 3000 rpm. As can clearly be
seen the most efficient cooling mixture was that constituted
by sodium chloride/acetone, in which the temperature
remained <7  C for 15 min, at least. Therefore, the cooling
bath system prepared from 33% sodium chloride with the
presence of acetone inside of bottle inserted was chosen to
continue the following experiments.
After setting the best cooling system, the suspension of
E. coli (50 mg mL1) was submitted to disruption at stirring/
cooling cycles of 60s/30s and agitation of 3000 rpm. Cell dis-
ruption (OD600), ASNase activity (U mL1) and protein
(mg mL1) detected into the supernatant are shown in
Figure 3. Therefore, it is clear that the highest enzyme activ-
ity released (8 U mL1) occurred after eight disruption
cycles. As the number of cycles augmented, the protein
releasing increased about 20% (Fig. 3). Nevertheless, the
enzyme activity remained constant after eight cycles, indicat-
ing the protein surplus would mainly result from extra dis-
ruption of cell fragments.
Escherichia coli cell disruption was also made by sonic-
ation, which is an alternative procedure to the stirring with
glass beads. In spite of both methods rely on their cell dis-
ruption capability to the mechanical shear forces they differ
on how these forces are generated. Through glass beads stir-
ring the rupturing force results from the friction between
cells and glass beads, whereas the sonication generates the
disruptive forces through the cavitation. This phenomenon,
in simple terms, consists on the formation of thousands of
micro bubbles into the cell suspension, which after collaps-
ing generate energy equivalent to a pressure of 300 MPa.[23]
Figure 4 shows the performance of disruption method by
sonication for E. coli cells, which presented similar behavior
to the parameters analyzed in the method using glass beads
(Fig. 3). It was observed that the cellular optical density
reached the value of 10, which shows the highest efficiency
of the cell disrupt, but in detrimental effects on the purity

Figure 3. Variation of Escherichia coli BL21 (DE3) cell disruption (), enzyme (^), and protein (D) releasing as the number of stirring/cooling cycles increased.
Figure 4. Variation of Escherichia coli cell disruption (), enzyme (䉫) and protein (D) releasing by the sonication procedure as the number of cavitation/resting
cycles increased.
of the crude extract obtained, due to the higher formation
of cell debris that contributed to the increase of protein con-
centration. There was also rapid release of ASNase (6 cycles,
75 s each cycle), relative to cycles performed using glass
beads stirring (9 cycles, 90 s per cycle). However, this time
was lower than that obtained by Feliu[11] for recombinant
b-galactosidase (6 min of minimal performance) may be due
to the cell location of the ASNase (periplasmic space), which
facilitates the effect of the releasing. Accordingly to this lat-
ter, and too initial hypo-osmotic conditions for the cells was
obtained significant ASNase activity in the initial time.
The suspension of L. muscorum was submitted to attri-
tion with 0.5-mm glass bead for 15 min performing a total
of ten stirring/cooling cycles of 1.5 min each one. According
to Ricci-Silva et al.,[24] agitation of 2,300 rpm for 6 min is a
suitable condition to obtain 60% of yeast cell disruption by
PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 711
glass beads, which is the minimum acceptable percentage
required for obtaining enzyme from disrupted yeast cells.
Considering this premise and data plotted in Figure 5, the
breaking conditions applied in this work were not able to
reach this minimum value. OD600 dropped from 57.53 ± 2.98
to 30.23 ± 0.71, resulting in almost 45% of disruption.
However, the protein concentration into the cell-free extract
ranged from 0.038 mg mL1 to 4.128 mg mL1 after 15 min
of breakage, which represents an increase about 99%.
Although the highest detectable enzymatic activity, which
occurred at the tenth cycle, was 0.27 ± 0.04 IU mL1, the
enzyme recovery decreased after the fourth cycle (6 min of
cell disruption) and the specific activity remains almost con-
stant (about 90.0  103 ± 0.03 IU mg1).
Sonication is a method for mechanical cell disruption that
has been used for yeast cells.[16,17,25] The effectiveness of
712 T. A. COSTA-SILVA ET AL.

Figure 5. Variation of Leucosporidium muscorum cell disruption (), enzyme (w), and protein (D) releasing as the number of stirring/cooling cycles increased. Cell
suspension (25 mg.mL1) was stirred at 3000 rpm for 60 s following 30 s of repose. OD600  102: optical density at 600 nm for 10–2 diluted sample.

Figure 6. Variation of Leucosporidium muscorum cell disruption (), enzyme (䉫), and protein (D) releasing by the sonication procedure as the number of cavitation/
resting cycles increased.

product release is correlated with sonication time and acous-
tic power.[26] In this work, we analyzed the total protein and
ASNase release during the sonication cycles (20 s for sonic-
ation followed by 40 s for cooling). By comparing the cell dis-
ruption (in terms of OD600 diminution) attained with glass
beads attrition and sonication, we observed that the cell dis-
ruption of about 45% occurred in both cases (Figs. 5 and 6).
On the other hand, the protein releasing by glass bead
method (4.12 ± 0.06 mg mL1) was about 37% higher than
that attained from sonication (2.59 ± 0.01 mg mL1).
Moreover, the disruption time consumed by the sonication
was the double of that required by the glass beads method.
The ASNase released by sonication was also lower than that
obtained through glass beads method (Figs. 5 and 6). The
maximum value of specific activity (0.07 ± 0.002 IU mg1) was
detected at the last cycle. However, this value was about 36%
lower after 6 min of cell disruption (57.1  103 ±
0.006 IU mg1) when compared to glass bead technique,
which reached 90.0  103 ± 0.03 IU mg1 at this time.
The poor disrupting performance presented by L. mus-
corum against glass beads and sonication was also
described by Bzducha-Wr obel et al.[17] Nevertheless,
large differences among the results reported in literature
may be due variations in cell breakage conditions
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 713

Figure 7. Variation of enzyme specific activity (䉲), total enzyme activity (䉫), and protein (D) releasing from Aspergillus terreus as the number of stirring/cooling
cycles increased using glass beads.

Figure 8. Variation of specific activity (䉲), total enzyme activity (䉫), and protein (D) releasing from Aspergillus terreus by the sonication procedure as the number
of cavitation/resting cycles increased.

(e.g., temperature, pH, duration of disruption, method of
preparing yeast suspension, composition of the medium,
and cell concentration) and different yeast cell strain. A
method for Saccharomyces cells disruption by ultrasonica-
tion was optimized and the most effectiveness purification
was obtained after 20 min of sound generation and power
of 600 W.[27] A comparative study of fungal cell disruption
for glucose-6-phosphate dehydrogenase recovery of yeast,
Rhodotorula gracilis, revealed more disruption effectiveness
using ultra-sonication method than glass beads.[16] Similar
comparison between these methods was investigated[17] and
the highest degree of cytosol component release
and purification of b-glucans was detected when
Saccharomyces cerevisiae yeast cell wall preparations were
submitted for 30 min of cell homogenization with zirco-
nium-glass beads.
Disrupting A. terreus cells through 4.0 mm glass beads
(Fig. 7) or sonication (Fig. 8) showed to be highly difficult
due to the cell wall thickness and hyphal arrangement of the
fungus vegetative body. Both characteristics lead to the for-
mation of fluffy pellets, which reduces the effectiveness of
shear forces either of attrition or cavitation origin. Figure 7
shows that at the fifth cycle the ASNase activity and specific
activity increased about 12-fold and 6-fold, respectively,
when compared to the initial values. The use of glass beads
with smaller diameters (0.5, 1.0, and 2.5 mm) revealed to be
less effective for the disintegration of filamentous fungi cells,
releasing little protein (data not shown).
714 T. A. COSTA-SILVA ET AL.
In the case of sonication, the highest enzyme specific
activity (4.61 ± 0.12 IU mg1) and total enzyme activity
occurred at the third and eight cycles, respectively (Fig. 8).
Undoubtedly, the sonication was more effective than glass
beads stirring on the disruption of fungal biomass.
Mechanical disruption by sonication was the most effective
cell disintegration method using A. oryzae NSK as the
source of glutamate decarboxylase (GAD). This method
yielded 1.99 U mg1 of GAD, which is 170% higher than the
value obtained when the freezing and grinding method
was used.[27]
Due to the larger cell diameters formed by A. terreus
compared with the other microorganisms (pellets of
approximately 3 mm), the freezing and grinding method was
applied only to these filamentous fungus cells. 2.82 ± 0.62 mg
of total protein was obtained and the specific activity was
2.91 ± 012 IU mg1. Struszczyk et al.[19] performed the
extraction of intracellular enzymes from the mycelium of
Mucor circinelloides by various methods (e.g., detergents,
freezing and grinding, and sonication). The extract obtained
by freezing and grinding displayed high activities of both
chitosanase (16.65 U mg1) and lipase (5.98 U mg1).
The scientific studies about disruption of fungal cells
have been, in general, poorly conducted and only few infor-
mation on this topic are available especially compared to the
amount of studies done using bacteria and yeast. Herein, we
used A. terreus as model to establish the most efficient
method of filamentous fungus cell lysis. The highest yield of
protein extraction from fungal cell was achieved by mechan-
ical treatment. The extracts produced by sonication dis-
played high specific activity of ASNase with total time
operating of 3 min. The use of glass beads stirring was an
efficient disruption method for releasing fungal ASNase, but
the specific activity was lesser (3.37 ± 0.12 IU mg1) with a
higher time operating (7 min). Freezing and grinding
method was the simpler and fast method used for ASNase
release. However, this procedure shown the lower specific
activity due to the no-specific mode of action on releasing
the cell constituent (all cellular constituents were broken by
grinding). The mechanical disruption procedures revealed to
be the most effective for the disintegration of filamentous
fungi cells, specifically, A. fumigatus and Penicillium
citrinum. Ultrasonication led to obtaining crude extracts
containing high concentrations of proteins and the enzyme
glucose-6-phosphate dehydrogenase.[16] The fungal cell wall
is unique to the fungi and is highly dynamic structure pri-
marily composed of chitin, glucans, mannans, and
Table 2. Osmotic shock method for releasing ASNase from Escherichia coli, Leucosporidium Muscorum, and Aspergillus terreus.
Total enzyme activity (IU)
ASNase source EDTA (mM) Hypertonic solutiona
Escherichia coli 0.1 1.15 ± 0.03
1.0 1.63 ± 0.04
Leucosporidium muscorum 0.1 ND
1.0 ND
Aspergillus terreus 0.1 0.13 ± 0.01
1.0 0.24 ± 0.01
a
Hypertonic solution I (0.5 M sucrose, 33 mM Tris-HCl pH 7.2, 1.0 or 0.1 mM EDTA).
b
Hypotonic solution II (0.5 mM MgCl2).
c
ND ¼ not detected.
glycoproteins. This structure composition provides an excel-
lent mechanical strength to environmental stresses (for
example: changes in osmotic pressure) and requires robust
techniques for release the intracellular products. Cell wall
structure in Aspergillus sp. (and others filamentous fungi)
contain 10–20% chitin,[28] which is responsible for cell ten-
sile resistance and rigidity, which implies that fungal cells
are, in general, resistant towards some disintegration proce-
dures usually applied for bacteria such as non-mechanical
cell disruption methods. Finally, mycelial growth in pellets
and the thickness of cell wall prevented the A. terreus cells
from immediate disintegration by no-mechanical methods.
The same results were reported by Royer et al.[29] using fila-
mentous fungus.
ASNase release by physical method
As the amount of protein present into the cell wall represent
4-8% of the total cell protein[30] and ASNase is located
mainly in the periplasmic space, so using a less disruptive
cell method, such as the osmotic shock (based on the
osmotic pressure unbalance between the periplasmatic space
and the external medium) would lead to an extract quite
less contaminated by proteins other than ASNase. Therefore,
it was applied the osmotic shock method as described by
Neu and Heppel[31] and Hamilton-Miller[32] modified by
using 0.1 mM or 1.0 mM EDTA. Basically, the method con-
sists on alternate cell submission to hypertonic and hypo-
tonic solution constituted by sucrose and EDTA. As can be
seen from Table 2, the ASNase was successfully extracted
only from E. coli. Particular characteristics in cell wall struc-
tures of each microorganism – probably related to the pres-
ence, type and amount of bivalent ions (such as Caþ2)
acting as stabilizers––would explain the low response pre-
sented by L. muscorum and A. terreus to the osmotic shock
procedure. A similar result was observed by Klimek-Ochab
et al.[16] when the disintegration of filamentous fungi
A. fumigates, Penicillium citrinum, and the yeast strain
Rhodotorula gracilis cells by osmotic shock failed for glu-
cose-6-phosphate dehydrogenase release.
ASNase release by chemical method
The extraction of ASNase from E. coli, L. muscorum and A.
terreus was also carried out by using compounds (SDS,
Tween 80, TRITON X-100, toluene and ethyl acetate)
Specific activity (IU mg1)
Hypotonic solutionb Hypertonic solution Hypotonic solution
1.93 ± 0.17 0.63 ± 0.01 1.76 ± 0.07
8.20 ± 0.40 0.88 ± 0.04 5.38 ± 0.08
ND NDc ND
ND ND ND
0.22 ± 0.01 0.16 ± 0.09 0.37 ± 0.04
0.44 ± 0.01 0.32 ± 0.06 0.87 ± 0.02
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 715

capable of destabilize the wall structure (through alteration
protein-protein interactions, membrane proteins solubiliza-
tion, disturbing the cell membrane lipopolysaccharides and
phospholipids) in order to disrupt the chemical bonds which
maintain the enzyme confined inside the periplasm space.[33]
Table 3 shows that ASNase was released from E. coli more
efficiently by the chemicals cited than from L. muscorum
and A. terreus. By the way, regarding the yeast and the fun-
gus negligible enzyme activity was extracted. This result cor-
roborates those described by Taubert et al.[34] regarding to
the extraction of glucose-6-phosphate dehydrogenase
(G6PDH) from Ganoderma applanatum and Pycnoporus
cinnabarinus.
Particularly to the non-ionic detergents (Tween 80 and
TRITON X-100) was observed that they were also inefficient
to release G6PDH from R. gracilis.[8] However, when those
authors used SDS for 20 min the G6PDH activity measured
into the supernatant was 4.04 ± 0.23x103 U mg1.
Meanwhile, as can be seen from Table 3, the extraction of
ASNase from L. muscorum with SDS was ineffective.
Probably the different genus of yeasts considered – which
implies on distinct cell wall structure—the particular cell
location of the cited enzymes (G6PDH into the cytoplasm
whereas ASNase in the periplasm space) and the different
susceptibility of enzymes against the detergent would explain
the results appointed.
There are several intracellular enzymes that are produced
and industrially commercialized: glucose oxidase for food
conservation, penicillin acylase for antibiotic production,
ASNase for leukemia treatment, b-galactosidase for milk
processing, and glucose-6-phosphate dehydrogenase for clin-
ical analysis.[12,35] Therefore, in the case of enzymes located
into the cytoplasm or periplasm space, the cell disruption
constitutes the first step of any downstream protocol.
Furthermore, depending on the disruption method chosen it
can interfere on the following unit operations selected for
carrying out the downstream, which would, at the end,
affect the yield and quality of product obtained. Table 4
presents the specific ASNase activities of E. coli, L. musco-
rum and A. terreus measured into the medium after apply-
ing several types of disruption methods.
From Table 4, we can see that both the specie of micro-
organism and the type of disruption method interfered on
the specific ASNase activity attained. On one hand, we
observe that the E. coli BL21 (DE3) cell was disrupted by
using any type of disruption method, preferentially the
osmotic shock, followed by sonication, chemical and glass
beads methods. On the other hand, all disruption methods
were inefficient for disrupting L. muscorum and quite less
effective for A. terreus cells. The latter, however, was dis-
rupted by sonication in some extension (about 50% lower
than E. coli).
The efficiency of mechanical disintegration methods
appears to be the best choice for the disintegration of micro-
bial cells in comparison to the other procedures. But, in
general, they are generalized in action and non-selective to
release the product. The co-release of contaminants and
micronization of the cell debris are the mainly disadvantages
presented by sonication and glass beads grinding, because
the molecule of interest must be separated from a complex
cocktail of other byproducts (i.e., nucleic acid, proteins, cell
constituents fragments). In addition, the heat generated by
mechanical energy dissipation needs to be removed by effi-
cient cooling system to retain the activity of thermo-
sensitive products; hence, temperature control is crucial and
obligatory. In this paper we used a cooling system which
allowed to keep the temperatures of disruption solutions
<10  C by all the operating time.
Furthermore the cell structure, the adequate operation
time of disruption procedure is other crucial variable

Table 3. Disruption of Escherichia coli, Leucosporidium muscorum, and Aspergillus terreus cells through the chemical method.
ASNase source
Escherichia coli Leucosporidium muscorum Aspergillus terreus
Total enzyme Specific Total enzyme Specific Total enzyme Specific
Chemical method activity (IU) activity (IU mg–1) activity (IU) activity (IU mg–1) activity (IU) activity (IU mg–1)
SDS 34.1 ± 0.12 6.3 ± 0.11 ND ND ND ND
Tween 80 37.6 ± 0.30 8.7 ± 0.06 ND ND 0.17 ± 0.01 0.43 ± 0.07
Triton X-100 31.2 ± 0.28 7.1 ± 0.22 ND ND 0.11 ± 0.04 0.27 ± 0.03
Toluene 37.0 ± 0.12 8.7 ± 0.10 ND ND ND ND
Ethyl acetate 37.7 ± 0.14 5.6 ± 0.11 ND ND ND ND
ND: no detected.

Table 4. Comparison of disruption methods used for microbial ASNase release.

Specific activity (IU mg1)a
Methods of disruption
Mechanical
ASNase Source Glass beads Sonication Freeze grinding Physical Chemical
Escherichia coli 5.09 ± 0.24 9.01 ± 0.24 – 9.12 ± 0.09 8.7 ± 0.10b
Leucosporidium muscorum 0.09 ± 0.04 0.07 ± 0.002 – ND ND
Aspergillus terreus 3.37 ± 0.12 4.6 ± 0.12 2.8 ± 0.62 0.87 ± 0.02 0.63 ± 0.12
a
Specific activity obtained at the best condition of each disruption method.
b
By use of 1.5% v/v toluene.
ND: No detected.
716 T. A. COSTA-SILVA ET AL.
influencing the release of intracellular constituents and con-
sequently, the process yield. In case of sonication for
example, the short time of exposure of fungal cell was
appropriate (overall operation time of 3.0 min), getting as
well as crude extract with high level of proteins and a high
specific activity of ASNase. This high value of specific activ-
ity at the beginning of disintegration cell process could be
related with the enzyme localization inside the fungal pellet.
Fungal ASNase is located at the periplasmic space and is
necessary few minutes of operation to reach this region and
release the product of interest. At prolonged periods of con-
tinuous sonication more cell structures can be disrupted and
more contaminants compounds and debris are produced.
Conclusions
The data presented lead to conclude that the mechanical
methods appeared to be the most effective for the disinte-
gration of the all microbial cells studies. Sonication proved
to be a better and efficient disruption procedure for E. coli
BL21 (DE3) and A. terreus CCT 7693 cells due to the high
values of total ASNase activity (higher efficiency) and spe-
cific activity (higher selectivity). This is the first report
related to the experimental comparison of L ASNase extrac-
tion procedures from different microorganisms, which can
also be used for extracting periplasm located enzymes from
other organisms.
Acknowledgments
The authors thank Dr Gisele Monteiro de Souza of Universidade de
S~ao Paulo—USP for Escherichia coli BL21 (DE3) and Dr Lara Dur~aes
Sette of Universidade Estadual Paulista Julio de Mesquita Filho—
UNESP for Leucosporidium muscorum both used in this work.
Funding
This work was supported by State of Sao Paulo Research Foundation
(FAPESP) [grant number 2013/08617-7], [grant number 2013/19584-2].
References
[1] Costa-Silva, T.A.; Carvalho, A.K.F.; Souza, C.R.F.; De Castro,
H.F.; Said, S.; Oliveira, W.P. Enzymatic Transesterification of
Coconut Oil Using Chitosan-Immobilized Lipase Produced by
Fluidized-Bed System. Energy Fuels 2017, 31, 12209–12216.
[2] Fernandes, M.V.R.; Costa-Silva, T.A.; Pfenning, L.H.; Costa-
Neto, C.M.; Heinrich, T.A.; Alencar, S.M.; Lima, M.A.; Ikegaki,
M. Biological Activities of the Fermentation Extract of the
Endophytic Fungus Alternaria alternata Isolated from Coffea
arabica L. Braz. J. Pharm. Sci. 2009, 45, 677–685.
[3] Lugani, Y.; Sooch, B.S. Development of Cell Disruption Strategy
for Enhanced Release of Intracellular Xylose Reductase from
Pseudomonas putida BSX-46. Int. J. Curr. Microbiol. App. Sci.
2017, 6, 3682–3697.
[4] Bystryak, S.; Santockyte, R.; Peshkovsky, A.S. Cell Disruption of
S. cerevisiae by Scalable High-Intensity Ultrasound. Biochem.
Eng. J. 2015, 99, 99–106.
[5] 
Colnik,  Leitgeb, M. Use of Non-
M.; Primozic, M.; Knez, Z.;
Conventional Cell Disruption Method for Extraction of
Proteins from Black Yeasts. Front. Bioeng. Biotechnol. 2016, 4,
1–12.
[6] Chisti, Y.; Moo-Young, M. Disruption of Microbial Cells for
Intracellular Products. Enzyme Microbiol. Technol. 1986, 8,
194–204.
[7] Geciova, J.; Bury, D.; Jelen, P. Methods for Disruption of
Microbial Cells for Potential Use in the Dairy Industry—a
review. Int. Dairy J 2002, 12, 541–553.
[8] Silhavy, T. J.; Kahne, D.; Walker, S. The Bacterial Cell
Envelope. Cold Spring Harb. Perspect. Biol. 2010, 2, a000414.
doi:10.1101/cshperspect.
[9] Kula, M. R.; Schutte, H. Purification Disruption of Proteins and
Microbial Cells. Biotechnol. Prog. 1987, 3, 31–42.
[10] Balasundaram, B.; Harrison, S.; Bracewel, D.G. Advances in
Product Release Strategies and Impact on Bioprocess Design.
Trends Biotechnol. 2009, 27, 477–485.
[11] Feliu, J. X.; Villaverde, A. An Optimized Ultrasonication Protocol
for Bacterial Cell Disruption and Recovery of b-Galactosidase
Fusion Proteins. Biotechnol. Tech. 1994, 8, 509–514.
[12] Narta, U.K.; Kanwar, S.S.; Azmi, W. Pharmacological and
Clinical Evaluation of L-Asparaginase in the Treatment of
Leukemia. Crit. Rev. Oncol. Hematol. 2007, 61, 208–221.
[13] Dhanam Jayam, G.; Kannan, S. The Various Sources of L-
Asparaginase. Int. J. Recent Sci. Res. 2014, 2, 342–346.
[14] Egler, R.A.; Ahuja, S.P.; Matloub, Y. L-Asparaginase in the
Treatment of Patients with Acute Lymphoblastic Leukemia.
J. Pharmacol. Pharmacother. 2016, 7, 62–71.
[15] Meghavarnam, A.K.; Janakiraman, S. Evaluation of Acrylamide
Reduction Potential of L-Asparaginase from Fusarium
Culmorum (ASP-87) in Starchy Products. LWT—Food Sci.
Technol. 2018, 89, 32–37.
[16] Klimek-Ochab, M.; Brzezi nska-Rodak, M.; Zyma nczyk-Duda,
E.; Lejczak, B.; Kafarski, P. Comparative Study of Fungal Cell
disruption-scope and limitations of the methods. Folia
Microbiol. (Praha) 2011, 56, 469–475.
[17] Bzducha-Wr obel, A.; Bła_zejak, S.; Kawarska, A.; Stasiak-
Roz_ a
nska, L.; Gientka, I.; Majewska, E. Evaluation of the
Efficiency of Different Disruption Methods on Yeast Cell Wall
Preparation for b-Glucan Isolation. Molecules 2014, 19,
20941–20961.
[18] Loureiro, C.B.; Borges, K.S.; Andrade, A.F.; Tone, L.G.; Said, S.
Biochemical Caracterization of Native and Pegylated Form of l-
Asparaginase Produced by Aspergillus terreus: Evaluation In
Vitro of Antineoplasic Activity. AiM 2012, 02, 138–145.
[19] Struszczyk, K.; SzczeR sna-Antczak, M.; Walczak, M.;
Pomianowska, E.; Antczak, T. Isolation and Purification of
Intracellular Chitosanolytic Enzymes of Mucor circinelloides.
Prog. Chem. 2009, 78, 16–116.
[20] Imada, A.; Igarasi, S.; Nakahama, K.; Isono, M. Asparaginase
and Glutaminase Activities of Micro-Organisms. J. Gen.
Microbiol. 1973, 76, 85–99.
[21] Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.;
Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.;
Olson, B.J.; Klenk, D.C. Measurement of Protein Using
Bicinchoninic Acid. Anal. Biochem. 1985, 150, 76–85.
[22] Peterson, M.E.; Daniel, R.M.; Danson, M.J.; Eisenthal, R. The
Dependence of Enzyme Activity on Temperature:
Determination and Validation of Parameters. Biochem. J. 2007,
402, 331–337.
[23] Saranya, N.; Devi, P.; Nithiyanantham, S.; Jeyalaxmi, S. Cells
Disruption by Ultrasonication. BioNanoScience 2014, 4,
335–337.
[24] Ricci-Silva, M.E.; Vitolo, M.; Abrahao-Neto, J. Protein and
Glucose 6-Phosphate Dehydrogenase Releasing from Baker’s
Yeast Cells Disrupted by a Vertical Bead Mill. Process Biochem.
2000, 35, 831–835.
[25] Liu, D.; Zeng, X.; Sun, D.; Han, Z. Disruption and Protein
Release by Ultrasonication of Yeast Cells. Innov Food Sci Emerg
J. 2013, 18, 132–137.
[26] Singh, R.S. A Comparative Study on Cell Disruption Methods
for Release of Aspartase from E. coli K-12. Indian J. Exp. Biol.
2013, 51, 997–1003.
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 717

[27] Yeng, A.L.Y.; Kadir, M.S.; Ghazali, H.M.; Rahman, R.N.R.A.R.; [31] Neu, H.C.; Heppel, L.A. The Release of Enzymes from
Saari, N. A Comparative Study of Extraction Techniques Escherichia coli by osmotic shock and during the formation of
for Maximum Recovery of Glutamate Decarboxylase spheroplasts . J. Biol. Chem. 1965, 240, 3685–3692.
(GAD) from Aspergillus oryzae NSK. BMC Res. Notes 2013, 6, [32] Hamilton-Miller, J.M. Damaging Effects of
526–529. Ethylenediaminetetra-Acetate and Penicillins on Permeability
[28] Bowman, S.M.; Free, S.J. The Structure and Synthesis of the Barriers in Gram-Negative Bacteria. Biochem. J. 1966, 100,
Fungal Cell Wall. Bioessays 2006, 28, 799–808. 675–682.
[29] Royer, G.; Yerushalm, L.; Rouleau, D.; Desrochers, M. [33] Stanbury, P.; Whitaker, A.; Hall, S. Principles of Fermentation
Continuous Decolorization of Bleached Kraft Effluents by Technology. 3rd ed. 2016.
Coriolus versicolor in the Form of Pellets. J. Ind. Microbiol. [34] Taubert, J.; Krings, U.; Berger, R.G. A Comparative Study on
1991, 7, 269–278. the Disintegration of Filamentous Fungi. J. Microbiol. Methods.
[30] French, C.; Keshavarz-Moore, E.; Ward, J.M. Development of a 2000, 42, 225–232.
Simple Method for the Recovery of Recombinant Proteins from [35] Semashko, T.V.; Mikha%lova,K R.V.; Eremin, A.N. Intracellular
the Escherichia coli Periplasm. Enzyme Microb. Technol. 1996, Glucose Oxidase from Penicillium funiculosum 46.1. Prikl
19, 332–338. Biokhim Mikrobiol. 2003, 39, 419–426.