Journal of Cleaner Production 127 (2016) 600e609

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Journal of Cleaner Production

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

Scalable production, fast purification, and spray drying of native

Pycnoporus laccase and circular dichroism characterization
Jiayang Liu a, *, Zhonghua Yu a, Xiangru Liao b, Junhe Liu a, Feijun Mao c,
Qingguo Huang d, **
a
Fermentation Technology Division, School of Bioengineering, Huanghuai University, Zhumadian 463000, China
b
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
c
Wuxi Jinkun Bio-technology Co., Ltd., Wuxi 214131, China
d
Department of Crop and Soil Sciences, University of Georgia, Griffin, GA30223, USA

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

Article history: Laccase has been emerged as a valuable green biocatalyst in cleaner production, which requires vast
Received 2 July 2015 amount of laccase at low production cost and with good properties. The current work evaluated a wild
Received in revised form strain Pycnoporus sp. SYBC-L3 as an efficient microbial cell factory for laccase production. High-cell
12 March 2016
density cultivation (HCDC) of L3 was developed in a 5-L bioreactor by adjusting the ratio of inoculum
Accepted 28 March 2016
to medium, achieving over 4-fold of both laccase activity (240 U/mL) and fungal biomass (20 g/L) than
Available online 11 April 2016
those obtained under routine procedures. Laccase productivity was further assessed at pilot-scale in a
500-L and a 5-ton bioreactor, where the highest activity of around 60 U/mL and 80 U/mL were produced,
Keywords:
Scalability
respectively. Cost percentage analysis for the 5-ton reactor demonstrated that labor was the highest
Laccase portion (50%) in total cost for industrial production in this case. Using a one-step purification scheme,
Purification laccase was purified to homogeneity from the culture broth with yield of 44% and 97% purity. Circular
High-cell density cultivation dichroism (CD) analyses suggested that the secondary structure of laccase was mainly composed of b-
Circular dichroism sheet that played a vital role in maintaining laccase activity, and also revealed the potential mechanisms
Powdered enzyme for the changes in laccase activities under varying conditions. Powdered laccase was efficiently prepared
from cell-free culture broth using spray drying technique with above 74% activity retained. This study
provides important information for an efficient and scalable production of laccase.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction laccase is expected worldwide, and the gap between production

Many eco-friendly enzymes have been used in industries to
promote cleaner production via enzymatic processes (Boruah et al.,
2016; Madara
sz et al., 2015; Skoronski et al., 2016). Laccase (EC
1.10.3.2) is a very useful green catalyst for industrial, environ-
mental, and medical applications due to its diverse capability in
converting a wide range of substrates through catabolic or anabolic
reactions (Jeon and Chang, 2013). As a green catalytic reagent,
laccase has been increasingly employed in cleaner production,
covering biofuel production, textile processing, biodegradation,
biografting (Liu et al., 2015; Maryan et al., 2015; Spina et al., 2015;
Thakur et al., 2016). As such, a rapid growth in the demand of
* Corresponding author. Tel.: þ86 18839648638.
** Corresponding author. Tel.: þ1 770 229 3302; fax: þ1 770 412 4734.
E-mail addresses: liujiayang5@gmail.com (J. Liu), qhuang@uga.edu (Q. Huang).
and demand will enlarge. It is thus of practical significance to seek
microbes as potent cell factories to produce laccase with high yield
at low cost.
The major obstacle of broad application is the presumed low
productivity by target organisms. Some strategies have been
attempted to improve laccase productivity, including medium and
process optimization, inducer supplementation, and ultrasonic
treatment (Jing and Wang, 2012; Wang et al., 2012). High cell
density cultivation (HCDC) has emerged as a research interest
among fermentation industries, which can shorten production cy-
cle and thus improve production efficiency (Lee, 1996). By adjusting
parameters, such as dissolved oxygen (Matsui et al., 2006), fed-
batch (Babaeipour et al., 2007), hyper-production of target prod-
ucts via HCDC can be achieved. There has not been a report on
HCDC production of laccase by fungus to the best of our knowledge.
In our previous experiments as well as in other reports, fungal
biomass was normally limited to a certain level under routine

http://dx.doi.org/10.1016/j.jclepro.2016.03.154
0959-6526/© 2016 Elsevier Ltd. All rights reserved.
 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609 601

cultivation procedure, for example 6 g/L in our previous study (Liu enzyme sample was measured using a SBA-40E Biosensing
et al., 2013), thereby limiting hyper-production of laccase. HCDC analyzer (Shandong Academy of Sciences, Jinan, China). Each
was therefore designed for the first time in this study by means of experiment was performed in triplicate and the data were used to
adjusting seed cultivation and inoculation to explore the possibility calculate the mean ± standard deviation (SD).
of robust laccase production.
Scalability of laccase production is one major factor that has to
2.2. Fermentation in 5-L STR under a routine or HCDC operation
be addressed for its industrial applications (Couto and Toca-
Herrera, 2007). The cost of laccase production in industrial bio-
To perform laccase production under a routine operation, the
reactors can be reduced by several folds in comparison to that at
fungus was firstly cultured on potato dextrose agar (PDA) plate at
bench scale (Osma et al., 2011). Previous studies reported laccase
30  C for 5 days and subsequently inoculated into growth medium
production with varied efficiency at different scales, most of which
containing 5% glucose, 1% yeast extract, and 0.5% peptone to gain a
were below 50 L (Couto and Toca-Herrera, 2007). Although a
seed culture under 200 rpm and 30  C. After 48 h of incubation,
multitude of higher fungi have been proven to be able to produce
600 mL of the inoculum was transferred into a 5-L STR bioreactor
laccase, specific fungus that is propitious to scaling up at desirable
(Baoxing Bioengineering Equipment Co., Ltd, Shanghai, China) for
level is relatively few. Among various ligninolytic fungi, the genus
laccase production in a 3-L optimized production medium as pre-
Pycnoporus attracted research interest for its overproduction of
viously described (Liu et al., 2013). The two major process param-
high redox potential laccase and as a strong contender for green
eters, pH and dissolved oxygen (DO), were online monitored and
biotechnology (Lomascolo et al., 2011). We in our previous study
recorded by a pH and a DO probe. The culture broth was periodi-
have achieved efficient laccase production by Pycnoporus in a 65 L
cally sampled every 12 h followed by centrifugation to separate
air-lift reactor (Liu et al., 2013), yet evaluation of laccase production
mycelia and crude enzyme for subsequent analysis. The fermen-
in larger bioreactors, such as in the 500-L or 5-ton reactors in this
tation parameters were set as follows: 30  C, agitation rate
study, is still of great significance. As far as we know, description of
200 rpm, constant aeration rate 3 L/min. The cultured fungus in
laccase production at such scales has not been reported to date.
bioreactor for 3 days was taken out for morphological observation
As an important spectroscopic technology, circular dichroism
using scanning electron microscope (SEM).
(CD) analysis has been extensively used in characterizing protein
As for HCDC operation, a minor modification was made on a
structures (Schneider et al., 1999). The near-UV CD spectrum
basis of the routine procedure, shown in Fig. 1. Specifically, a 3 L of
mainly reflects the tertiary structure while far-UV CD spectrum can
seed culture was obtained by growing the fungus in the 5-L STR
provide information on the secondary structure of a protein
bioreactor (phase I) and then 1 L of optimized medium was added
(Greenfield and Fasman, 1969). The properties of laccase produced
in the seed culture which was further cultivated for 7 days (phase
by Pycnoporus sp. SYBC-L3 has been previously studied in both
II), while all other conditions were maintained the same as above
crude and purified forms, which exhibited excellent thermosta-
mentioned in the routine operation.
bility, acidic pH optima, and metal tolerance (Liu et al., 2012, 2013).
It is interesting to explore the underlying mechanisms for these
properties in terms of its secondary structures, which have how- 2.3. Fermentation at pilot-scale in a 500-L or 5-ton bioreactor
ever not been studied before. Therefore a systematic characteriza-
tion of the Pycnoporus laccase under various conditions, e.g., In order to produce laccase in 500-L STR, a secondary seed
temperature, pH, and some heavy metal ions, was performed using culture was prepared in a 50-L bioreactor (working volume 35 L) for
far-UV CD spectral analysis in this study. 2 days under conditions: agitation 200 rpm, temperature 30  C,
The specific goals of this study were: 1) to develop a HCDC constant aeration rate 10 L/min, constant reactor pressure
strategy for fungal cultivation to enhance laccase production via 0.05 Mpa. The seed culture was then transferred in a 300 L of
fermentation in bioreactors at different scales, 2) to purify laccase production medium (glucose was replaced with the same gram of
from the culture broth with an one-step scheme and explain the maltose) in a 500-L reactor for laccase production under the
catalytic properties of laccase by CD spectral analysis, and 3) to following conditions: agitation 200 rpm, temperature 30  C, con-
prepare powdered laccase using a spray-drying technique. The stant aeration rate 6 m3/h (100 L/min), constant reactor pressure
results will provide experimental data for industrial production 0.05 Mpa. A volume of 30 mL antifoaming agent was added into the
and application of laccases.

2. Materials and methods

2.1. Chemicals, fungus, and assays

Laccase substrate 2,6-Dimethoxyphenol (DMP) was obtained

from Sigma (St. Louis, MO), and other chemicals of analytical-grade
were purchased from Sinopharm Chemical Reagent Co., Ltd. Wild
fungus, Pycnoporus sp. SYBC-L3, was a stock culture for laccase
production in our laboratory (Liu et al., 2013). Laccase activity was
determined by monitoring the increase in absorption at 469 nm in
100 mM phosphate-citrate buffer (pH 3.5) with DMP
(ε ¼ 49.5 mM1cm1) as substrate, where one unit of laccase ac-
tivity was defined as the amount of enzyme required to oxidize
1 mol of DMP per min (Litthauer et al., 2007). Protein content was
determined using bovine serum albumin (BSA) as standard ac-
cording to the method of Bradford (1976). The vacuum filtered or
centrifuged mycelia were oven dried at 80  C to constant weight for Fig. 1. Schematics for routine submerged fermentation (A) and HCDC (B) for laccase
fungal biomass determination. Glucose concentration in crude production in 5-L STR.
602 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609
production medium prior to steam sterilization (121  C, 30 min).
Fermentation parameters, such as pH, temperature, agitation,
aeration, and dissolved oxygen were controlled and/or recorded
on-site during seed cultivation, while laccase activity, fungal
biomass, and residual sugar was determined by taking 20 mL
aliquot of culture medium from the reactor every day. Content of
residual sugar was determined using dinitrosalycyclic acid (DNS)
method with glucose as standard (Ghose, 1987). After each sam-
pling, the volume of medium was not adjusted to its original vol-
ume in each batch run.
In order to scale up laccase production in a 5-ton STR, a sec-
ondary seed culture was prepared likewise but in a 2-ton bioreactor
(working volume 1 ton) for 54 h under conditions: agitation
280 rpm, temperature 30  C, constant aeration rate 0.4 vvm, con-
stant reactor pressure 0.05 Mpa. The seed culture was then trans-
ferred in 2.5 ton production medium (glucose was replaced with
the same gram of maltose) in a 5-ton reactor for laccase production
under the following conditions: agitation 260 rpm, temperature
30  C, constant aeration rate 0.38 vvm, constant reactor pressure
0.05 Mpa. A volume of 300 mL antifoaming agent was added into
the production medium prior to steam sterilization (121  C,
30 min). Laccase production cost was analyzed based on the actual
consumption in this batch operation, with all five major compo-
nents included: feedstock, electricity, steam, manpower and
equipment depreciation. The cost of each component to carry
through the entire operation was calculated. The costs of all com-
ponents were the actual local prices, and the labor cost was esti-
mated by the total manpower input and the actual salary paid to
each worker.
2.4. Purification, gel electrophoresis, and capillary electrophoresis
For laccase purification, culture broth was collected after 6 days
of cultivation and centrifuged at 8000 g for 10 min to remove fungal
biomass and then the supernatant was filtered through a 0.45 mm
cellulose nitrate membrane by a vacuum pump. Afterward, the
filtrate was loaded on a DEAE 52 anion exchange chromatography
column pre-equilibrated with citric-phosphate buffer (pH 6.0,
20 mM) and then eluted with a linear gradient (0e1 M NaCl in
500 mL buffer). The flow rate was set at 2 mL/min and fractions of
every 10 mL were pooled in tubes for the subsequent laccase ac-
tivity assay and electrophoresis analysis. Eluate in each tube was
further subjected to native polyacrylamide gel electrophoresis
(Native-PAGE) for active staining with 1 mM DMP as substrate or
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) for homogeneity verification with 0.2% Coomassie brilliant
blue G-250. Purity of laccase was also verified by capillary elec-
trophoresis using a P/ACE 5000 system (Beckman Instruments,
Fullerton, CA, USA) equipped with a UV detector (Bornscheuer
et al., 1994).
2.5. UVevis and CD spectra analysis of laccase
A spectrophotometer (U-3010, HITACHI, Japan) was employed to
record UVevis spectra (190e700 nm) of purified laccase. As for CD
spectra, the elutions from DEAE 52 with laccase activity in peak A
were pooled and powdered with a freeze-dryer (Labconco, USA).
Then the powdered laccase was dissolved in 10 mM sodium
phosphate buffer (pH 6.0) to prepare concentrated laccase which
was further dialyzed against deionized water for 48 h. The dialyzate
was used as control sample (correcting the baseline of the spec-
trum) and the reserved pure laccase (protein content of 0.02 mg/
mL) was scanned for CD spectra using a MOS-450 circular dichro-
ism spectropolarimeter (Bio-Logic, France). The far-UV CD spectra
of purified laccase were determined from wavelength
190e250 nm at 25  C. The scan speed was set at 60 nm/min with a
response of 1 s and band width at 1 nm adapted from Salony et al.
(2008). The effect of temperature on CD spectra of laccase was
measured in a quartz cuvette of 1 cm path length, while the effect of
pH and metal ions on CD spectra was measured in a quartz cuvette
of 0.1 cm path length. Every experiment was carried out in dupli-
cate and the average value was used for calculation of secondary
structure using the software K2d (Salony et al., 2008).
2.6. Powder laccase preparation by spray drying
The culture broth that was collected from selected production
procedures was firstly filtered through 8-layer gauze to remove
fungal biomass and then subjected to spray drier (Uniform Drying
Co., Ltd.,Changzhou, China) without further concentration. Two
batches were separately carried out with the following conditions:
inlet temperature 200  C, outlet temperature 90  C, flow rate of 1 L/
h or 2 L/h. After spray drying, the powdered laccase was collected
from the collecting vessel and the inner wall of the pipeline. The
activity of liquid or powdered laccase was determined respectively
to calculate the activity recovery.
3. Results and discussion
3.1. Laccase production in 5-L bench-scale STR under routine
operation
As shown in Fig. 2 for laccase production in 5-L STR, pH value of
culture media decreased slightly from its initial point (4.5) to the
lowest number (3.8) in the first 2 days and then gradually rose up to
6.5 during the following cultivation period. This trend is in accor-
dance with that in our previous report in an air-lift reactor (Liu
et al., 2013) and also with that by Stereum ostrea and Phaner-
ochaete chrysosporium for laccase production (Praveen et al., 2011),
but slightly different from that by Coriolus versicolor, which
continuously dropped to pH 3 from the initial pH 6 during the
entire fermentation course (Que et al., 2014).
Dissolved oxygen (DO) can greatly impact the growth of free cell
and subsequent secretion of extracellular laccase. We in the present
study adopted a constant ventilation rate which resulted in variable
DO level (Fig. 2A), while some studies chose adjusted ventilation
approach to maintain constant DO level (e.g. 30%) throughout the
fermentation process (Colao et al., 2006; Que et al., 2014). Fungal
biomass and laccase reached the highest value of approximately
3.5 mg/mL on day 2 and 60 U/mL on day 7, respectively (Fig. 2B&C),
which were comparable with that obtained (6 mg/mL on day 3 and
72 U/mL on day 6) in an air-lift reactor (Liu et al., 2013). Que et al.
(2014) also performed fungal cultivation in a 5-L bioreactor and
produced the highest fungal biomass of 30 mg/mL on day 7 and
laccase activity of 7.5 U/mL on day 5, respectively.
Along with the rapid fungal growth, glucose content dramati-
cally decreased (Fig. 2B). Extracellular laccase accumulated gradu-
ally only in the late phase of cultivation presumably because of the
release of intracellular laccase caused by cell lysis (Bose et al., 2007).
Protein content versus time course kept a similar pace with laccase
production, but was produced in low quantities with its maximum
of 0.24 mg/mL at day 6 or 7. Recombinant laccase was expressed
and produced in Aspergillus niger, bringing about the highest ac-
tivity of 42 U/mL and a specific activity of 50 U/mg protein
(Mekmouche et al., 2014), which were still lower than those in our
study (60 U/mL and 240 U/mg, respectively).
As seen in the pictures shown in Fig. 3A & B, growth of Pycno-
porus sp. SYBC-L3 in bioreactor tended to form trophosome in the
form of mycelial pellets (F z 2 mm) during liquid cultivation under
agitation, as observed in other fungus such as C. versicolor and
 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609 603

(A) 8 140
7 120
6
100

Disolved oxygen (%)

5
80
pH value

4
60
3
40
2
1 20

0 0
0 24 48 72 96 120 144 168 192
Cultivation time (h)
4 14
(B)
12
3
10 Glucose content (g/L)
Fungal biomass (g/L)

8
2
6
Fig. 3. Cultivation of Pycnoporus sp. SYBC-L3 in 5-L STR (A), fungal pellets (B), SEM of
4 fungus grown on PDA plate (C) and in 5L STR (D).
1
2
speed (150 rpm) and inoculum (105 spore/mL) can give rise to the
0 0 highest enzyme production with pellet size of 2.78 mm, which was
0 24 48 72 96 120 144 168 192 similar to our observation (pellet size 1e2 mm)
Cultivation time (h)
(C) 70 0.5 3.2. Laccase production in 5 L bench-scale STR under HCDC
condition
60
0.4 HCDC strategy was designed for Pycnoporus sp. SYBC-L3 (Fig. 1)
Protein content (mg/mL)
Laccase activity (U/mL)

50 and the result of laccase production was illustrated in Fig. 4. A 6-

40
30
20
10
0 0.0
0 24 48 72 96 120 144
Cultivation time (h)
Fig. 2. Fermentation behavior of Pycnoporus sp. SYBC-L3 in 5-L STR: pH and DO (A),
biomass and sugar consumption (B), laccase activity and protein content (C).
Tetraploa aristata (Que et al., 2014; Saparrat et al., 2002). The fungus
actually exhibited different morphological characteristics at
different stages during its growth and development, i.e., arthro-
spores were produced by means of fragmentation in later phase
(Fig. 3C) under static cultivation on PDA plate, while stirred liquid
cultivation hardly generated single spores during the entire culti-
vation (Fig. 3D). Obviously, the morphological form of hypha can
exert significant effect on metabolite accumulation, like laccase and
exopolysaccharides (Que et al., 2014). Jime
nez-Tobon et al. (1997)
studied the effect of inoculum and rotate speed on both pellet
size and MnP production and found that higher inoculum can result
in smaller pellet but higher enzyme activity, and appropriate rotate
0.3 fold increase in the highest fungal biomass (20 g/L on day 5) was
observed under HCDC but requiring 3 more days than the routine
operation (Fig. 3B). After a two-phase manipulation, the highest
0.2 laccase production (240 U/mL) was achieved on day 8, which is
about 4-fold of that under routine procedure and comparable to
0.1 that by Trametes (280 U/mL) within 6 days, the highest that have
been reported at flask scale to our knowledge (Ling et al., 2015). To
overcome limitations that hamper applicable exploitation due to
low enzyme production from native hosts, heterologous expression
168 192 in some efficient hosts is commonly employed for anticipated
hyper-production. Colao et al. (2006) heterologously expressed
Trametes trogii laccase gene in Pichia pastoris with a protein yield of
0.017 mg/mL and an activity of 2.52 U/mL after 8-d fermentation,
which were far below than those in our result, i.e., 1 mg/mL and
240 U/mL, respectively. It is worth noting that the maximum lac-
case activity under HCDC could double to over 500 U/mL if the
preferable substrate ABTS, instead of DMP, was adopted for activity
assay (Liu et al., 2012).
3.3. Laccase production in pilot-scale 500-L STR
For fungal fermentation in 500-L STR for laccase, the first-order
seed culture from flask was inoculated and amplified in 50-L STR
for the second-order seed culture, and the growth behaviors of
three separate batches were described in Fig. 5. It can be seen that
rapid fungal biomass build-up took place in 2-d cultivation. The pH
value of growth medium experienced slight fluctuation but was in
604 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609

(A) 8 140 reached 60% on day 4, which was higher than that (40%) in 5-L
(phase I) (phase II) reactor (Fig. 1A) because of enhanced ventilation in 500-L STR.
7 120 The maximum biomass was approximately 10 g/L on day 3, about
6 3-fold of that (3.5 g/L) in 5-L STR. An interesting phenomenon that
100

Disolved oxygen (%)

the residual sugar content (determined as reducing sugar)
5 increased rather than decreased was observed in the first 3-day
80
pH value

(Fig. 6D), possibly owing to increased glucose molecule derived

4
60 from maltose hydrolysis by fungal extracellular hydrolytic en-
3 zymes. Up scaling from 5-L to 500-L STR, the laccase production
40 maintained to the similar level of about 60 U/mL (Fig. 6E). A robust
2
daily output of laccase achieved on day 5e7, close to 12 U/mL/
1 20 d (Fig. 6F). During the 8-d cultivation, about 50 L of culture medium
was lost from the starting volume of 300 L as a result of evapora-
0 0 tion, suggesting evaporation rate of around 8 L per day.
0 24 48 72 96 120 144 168 192 216 240
Cultivation time (h)

(B) 25 60 3.4. Laccase production in 5-ton production-scale STR

(phase I) (phase II)

20 48 Laccase productivity was further evaluated in a 5-ton STR, in

which four major process parameters, namely laccase, sugar, pH,
Glucose content (g/L)
Fungal biomass (g/L)

and biomass, were investigated. As seen in Fig. 7A, the four vari-
15 36 ables followed similar trends in general as those in 5-L or 500-L STR
(Figs. 2 and 6). A notable difference was the delayed maximal lac-
10 24 case activity (83 U/mL) on day 9, which might be due to the fact that
the first-order fungal seed was directly inoculated in the 5-ton
reactor production, instead of second-order seeding. Laccase
5 12 accumulation in the late phase in 5-ton STR appeared to be more
rapid relative to that in 5-L STR. Reducing sugar content dropped
0 0 steadily from the starting point of 25 g/L until day 7, reaching less
0 24 48 72 96 120 144 168 192 216 240 than 2.5 g/L and then maintained unchanged for the following 4
days. The residual reducing sugar in culture broth was supposed to
Cultivation time (h)
be partly from the sugars in laccase for glycosylation. The fungus
(C) 250 1.2 exhibited robust mycelial growth since the transferring of the first-
(phase I) (phase II)
order seed culture, achieving the highest biomass of near 10 g/L on
1.0 day 3, and then decreased over the remaining cultivation time. The
200
Protein content (mg/mL)

pH value of culture broth underwent three phases: a dramatic drop

Laccase activity (U/mL)

0.8 from the initial pH 5.5 to pH 3.5 in the first day, maintaining pH 3.5
150 for the subsequent 4 days, and then rising up to the final value of
0.6 about pH 7 after day 6.
100 The cost percentage of this batch was roughly calculated on a
0.4 basis of actual and predicted consumption of five essential con-
stituents, shown in Fig. 7B. The highest percentage (50%) in the
50 total cost was labor in the form of salary which might vary with
0.2
different working district. Human power was mainly used to
0 0.0 manage microorganisms, measuring procedure parameters, as well
0 24 48 72 96 120 144 168 192 216 240 as operating the fermentation process. Electricity accounted for 36%
of the total cost, the second contribution after labor. Depreciation of
Cultivation time (h)
the equipment was about one-tenth of the total cost. Feedstock was
Fig. 4. Fermentation behavior of Pycnoporus sp. SYBC-L3 in 5-L STR under HCDC: pH not a major cost factor, accounting for less than 5% of the total cost.
and DO (A), biomass and sugar consumption (B), laccase activity and protein content The lowest percentage was the consumption of steam, which was
(C). phase I: seed cultivation, phase II: laccase production. only 1%. The actual production cost was not revealed here in order
to avoid possible conflict of commercial interest, however our
estimated price for laccase by L3 in 65-L bioreactor was far below
the range of 4.5e6.0 (Fig. 5A). Steep decline in DO from 100% to
than those commercially available (Liu et al., 2013). By examining
almost zero indicated the surge in biomass amount (Fig. 5B). The
26 different culture medium and their corresponding laccase pro-
second-order seed culture propagated slowly in the first 10 h and
duction in flask under submerged fermentation, Osma et al. (2011)
then dramatically increased in the following 10 h and finally leveled
concluded that the culture medium, in all cases, was the largest
off at 8e10 g/L after 20 h cultivation (Fig. 5C). As for up-scaled
component (90%) in the total cost, followed by the cost in operation
production in 500-L STR (Fig. 6), the pH value, DO, biomass, lac-
(10%). In real pilot-scale fermentation, however, the situation may
case activity versus time course followed similar trends to those in
be greatly different depending on the current feedstock, power and
5 L STR (Fig. 2). The pH value of culture broth remained relatively
labor costs, as discussed above. It is worth noting that the we
stable (4e5) throughout the cultivation for all data points recorded,
believe the cost of laccase production can be further reduced by
while malfunction of the pH and DO electrodes occurred in the first
further enhancing laccase productivity using certain strategies at
batch (indicated as black square in Fig. 6A and 6B). The lowest DO
pilot-scale, such as HCDC, stage pH-control, and fed-batch method.
 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609 605

6.0 (B) 100 (C) 12

(A)
5.5 80 10

Fungal biomass (g/L)

Disolved oxygen (%)
8
5.0 60

pH value
6
4.5 40
4
4.0 20 2

3.5 0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Cultivation time (h) Cultivation time (h) Cultivation time (h)

Fig. 5. Mycelial growth behavior of Pycnoporus sp. SYBC-L3 in 50-L STR for second order seed culture: batch 1 (black square), batch 2 (red circle), batch 3 (blue triangle). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

7 100 15
(A) (B) (C)

6 80 12

Fungal biomass (g/L)

Disolved oxygen (%)

5 60 9
pH value

4 40 6

3 20 3

2 0 0
0 48 96 144 192 0 48 96 144 192 0 48 96 144 192
Cultivation time (h) Cultivation time (h) Cultivation time (h)

25 80 (F) 15
(D) (E)
Laccase productivity (U/mL/d)

20 64 12
Laccase activity (U/mL)
Residual sugar (g/L)

15 48 9

10 32 6

5 16 3

0 0 0
0 48 96 144 192 0 48 96 144 192 0 48 96 144 192
Cultivation time (h) Cultivation time (h) Cultivation time (h)

Fig. 6. Fermentation behavior of Pycnoporus sp. SYBC-L3 in 500-L STR for laccase production: batch 1 (black square), batch 2 (red circle), batch 3 (blue triangle). (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. Fast purification of laccase The purified laccase did not show the blue color by naked eye,
the typical color for blue laccase with absorption peak at around
Purification of laccase from the culture broth using ion- 600 nm (Eggert et al., 1996). When subjected to UVevis spectro-
exchange chromatography was performed and the profiles are photometer, a broad shoulder peak showed up at wavelength
shown in Fig. 8. As the NaCl concentration increased linearly from 300 nm for purified laccase combined from tube 2 and 3, which is
0 to 1 mol/L, two absorption peaks (OD 280) of B and C showed up in very similar to some previous reports (Lu et al., 2007; Wang et al.,
order, corresponding to tube 3 and 8, respectively. Laccase activity 2010), indicating the presence of type III binuclear Cu (II) pair
in each tube from 2 to 10 was examined by Native-PAGE (Fig. 8B), (Eggert et al., 1996). The purified laccase was also confirmed by
which suggested that the elutions in tube 2 and 3 had the highest capillary electrophoresis and the purity of laccase was found to be
activity. Elutions in tube 2 and 3 were also applied to SDS-PAGE around 97.83% (major peak at 2.013 min), shown in Fig. 9. Normally,
(Fig. 8C) and a single band was observed for each tube, indicating at least three successive steps were required to purify laccase from
a purified form of laccase was obtained. Elutions in tube 2 and 3 white-rot fungi: precipitation, ion exchange, and gel filtration or
were then combined to calculate the laccase recovery from crude affinity chromatography (Litthauer et al., 2007; Lu et al., 2007;
laccase, which was around 72%. Wang et al., 2010), while only very few studies reported simpler
606 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609

Fig. 7. (A) Fermentation behavior of Pycnoporus sp. SYBC-L3 in 5-ton STR for laccase
production: laccase activity (open circle), reducing sugar (solid square), pH value (open
square), and fungal biomass (solid circle). (B) Cost percentage analysis of laccase
production performed in 5-ton STR. Total cost (100%) of the batch was the sum of five
essential parts, namely feedstock (medium, H2O, and antifoam), electricity (bioreactor,
compressor, and water pump), steam (sterilization and heating), labor (culture pres-
ervation room, inspection center, and production workshop), and depreciation
(including equipment purchase fee).

purification procedures, such as by phenyl-Sepharose High Per-

formance column (Garcia et al., 2006). Although recombinant
proteins (e.g., laccase) have been thought to require simple purifi-
cation procedures (Erjavec et al., 2012), published data revealed
that several steps were still needed (Colao et al., 2006). Therefore,
the fact the laccase produced from Pycnoporus sp. SYBC-L3 can be
readily purified in a one-step column procedure is a great advan-
tage, and this is probably because the laccase is usually excreted as
a major extracellular enzyme by genus Pycnoporus (Liu et al., 2013;
Lomascolo et al., 2011).

3.6. CD spectra of laccase

The Far-UV CD spectra of the purified laccase are displayed in

Fig. 10. The CD spectra showed one positive peak around 198 nm
and one negative trough around 218 nm, indicating the typical
signature for a-helix and b-sheet structure, respectively (Salony
et al., 2008; Schneider et al., 1999). Similarly, Bukh and Bjerrum
(2010) reported that Coprinus cinereus laccase showed CD spectra
with maximum absorption at 199 nm and minimum at 218 nm. The
content of secondary structure calculated from the experimental
CD values in the purified laccase is shown in Table 1. The laccase
consisted of approximately 3% a-helix, 50% b-sheet, and 47% Fig. 8. (A) Purification of laccase on DEAE-sepharose 52. Abs 280 (line with triangle),
random coil, which is similar to that in C. cinereus laccase (6% a- laccase activity (line with circle), NaCl concentration (line). (B) Native-PAGE of laccase
helix, 40% b-sheet, and 54% random coil) (Bukh and Bjerrum, 2010). collected in corresponding tube from DEAE column. Laccase activity was stained with
In contrast, fungal laccase with higher a-helix content (68%), e.g. C. DMP in phosphorus buffer. The lane number above each reddish band was for each
tube number in (A). (C) SDS-PAGE of crude laccase and purified laccase. lane 1: mo-
versicolor laccase, has been documented as well (Que et al., 2014).
lecular mass markers (94kD: TaqDNA Polymerase; 62kD: T4 DNA Ligase; 45kD: Al-
The potential mechanism of thermostability and inhibition of bumin from chicken serum; 33kD: His-Tag fusion protein). lane 2 and lane 3: laccase in
some metal ions on laccase was explored using CD analysis under the tube number 2 and 3 from DEAE 52. Lane 4: crude laccase in culture broth. (D)
different conditions. As shown in Table 1, no significant change in Absorbance spectrum of purified laccase by UVevis spectrophotometer, where tube 2
the secondary structure was observed with temperature increasing and 3 were combined.

from 5 to 55  C. However, when temperature was above 75  C, the

 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609 607

Table 1
Predicted secondary structure of purified laccase under various conditions.

Influencing factor Alpha helix/% Beta sheet/% Random coil/%

Control 3 50 47
5 C 3 50 47
25  C 3 50 47
55  C 3 50 47
75  C 46 23 31
AgNO3 (1 mM) 11 41 48
AgNO3 (5 mM) 13 40 47
AgNO3 (50 mM) 0 4 96
FeSO4 (1 mM) 10 43 47
FeSO4 (5 mM) 9 42 48
FeSO4 (50 mM) 4 48 48
FeCl3 (1 mM) 4 49 47
FeCl3 (5 mM) 3 50 47
FeCl3 (50 mM) 3 49 47

3.7. Preparation of powder laccase

Fig. 9. Purity verification of laccase by capillary electrophoresis.
absorbance peak around 208 nm and 220 nm decreased (figure not
shown), suggesting thermal denaturing (Table 1), resulting in the
loss of activity. Likewise, de Wilde et al. (2008) confirmed less
thermal stability of Melanocarpus albomyces laccase expressed in
rice than in Trichoderma reesei using CD analysis with temperature
in the range of 30e90  C. When exposed to various pH values at the
same solute concentration (0.1 M), no evident change in the CD
spectra was observed for all pH values below 6.5, even though both
the purified and crude laccase lost most of its activity toward DMP
when pH is above 4.5 (Liu et al., 2012, 2013). The spectra of laccase
exposed to Fe2þ and Fe3þ (from 1 to 50 mM) did not show
noticeable changes (Fig. 10B), which were also reflected in the
relative content of secondary structure (Table 1). However, the
presence of Agþ ranging from 5 to 50 mM remarkably altered CD
spectra with messy curved lines emerged (figure not shown),
suggesting a dramatic decrease of b-sheet content in laccase
(Table 1). The analysis of CD spectral changes strongly indicated the
different inhibition mechanisms of Fe2þ/Fe3þ and Agþ on laccase.
Although CD analytical technique has been utilized for secondary
structures of proteins (Allendorf et al., 1985; Salony et al., 2008),
studying CD changes in response to varied conditions is relatively
few. This study represents the first attempt to collect series of CD
data for laccase exposed to different conditions, providing insight
into the differences in enzymatic properties.
After liquid cultivation in bioreactor, supernatant without fungal
biomass was subjected to spray drying to produce powdered lac-
case and the details are listed in Table 2. Laccase powder was
successfully produced with high activity in both two batches with
recovery as high as 74.6% and 82.2%, respectively. The prepared
laccase powder is uniform and smooth, looked yellowish and
smelled like peptone. It should be noted that the drying condition
was our first trial, and the final yields may be further improved if
the drying condition, such as inlet and outlet temperature and the
flow rate, are optimized. There has been an argument that pro-
duction of laccases in their native hosts is often not economically
feasible and natural laccases may not withstand the harsh condi-
tions processes (Erjavec et al., 2012). Our results in this study
however clearly prove that certain wild fungus, like Pycnoporus sp.
SYBC-L3, can serve as an excellent cell factory for laccase produc-
tion with outstanding properties and economic viability.
4. Conclusion
Laccase production was successfully achieved by high cell den-
sity cultivation at various scales. The highest laccase production
was 80 U/mL in a 5-ton bioreactor, where the labor cost was found
to be the highest among five cost components. An one-step puri-
fication scheme was proven easy and efficient. Circular dichroism
revealed the secondary structure of and explained some properties

(A) 20 pH2.5 pH3.0 (B) 20

pH3.5 pH4.0 control
pH4.5 pH5.5 FeSO4/1 mmol/L
Molar Ellipticity (deg.cm .dmol )

Molar Ellipticity (deg.cm .dmol )

-1

-1

pH6.5 FeSO4/5 mmol/L

10 10
FeSO4/50 mmol/L
2

0 0

-10 -10

-20 -20
190 200 210 220 230 240 250 190 200 210 220 230 240 250
Wavelength (nm) Wavelength (nm)

Fig. 10. UV-CD spectrum of laccase affected by varying pH (A) and FeSO4 (B) at 25  C. Protein concentration was 0.5 mg/ml. CD spectrum was measured in a quartz cuvette of 0.1 cm
path length and the value of ellipticity was converted to molar ellipticity.
608 J. Liu et al. / Journal of Cleaner Production 127 (2016) 600e609
Table 2
Preparation of powdered laccase using spray-dryer.
Description Batch 1
Experiment site Engineering center at School of Food Science, Jiangnan University, Wuxi, China
Drying condition Inlet T 200  C, outlet T 90  C, flow rate 1 L/h
Total volume of crude liquid laccase 4L
Total activity of crude liquid laccase 288000 U
Total weight of powdered laccase 3.28 g
Total activity of powdered laccase 215000 U
Specific activity of powdered laccase 65548.7 U/g
Activity recovery 74.6%
of the laccase, like thermostability. A spray-drying technique was
employed to prepare powdered laccase with over 74% activity
recovered. By investigating productivity, scalability, purification,
property, and powder preparation as well as production cost, the
laccase by wild Pycnoporus fungus exhibited a great potential for
green and clean applications.
Acknowledgments
This study was supported in part by Scientific Research Foun-
dation for Advanced Talents of Huanghuai University
(1000.12.01.1342). We would like to thank Dr. Xiaolian Zhan (Wuxi
Jinkun Biotechnology Co., Ltd.) for providing 500 L bioreactor and
general manager Guobao Jiao (Henan Yangshao Bioproducts Co.,
Ltd.) for 5 ton bioreactor and engineer Huaqiang Xi for collecting
data in cost analysis. We also thank Changzhou Uniform Drying Co.,
Ltd for generous help in preparation of powdered laccase.
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