Process Biochemistry 51 (2016) 759764

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Agarose hydrolysis by two-stage enzymatic process and bioethanol

production from the hydrolysate
Young Bin Seo a,1 , Juyi Park a , In Young Huh a , Soon-Kwang Hong b , Yong Keun Chang a,
a
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
b
Division of Bioscience and Bioinformatics, Myung-Ji University, Yongin 449-728, Republic of Korea

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

Article history: A two-stage enzymatic process was developed for agarose hydrolysis without acid pretreatment. In the
Received 8 September 2015 rst stage, agarose was hydrolyzed to produce neoagarobiose using an optimized dosage of AgaG1 and
Received in revised form 12 March 2016 DagB at pH 7.0 and 40 C. Agarose gelation was avoided by momentarily elevating the reaction tem-
Accepted 14 March 2016
perature for the rst 10 min, instead of employing a pretreatment step with acid prior to the enzyme
Available online 17 March 2016
reaction. In the second stage, neoagarobiose was further hydrolyzed to produce galactose using neoa-
garobiose hydrolase (NABH) at pH 7.0 and 35 C. The overall yield of galactose from agarose was 88%
Keywords:
of the theoretical maximum. The crude galactose solution produced from agarose hydrolysis was used
Agarose
Enzymatic hydrolysis
directly for ethanol production by yeast to demonstrate its potential. In overall, 20 g/l of agarose could
Galactose be converted to 3.71 g/l of ethanol.
Bioethanol 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Ethanol can be produced by fermentation from various types
of biomass including starch, lignocelluloses, and marine algae
biomass. The bioethanol produced from marine algae has several
advantages over that based on starch and lignocellulosic biomass. It
does not compete with human foods, does not cause forest denuda-
tion, has a low content of lignocellulose, and can x a larger amount
of CO2 per unit mass [1,2].
The main constituent of red algae, one of the marine algae
biomass, is agarose. Agarose is a linear chain of alternating
residues of 3-O-linked -d-galactopyranose and 4-O-linked 3,6-
anhydro--l-galactose [3]. Agarose can be hydrolyzed chemically
or enzymatically. During chemical hydrolysis, galactose from the
galactosyl residue is retained, while the anhydrogalactose formed
is further converted to 5-hydrooxymethyl furfural (5-HMF) and
then to levulinic acid (LA), both of which are known to be toxic
to many microorganisms including yeast to lower fermentation
efciency [4,5]. Therefore, the resulting crude galactose solution
could be used as the medium for ethanol production only after
a pretreatment step, for example, by nanoltration and electro-
dialysis for their removal [6]. In contrast, an enzymatic hydrolysis
Corresponding author.
E-mail address: ychang@kaist.ac.kr (Y.K. Chang).
1
Current address: SK innovation co., 325, Exporo, Yuseong-Gu, Daejeon 305-712,
Republic of Korea.
process does not produce 5-HMF and LA, and leaves 3,6-anhydro-l-
galactose intact. The resulting crude galactose solution, containing
no toxic compounds, can be used directly to produce ethanol by
fermentation.
Enzymatic hydrolysis of agarose is known to include three steps,
in principle: (1) agarose liquefaction by -agarase I to neoagaro-
oligosaccharides; (2) neoagaro-oligosaccharides degradation by
-agarase II to neoagaoriobiose; and (3) neoagarobiose decom-
position by -neoagarobiose hydrolase (NABH) to galactose and
anhydrogalactose [7].
In a series of efforts by our research group to search for
enzymes needed for agarose degradation, the rst involved AgaG1,
screened from Alteromonas sp. GNUM1. This enzyme is an endo-
type -agarase, which hydrolyzes agarose to neoagarobiose and
neoagarotetroase. It was expressed in Escherichia coli BL21 [810].
The second enzyme, DagB, an exo-type -agarase which hydrolyzes
agarose and neoagarotetraose to neoagarobiose, was screened
from S. coelicolor A3(2) and expressed in S. lividans TK24 [11].
Finally, a newly screened microbial strain of Alcanivorax sp. A28-3
(KCTC12788BP) was found to produce NABH, although its amino
acid sequence is yet to be identied. When neoagarobiose was
treated with the cell lysate of Alcanivorax sp. A28-3, galactose and
anhydrogalactose were produced as the nal products [12].
In this study, a two-stage process for the saccharication of
agarose to galactose and anhydrogalactose was developed and opti-
mized. In the rst step, agarose was degraded to neoagarobiose
using a mixture of AgaG1 and DagB, produced by a recombinant
E. coli and a recombinant S. lividans, respectively. In the second step,

http://dx.doi.org/10.1016/j.procbio.2016.03.011
1359-5113/ 2016 Elsevier Ltd. All rights reserved.
760 Y.B. Seo et al. / Process Biochemistry 51 (2016) 759764
neoagarobiose was decomposed to galactose and anhydrogalactose
using the cell lysate of Alcanivorax sp. A28-3. The resulting galactose
solution was used by Saccharomyces cerevisiae KL17 (KFCC11493P),
a galactose-utilizing yeast strain newly screened by our group for
ethanol production.
2. Materials and methods
2.1. Strains and media
E. coli BL21 and pMoPac1 were used as the host and plas-
mid for the expression of agaG1 gene, respectively [10]. E. coli
BL21/pMoPac1-AgaG1 was grown on the Luria-Bertani (LB)
medium containing 35 mg/l of chloramphenicol.
S. lividans TK24, which was obtained from The John Innes Foun-
dation (United Kingdom) and pUWL201PW [13] were used as
the host and vector for the expression of DagB, respectively. The
S. lividans TK24/pUWL201PW-DagB culture was performed in a
R2YE medium with no sucrose, containing per liter: 0.25 g K2 SO4 ,
10.12 g MgCl2 6H2 O, 10 g glucose, 0.1 g casamino acids, 5 g yeast
extract, 10 ml of 0.5% K2 HPO4 , 80 ml of 3.68% CaCl2 2H2 O, 15 ml
of 20% l-proline, 100 ml of 5.73% N-tris(hydroxymethyl) methyl-2-
aminoethanesulfonic acid (TES; pH 7.2), and 2 ml of trace elements
solution [14]. For stable maintenance of the plasmid, 50 mg/l of
thiostrepton was used [11].
Alcanivorax sp. A28-3 (KCTC12788BP) was used for the produc-
tion of NABH. The strain was cultured in an articial sea water
media (ASW-YP) containing per liter 6.1 g Trizma base (pH 7.2),
12.3 g MgSO4 , 0.74 g KCl, 0.13 g (NH4 )2 HPO4 , 17.5 g NaCl, 0.14 g
CaCl2 , 0.2 g yeast extract, 3.0 g bacto peptone, and 2.0 g agar.
S. cerevisiae KL17 (KFCC11493P) was used for the production of
ethanol, and was cultured in an YPG medium containing per liter:
10 g yeast extract, 20 g peptone, and 10 g galactose, or in an YPEH
medium containing per liter: 10 g yeast extract and 20 g peptone,
and actual enzyme hydrolysate of agarose.
2.2. Preparation of enzyme solutions
2.2.1. AgaG1
E. coli BL21/pMoPac1-AgaG1 culture was performed in bafed
Erlenmeyer ask. Cells from the cell stock were used to inoculate
10 ml of LB medium for the seed culture. The culture was performed
at 37 C and 200 rpm. After overnight culture, 2% of the seed culture
broth was inoculated into 200 ml of LB medium. When the culture
OD600 reached 1.2, the temperature was decreased to 18 C and
then AgaG1 expression was induced using 1.0 mM of IPTG. The cul-
ture was performed for 8 h at 18 C after induction. The harvested
cells were disrupted by using a homogenizer (Model 500, Fisher
scientic, USA) to recover AgaG1 inside the cells. Cell debris was
removed by centrifugation for 20 min at 5000 g-force. The super-
natant was used for the hydrolysis of agarose.
2.2.2. DagB
S. lividans TK24/pUWL201PW-DagB was grown in a bafed
Erlenmeyer ask. Cells from the cell stock were used to inoculate
10 ml of R2YE medium for the seed culture. The culture was per-
formed at 28 C and 200 rpm. After 2 days of seed culture, 2% of the
seed culture broth was inoculated into 200 ml of R2YE medium.
After 2 days of culture, cells were removed by centrifugation for
20 min at 5000 g. The supernatant was used for the hydrolysis of
neoagarotetraose.
2.2.3. NABH
Alcanivorax sp. A28-3 was cultivated in an Erlenmeyer ask.
Cells from the cell stock were used to inoculate 10 ml of ASW-
YP medium for the seed culture at 28 C and 200 rpm. After 1 day
of seed culture, 2% of the seed culture broth was inoculated into
200 ml of ASW-YP medium. After 2 days of culture, the harvested
cells were disrupted by using a homogenizer (Model 500, Fisher
scientic, USA) to recover NABH inside the cells. Cell debris was
removed by centrifugation for 20 min at 5000 g. The supernatant
was used for the hydrolysis of neoagarobiose.
2.3. Two-stage hydrolysis of agarose
In the rst stage, 10 or 20 g/l of agarose (Lonza, Switzerland)
was hydrolyzed to neoagarobiose by using a mixture of AgaG1 and
DagB, both of which had the same optimum condition of pH 7.0, and
40 C [10,11]. The enzyme reaction was performed pH 7.0, 40 C,
and 100 rpm for 12 h in an Erlenmeyer ask containing 100 ml of
reaction mixture. Tris-HCl buffer at 50 mM was used. To avoid the
problem of agarose gelation, the reaction was started at 46 C. The
temperature was lowered to 40 C after 10 min, when gelation was
no longer a problem. In the second stage, neoagarobiose produced
in the rst stage was treated with the NABH solution at pH 7.0,
35 C, and 100 rpm for 12 h in a ask containing 100 ml of reaction
mixture. Tris-HCl buffer was used.
2.4. Ethanol production
S. cerevisiae KL17 (KFCC11493P) was used. It had been newly
screened from the soil by our group, and exhibited efcient uptake
of galactose to produce ethanol [15]. Seed culture was carried out
in 10 ml of the YPG medium. The main culture was inoculated with
2% seed culture broth. As required, 100 ml of YPG medium, YPEH
medium, or YPGH (YPG media + 0.5% of 5-HMF) medium was used
for the main culture. All the yeast cultures were carried out at 30 C
and 200 rpm.
2.5. Analysis
The agarase activity was measured by the dinitrosalicylic acid
(DNS) method as previously described [10]. One unit of enzyme
activity was dened for both AgaG1 and DagB as the amount
of enzyme liberating 1 mole of reducing sugar per minute, at
pH 7.0 and 40 C. One unit of NABH activity was dened as the
amount of enzyme liberating 1 mole of galactose per minute,
at pH 7.0 and 35 C. Neoagarobiose and neoagarotetraose were
quantied by using a high-performance liquid chromatograph
(HPLC) (Waters, USA), with an Asahipak NH2P-50 4E column
(250 4.6 mm, Shodex, Japan) and an ELSD detector (Sedex 75,
Sedere, France) under conditions described previously [16]. Galac-
tose and ethanol were quantied by using an YSI 2700 Select
Biochemistry Analyzer (YSI, USA). The agarose conversion to
neoagarobiose and neoagarobiose conversion to galactose, were
estimated from the amount of these two compounds produced by
the reaction using the following conversion factors.
The amount of agarose converted to neoagarobiose:
agaroseconverted(g) = neoagarobioseproduced(g)/f biose/agarose
(1)
where fbiose/agarose = 1.06 (=324/306).
The amount of neoagarobiose converted to galactose:
neoagarobioseconverted(g) = galactoseproduced(g)/f galactose/biose
(2)
where fgalactose/biose = 0.56 (= 180/324).
 Y.B. Seo et al. / Process Biochemistry 51 (2016) 759764
Fig. 1. Agarose degradation by AgaG1 or DagB for 9 h.
3. Results and discussion
3.1. Preparation of enzyme solutions
By cultivating E. coli BL21/pMoPac1-AgaG1, an intracellular
AgaG1 activity of 5.9 U/ml-broth was obtained. The cell lysate,
prepared as described earlier, was concentrated about 10 fold by
ultraltration and then a required amount was used for each exper-
iment. From S. lividans TK24/pUWL201PW-DagB, an extracellular
DagB activity of 0.75 U/ml was obtained. The cell-free broth was
ultra-ltered to concentrate the DagB, before being used for exper-
iments. By cultivating Alcanivorax sp. A28-3, an intracellular NABH
activity of 0.025 U/ml-broth was obtained. The cell lysate, prepared
as described earlier, was concentrated about 20 fold before being
used for experiments.
3.2. Optimization of AgaG1 and DagB dosage for neoagarobiose
production
As mentioned earlier, the nal products of AgaG1 from agarose
hydrolysis were neoagarotetraose and neoagarobiose, while that
from DagB was neoagarobiose only [10,11]. Theoretically, agarose
can be hydrolyzed to neoagarobiose only by DagB. However, the
main problem with DagB is that it (an exo-type enzyme) is not ef-
cient in degrading oligomers because it attacks only one end of the
chain. On the other hand, AgaG1 (an endo-type enzyme) was found
more efcient than DagB in attacking oligomers larger than neoa-
garotetraose at the same dosage, as shown in Fig. 1. For this reason,
the use of a mixture of these two enzymes was considered an ef-
cient way to produce neoagarobiose from agarose and at the same
time, to minimize the dosage of DagB, which is very expensive to
produce. In such systems, the main contribution of AgaG1would
be the conversion of agarose to neoagarotetraose, which would
be subsequently degraded to neoagarobiose by DagB. Fortunately,
such simultaneous usage of these enzymes was possible because
both of them were found to have the same optimum conditions
(pH 7.0 and 40 C) [10,11].
761
In determining the dosages of these two enzymes required for
complete hydrolysis of 10 g/l agarose to neoagarobiose in 12 h, it
was assumed for the convenience of analysis that agarose hydrol-
ysis occurred in the following sequence:
Assumption (1) Neoagarotetraose is generated from agarose by
AgaG1, and
Assumption (2) DagB degraded neoagarotetraose to produce
neoagarobiose.
First, the kinetics of neoagarotetraose hydrolysis by DagB was
identied. The rate of neoagarotetraose hydrolysis was measured
for different concentrations of DagB (Fig. 2a). The neoagarote-
traose degradation rate was observed to follow rst-order kinetics
(Fig. 2b) as represented by Eq. (3).
d[N4]/dt = f ([E])[N4] (3)
where [N4] is concentration of neoagarotetraose, and f([E]) the rst-
order reaction rate coefcient as a function of DagB, [E].
The equation was integrated with [N4] = [N4]0 at t = 0 to give
ln[N4] = ln[N4]0 f ([E])t (4)
By plotting the experimental data, based on Eq. (4) (Fig. 2b), f([E])
was found to linearly dependent on DagB dosage as presented in
Fig. 2c and Eq. (5).
f ([E]) = 0.021 + 2.02[E](hr1 ) (5)
In the previous work, 10 g/l of agarose was completely degraded
by AgaG1 to 3.8 g/l and 6.4 g/l of neoagarobiose and neoagarote-
traose, respectively [10]. This meant that 6.4 g/l of neoagarotetraose
needed to be hydrolyzed by DagB when dealing with 10 g/l agarose.
Using Eq. (4) and (5), the required DagB dosage for the complete
hydrolysis of 6.4 g/l neoagarotetraose in 12 h was calculated to be
0.133 U/ml.
Second, different dosages of AgaG1, along with 0.133 U/ml of
DagB, were applied to degrade 10 g/l of agarose to the nal product,
neoagarobiose. As shown in Fig. 3, the neoagarobiose production
rate increased monotonically as the AgaG1 dosage increased (in
the range tested: 0.3751.500 U/ml). However, the dosage effect
was negligible at > 1.250 U/ml. When the AgaG1 dosage was 1.25
762 Y.B. Seo et al. / Process Biochemistry 51 (2016) 759764
Table 1
Neoagarobiose and neoagarotetraose formation from agarose hydrolysis by DagB and AgaG1.
AgaG1 dosage (U/ml) DagB dosage (U/ml) Reaction time (h)
0.375 0.133 12
0.625 0.133 12
0.875 0.133 12
1.250 0.133 9 10.02 0.02
1.500 0.133 9 10.05 0.00
or 1.50 U/ml, the agarose hydrolysis was nearly complete in 9 h.
With 0.875 U/ml of AgaG1, the reaction ended at 12 h, whereas
with 0.375 U/ml and 0.625 U/ml of AgaG1, the reaction was not
completed in 12 h. This was clear from a noticeable amount of neoa-
garotetraose remaining in the reaction mixture (Table 1). Unless
otherwise mentioned, 0.875 U/ml of AgaG1 and 0.133 U/ml of DagB
were used in the subsequent experiments.
3.3. Two-stage process of enzymatic hydrolysis of agarose
In the rst stage, 0.875 U/ml AgaG1 and 0.133 U/ml DagB were
used for the hydrolysis of 10 g/l agarose, at pH 7.0 and 40 C for
12 h. In the second stage, NABH was used for the degradation of
neoagarobiose to galactose and anhydrogalactose, under the opti-
mum conditions of pH 7.0 and 35 C [12]. Fig. 4 shows the time
proles of galactose, neoagarobiose, and neoagarotetraose during
the rst stage, when 9.7 g/l of neoagarobiose was produced from
10 g/l agarose. This indicated that 92% of the agarose added was
converted to neoagarobiose. In the second stage, the galactose con-
centration increased monotonically with reaction time, and its nal
concentration at 12 h was 5.2 g/l. This corresponded to 88% of the
galactose yield from agarose.
In the two-stage enzymatic process, the most critical step was
the rst stage of agarose liquefaction (primarily done by AgaG1),
lowering the viscosity or gelation tendency of the agarose solu-
tion. It was reported that an increase in the solution viscosity
caused decreasing enzyme activity due to decreased motion of
the enzymes in solution [17]. To avoid gelation or high-viscosity
problems, Kim et al. proposed an agarose pretreatment method
with mild acid (e.g., acetic acid) to degrade agarose partially
before enzymatic hydrolysis [18]. However, it is known that acids
cleave agarose bonds randomly [19], generating products which
-agarases cannot recognize, and which could cause a signicant
decrease in yield. Moreover, the use of acid entails a neutralization
step to adjust the pH to the optimum level for the subsequent step
of enzymatic hydrolysis. Such neutralization generates a signicant
amount of salts, which could potentially inhibit microbial activity
in the subsequent fermentation step. In this study, it was found
that enzymatic hydrolysis of agarose could be performed with no
acid pretreatment. Instead, the gelation problem was avoided by
keeping the temperature at 45 C for 10 min at the very beginning
of the reaction in the rst stage considering the gelling tempera-
ture is known to be 4345 C. Then, the temperature was lowered
and maintained at 40 C for the rest of the reaction. It was expected
that agarose molecules were randomly cleaved mainly by the action
of AgaG1 with a rapid decrease in the viscosity and thus gelation
tendency during the 10-min period of operation at 45 C [10]. In
other words, the gelation problem could be avoided at the cost of
briey sacricing enzyme activity, which was expected to cause no
signicant loss in the overall efciency of the process. In fact, the
AgaG1 activity was only about 5% less than that at the optimum
temperature of 40 C. Moreover, operation at 45 C for a period as
short as 10 min was expected to result in no signicant perma-
nent deactivation of AgaG1. It was found in the previous study that
AgaG1 activity could recover to about 95% of its original level after
exposure to 45 C for 10 min [10].
Neoagarobiose produced (g/l) Neoagarotetraose produced (g/l)
9.56 0.25 0.58 0.05
9.84 0.02 0.30 0.02
9.97 0.03 0.12 0.00
0.12 0.02
0.16 0.02
In this study, after the NABH treatment a galactose yield of
88% was obtained, which was signicantly higher than that (79%)
reported by the group using acid pretreatment [18]. In addition,
they calculated the yield based on the data obtained by the DNS
method only, with no actual measurement of the galactose con-
centration. This carried a high probability of overestimation.
3.4. Ethanol production
For the preparation of agarose hydrolysate for ethanol produc-
tion, a higher concentration of agarose (20 g/l) was used to obtain
a more concentrated galactose solution than before with 10.0 g/l
galactose. Other than galactose, the other required nutrients (such
as yeast extract) were added to the enzyme hydrolysate of agarose
to make YPEH medium for ethanol production by S. cerevisiae
KL17. The galactose concentration in the YPEH medium (9.2 g/l)
was slightly lower than that of the original agarose hydrolysate
solution (10.0 g/l) due to the dilution caused by the nutrient sup-
plementation step. For the purpose of comparison, a synthetic
galactose medium containing 10.0 g/l galactose (YPG) and YPG con-
taining 5 g/l 5-HMF (YPGH) were also tested for ethanol production.
As shown in Fig. 5, the galactose consumption rate and ethanol
production rate in the YPEH medium showed patterns similar to
those of the YPG medium, but showed a pattern of high inhibition
in the YPGH medium. For the YPG medium, 9.75 g/l of galactose
was consumed in 12 h to produce 3.85 g/l of ethanol, providing
an ethanol yield of 0.39 (g/g) (77% of theoretical yield). When
the YPEH medium was used, 8.76 g/l of galactose was completely
metabolized to produce 3.71 g/l of ethanol with an ethanol yield
of 0.42 (g/g) (83% of theoretical yield). As an example, yeast cul-
ture was performed using the crude galactose solution produced
by the two-stage enzymatic process, to demonstrate its usability as
a carbon source for the fermentation processes. As shown in Fig. 5,
the YPEH medium prepared from the crude galactose solution was
found to offer a good environment for the growth of S. cerevisiae
KL17, and for ethanol production. In the YPEH medium, galactose
was metabolized completely, and the ethanol yield was 83% of the
theoretical yield, while in YPG, a synthetic medium made from pure
galactose, the yield was 77% of theoretical. The higher amount of
ethanol produced with YPEH medium than YPG medium was con-
sidered due to its complex nature. It was believed to contain, as
originated from a crude hydrolysate solution, various unidentied
materials that might have been utilized by the yeast as carbon
source in addition to galactose. The possibility that the existence
of anhydrogalactose in the YPEH medium could have positively
contributed to ethanol production was slim because S. cerevisiae
is known to be unable to metabolize anhydrogalactose [7].
Both galactose consumption and ethanol production signi-
cantly declined in YPGH medium due to the toxic effects of 5-HMF,
one of the major toxic byproducts from the chemical hydrolysis
of agarose (Fig. 5). For this reason, the chemical hydrolysate of
agarose needed to undergo a detoxication step before being used
for ethanol fermentation [6], while enzyme hydrolysate containing
no inhibitory byproducts, and could be used directly, as done in this
study.
 Y.B. Seo et al. / Process Biochemistry 51 (2016) 759764 763

Fig. 3. Effect of AgaG1 dosage on neoagarobiose formation from agarose at pH 7.0

and 40 C at a xed DagB dosage of 0.133 U/ml.

Fig. 4. Two-stage enzymatic hydrolysis of agarose: In the rst stage, 10 g/l of agarose
was hydrolyzed to neoagarobiose by using AgaG1 and DagB mixture at pH 7.0 and
40 C. In the second stage, neoagarobiose was hydrolyzed to galactose by using NABH
at pH 7.0 and 35 C.

Fig. 2. Effect of DagB dosage on neoagarotetraose degradation rate at pH 7.0 and

40 C. (a) neoagarotetraose concentration proles (b) semi-log plot of neoagarote-
traose concentration proles (c) dependency of reaction rate coefcient on DagB
dosage.

Galactose has several applications. The ongoing and most

important one is for the synthesis of d-tagatose, a low-calorie
sweetener [20]. Galactose is also used for the synthesis of isopropyl
-d-1-thiogalactopyranoside also [21]. As mentioned earlier, anhy-
drogalactose is formed together with galactose during enzymatic Fig. 5. Ethanol production by Saccharomyces cerevisiae KL17. YPG: medium contain-
hydrolysis. Unfortunately, there have been no reports of anhy- ing synthetic galactose (control), YPEH: medium based on enzymatic hydrolysate,
YPGH: medium containing galactose and 5-HMF.
drogalactose utilization by microorganisms, although indirect
764 Y.B. Seo et al. / Process Biochemistry 51 (2016) 759764
evidence was observed by our group that some marine microor-
ganisms metabolize it (data not given). No cases of anydrogalactose
application have been reported, either. It would be a signi-
cant contribution to enhanced efciency of substrate utilization
if genetically engineered microorganisms could be developed that
metabolize anhydrogalactose.
4. Conclusions
A two-stage enzymatic process was developed and optimized
for agarose hydrolysis using a mixture of AgaG1 and DagB in the
rst stage, and NABH in the second stage. In the rst stage, the
agarose gelation problem was avoided by an adjustment of reac-
tion temperature requiring no pretreatment with acid. Agarose
could be hydrolyzed to galactose with a yield of 88% of theoreti-
cal maximum. The crude galactose solution produced from agarose
hydrolysis was used directly for ethanol production by yeast, with-
out cell-growth inhibition, to demonstrate its potential as a carbon
source for fermentation.
Acknowledgements
This work was supported by the New & Renewable Energy
Technology Development Program of the Korea Institute of Energy
Technology Evaluation and Planning (KETEP) grant funded by
the Korean government Ministry of Knowledge Economy (No.
2009301009001B) and the Advanced Biomass R&D Center (ABC)
of the Global Frontier Project funded by the Ministry of Education,
Science and Technology (ABC-2014-047211).
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