Exploring the possibility of using a thermostable mutant of β-glucosidase for rapid hydrolysis of quercetin glucosides in hot water

Article abstract of DOI:10.1039/b920195p

The antioxidant quercetin was extracted from yellow onion waste and converted to its aglycone form by a combination of subcritical water extraction and enzymatic hydrolysis. The hydrolytic step was catalysed by a double residue (N221S, P342L) mutant of the thermostable β-glucosidase (TnBgl1A), isolated from the thermophile Thermotoga neapolitana and cloned and produced in E. coli. The activity of wt TnBgl1A was shown to be dependent on the position of the glucosylation on the quercetin backbone, favouring hydrolysis of quercetin-4′-glucoside over quercetin-3-glucoside. The mutated variant of the enzyme harboured a mutation in the +2 sub-site (N221S) and showed increased catalytic efficiency in quercetin-3-glucoside hydrolysis and also to a certain extent hydrolysis of quercetin-4′-glucoside. The mutated enzyme was used directly in yellow onion extracts, prepared by subcritical water extraction, resulting in complete hydrolysis of the glucosylated flavonoids quercetin-3,4′-diglucoside, quercetin-4′-glucoside, quercetin-3-glucoside, isorhamnetin-4′-glucoside and isorhamnetin-3, 4′-diglucoside. To complete hydrolysis within five minutes, 3 mg of TnBgl1A-N221S was used per gramme of onion (dry weight). A life cycle assessment was done to compare the environmental impact of the new method with a conventional solid-liquid extraction-and-hydrolysis method utilising aqueous methanol and hydrochloric acid. Comparison of the methods showed that the new method is preferable regarding primary energy consumption and global warming potential. Another advantage of this method is that handling of toxic chemicals (methanol and HCl) is avoided. This shows that combined subcritical water extraction/enzyme hydrolysis is both a fast and sustainable method to obtain quercetin from onion waste.

Full text of DOI:10.1039/b920195p

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/greenchem | Green Chemistry
Exploring the possibility of using a thermostable mutant of b-glucosidase
for rapid hydrolysis of quercetin glucosides in hot water
a
c
b
b
c
a
Sofia Lindahl, Anna Ekman, Samiullah Khan, Christina Wennerberg, P a˚ l B o¨ rjesson, Per J. R. Sj o¨ berg,
b
a
Eva Nordberg Karlsson and Charlotta Turner*
Received 11th June 2009, Accepted 28th September 2009
First published as an Advance Article on the web 4th November 2009
DOI: 10.1039/b920195p
The antioxidant quercetin was extracted from yellow onion waste and converted to its aglycone
form by a combination of subcritical water extraction and enzymatic hydrolysis. The hydrolytic
step was catalysed by a double residue (N221S, P342L) mutant of the thermostable b-glucosidase
(
TnBgl1A), isolated from the thermophile Thermotoga neapolitana and cloned and produced in
E. coli. The activity of wt TnBgl1A was shown to be dependent on the position of the
glucosylation on the quercetin backbone, favouring hydrolysis of quercetin-4¢-glucoside over
quercetin-3-glucoside. The mutated variant of the enzyme harboured a mutation in the +2 sub-site
(
N221S) and showed increased catalytic efficiency in quercetin-3-glucoside hydrolysis and also to a
certain extent hydrolysis of quercetin-4¢-glucoside. The mutated enzyme was used directly in
yellow onion extracts, prepared by subcritical water extraction, resulting in complete hydrolysis of
the glucosylated flavonoids quercetin-3,4¢-diglucoside, quercetin-4¢-glucoside,
quercetin-3-glucoside, isorhamnetin-4¢-glucoside and isorhamnetin-3,4¢-diglucoside. To complete
hydrolysis within five minutes, 3 mg of TnBgl1A_N221S was used per gramme of onion (dry
weight). A life cycle assessment was done to compare the environmental impact of the new
method with a conventional solid–liquid extraction-and-hydrolysis method utilising aqueous
methanol and hydrochloric acid. Comparison of the methods showed that the new method is
preferable regarding primary energy consumption and global warming potential. Another
advantage of this method is that handling of toxic chemicals (methanol and HCl) is avoided. This
shows that combined subcritical water extraction/enzyme hydrolysis is both a fast and sustainable
method to obtain quercetin from onion waste.
Introduction
other flavonoids from waste and by-products since they possess
antioxidative properties – studies have shown that they have pos-
The agricultural, forestry, and food industries produce tonnes
of waste materials and by-products every year, such as onion
skin, apple peel, carrot waste and birch bark. In the case of
onion, the amount of onion waste annually produced within the
4
itive effects against, for example, cancer and neurodegenerative
5
diseases. These compounds are mainly present in glycosylated
forms in fruits and vegetables. In onion, they mainly occur as
quercetin-4¢-glucoside (Q-4¢) and quercetin-3,4¢-diglucoside (Q-
1
European Union is almost half a million tons. The produced
3,4¢) (Fig. 1), but other types of quercetin glucosides as well as
waste and by-products are today used in animal feed, or for
composting, incineration or anaerobic digestion. However, these
waste materials and by-products contain high-value compounds
such as antioxidants that can be extracted before the waste
reaches its final destination. Waste and by-products from fruits
and vegetables are rich in antioxidants such as polyphenols,
which have uses as additives in pharmaceuticals, food products
and cosmetics. A large group of polyphenolic compounds
are flavonoids. Quercetin (Q) is an example of a high-value
the aglycone form (Fig. 1) are also present, but at much lower
concentrations.
6
2,3
flavonoid, and can for example be extracted from onion.
Hence, it is of interest to be able to extract quercetin and
Fig. 1 Chemical structures of quercetin (R, R¢ = H), quercetin-3-
glucoside (R = glucose and R¢ = H), quercetin-4¢-glucoside (R = H
and R¢ = glucose) and quercetin-3,4¢-diglucoside (R, R¢ = glucose).
a
Uppsala University, Department of Physical and Analytical Chemistry,
Analytical Chemistry, P.O. Box 599, SE-751 24, Uppsala, Sweden.
E-mail: Charlotta.Turner@kemi.uu.se
There are a number of different methods published on how to
extract quercetin and quercetin glycosides from onion, of which
the most commonly used technique is solid–liquid extraction
b
Lund University, Department of Biotechnology, P.O. Box 124, SE-221
0
0, Lund, Sweden
c
Lund University, Department of Technology and Society, P.O. Box 118,
SE-221 00, Lund, Sweden
6,7
with aqueous methanol as extraction solvent. The extraction
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 159–168 | 159

View Article Online
of quercetin and quercetin glycosides is often combined with
acid hydrolysis of the extracted glycosides. To catalyse the
hydrolysis reaction, hydrochloric acid at high concentration is
and characterised to different environmental impact categories,
of which Global Warming Potential (GWP) is one example. LCA
can be used for several different purposes such as product and
process development and for comparison of the environmental
impact of different products and manufacturing processes. LCA
7-10
commonly used. Deglycosylation of the quercetin glycoside
mixture obtained after extraction is advantageous for two
reasons. Firstly, the glycosides are present in an extract at various
concentrations, and by hydrolysing them to the quercetin agly-
cone, the quantitative determination is simplified. Secondly, the
antioxidising power is in general higher for the deglycosylated
17
is standardised according to ISO 14044.
A long-term aim of our research is to develop a sustainable on-
line extraction-biocatalysis system with high efficiency that can
be used directly at the site of an industry that produces the waste,
or alternatively at a plant taking care of waste products. As a
first step, the aim of this study was to optimise the hydrolysis
reaction with quercetin glucosides at an analytical level, and
to improve the conversion of Q-3 to quercetin. A mutated b-
glucosidase (TnBgl1A_N221S) was utilised in the hydrolysis
and compared to the wild type (wt) enzyme, TnBgl1A. The
optimised subcritical water extraction and enzymatic hydrolysis
method was subsequently applied to a yellow onion extract,
and the environmental performance of the developed processes,
including greenhouse gas performance and energy efficiency, was
assessed from a life cycle perspective.
11
form, which we have also measured for quercetin species using
the DPPH (2,2-diphenyl-1-picrylhydrazyl) method (data not
shown). Hence, by hydrolysing the quercetin glucoside mixture,
the potential value of the material is increased. A problem with
acid-catalysed hydrolysis is that the conditions are harsh, result-
ing in degradation of compounds such as phenolic acids in the
8
extract, and even degradation of the target molecule quercetin.
Another drawback is that the acidity of the sample can cause
problems in the analysis step. This is commonly performed
by high performance liquid chromatography (HPLC), and the
HPLC-column may be damaged by the extremely low pH of the
sample. To overcome these problems, enzymatic hydrolysis is an
3,10,12
alternative.
b-Glucosidases catalyse hydrolysis of glucose
Results and discussion
linked to either a carbohydrate or non-carbohydrate moiety,
and these enzymes have previously been shown to hydrolyse
This paper focuses on use of the thermostable b-glucosidase, wt
TnBgl1A, and a mutated variant of the same enzyme (termed
TnBgl1A_N221S), as catalysts for deglucosylation of quercetin
glucosides in a high-temperature step, coupled off-line to an
environmentally sustainable, pressurised hot water method to
extract the quercetin species. Previous work showed that the wild
type, TnBgl1A, and a differently folded glucosidase (TnBgl3B),
both efficiently hydrolysed the major quercetin glucoside in
onion extracts, glucosylated at the 4¢-position of the quercetin
3,10,13
quercetin glucosides.
3
Turner et al. have described a subcritical water extraction
method, followed by hydrolysis by thermostable b-glucosidase
from Thermotoga neapolitana that yielded quercetin in the
aglycone form from a yellow onion extract. The pressurised
◦
liquid extraction with water as a solvent was run at 120
C
and 50 bar, and the following enzymatic hydrolysis in water
◦
at 90 C and pH 5.0. This method gave the same yield as
a conventional solid–liquid extraction method, although one
limitation in the hydrolysis step was that the enzyme had a low
conversion rate of quercetin-3-glucoside (Q-3) to the aglycone
form (Q), which was speculated to be due to steric hindrance
3
molecule (Q-4¢). However, the Q-3 in the extract was not
3
efficiently deglucosylated under the conditions used. To further
investigate the possibility of the enzyme to hydrolyse quercetin
species glucosylated at the 3-position (Fig. 1), this work involves
deglucosylation studies of standards of quercetin glucosides
(
Fig. 1). It is well known that water at elevated temperature and
14,15
pressure is an efficient, less polar solvent, similar to methanol.
(
Q-4¢, Q-3,4¢, and Q-3, see Fig. 1) over time with a special
Use of a thermostable enzyme may allow a combination of the
extraction with the hydrolysis step, without extensive cooling
in-between. Moreover, the hydrolysis reaction will go faster due
to increased diffusion rates. Other advantages using subcritical
water extraction combined with enzymatic hydrolysis compared
to conventional methanol extraction and acid hydrolysis include:
emphasis on Q-3, using both the wt and a variant of TnBgl1A
mutated at two positions (residues 221 (N→S) and 342 (P→L).
Only one (residue 221) of the two mutated residues was judged
to be important for specificity, as it is located at the substrate
binding site (+2 sub-site), while the other (residue 342) is
found on the surface on the reverse side (opposite the active
(
i) lower environmental load, as water is a more sustainable
18
site) of the enzyme. This variant was chosen from a set
solvent than methanol, and hence no production or destruction
facilities are needed; (ii) use of the onion waste as animal feed or
as feedstock in biogas production after extraction and hydrolysis
is still possible, since only a small fraction of the biomass is
removed, and (iii) a significantly shorter total processing time
per sample.
of mutants, as it both showed the highest turnover of the
substrate para-nitrophenyl-b-D-glucopyranoside (pNPG), and
based on a simple screening experiment, also higher conversion
of Q-3 to quercetin. Finally, the mutated variant is utilized
coupled with subcritical water extraction of onion waste, and
the environmental performance of the method is evaluated.
18
However, it is important to not just analyse qualitative pa-
rameters, like the exchange of a solvent with another, but to also
quantify the environmental load of the method. For this purpose
a standardised life cycle assessment (LCA) is an excellent tool
allowing quantification of primary energy consumption and
emissions. In LCA, fluxes of materials and energy are followed
from the extraction of raw materials, production and handling of
the product when it has become waste. The flows are quantified
HPLC-UV analysis
In order to follow the hydrolysis process, high selectivity in
the detection method is needed. Direct spectrophotometric
detection could be employed if (for example) in the hydrolysis
of pNPG to p-nitrophenol (pNP), the substrate and products
have clearly different absorption maxima. For quercetin and
16
1
60 | Green Chem., 2010, 12, 159–168
This journal is © The Royal Society of Chemistry 2010

View Article Online
quercetin glucosides there is no such difference in absorption
maxima, and a separation step prior to detection with UV is
necessary (although this is time-consuming).
The number of samples to be analysed with HPLC-UV
is high, and to minimise the analysis time of each sample
an isocratic elution profile was selected, with methanol–water
60 min. Enzymatic hydrolysis of the double glucosylated form
(Q-3,4¢), also showed that the 4¢-glucosylation was completely
hydrolysed within the 5 min interval, after which the remaining
Q-3 was slowly deglucosylated to yield approximately 70% in
the aglycone form (Fig. 2c). This shows that hydrolysis of the
4¢-glucosylation is clearly favoured by wt TnBgl1A. Moreover,
presence of Q-3 is not disturbing the deglucosylation at the 4¢-
position, and finally hydrolysis of Q-3 is possible but slow, which
may be due to low affinity or steric hindrance for efficient binding
of Q-3 in the active site. The homologous human b-glucosidase
(
50 : 50) containing 0.13 M (0.5 vol%) formic acid as mobile
phase. The isocratic elution profile used resulted in base-line
separation of the analytes: Q-3,4¢; Q-3; Q-4¢; and quercetin (see
Fig. 5b).
(
hCBG), which has high specificity for Q-4¢ and quercetin-7-
19
glucoside, does not hydrolyse 3-linked flavonoid glucosides,
Hydrolysis of quercetin glucosides by wt TnBgl1A
hence hydrolysis of this substrate by TnBgl1A suggests an
In Fig. 2a,b, the relatively fast conversion of Q-4¢ by wt TnBgl1A
is clearly shown. Using a 1 : 100 enzyme:substrate molar ratio,
Q-4¢ was completely converted to Q within 5 min, while the
same conditions for Q-3 resulted in only 70% conversion within
interesting difference in the catalytic site.
Thermal stability and solubility of quercetin species
Self-hydrolysis of quercetin glucosides at high temperature was
studied, and could be excluded since incubations of the quercetin
◦
glucosides at 95 C for 1 h (the highest expected temperature
for the enzyme catalysis), showed no thermal degradation of
the glucosides (data not shown), and the biocatalyst was hence
necessary.
A drawback with production of the more potent antioxidant
Q (the deglucosylated form), is a decreased water solubility,
and initial hydrolysis data showed that this decrease led to
losses of the produced quercetin (data not shown). To avoid
this, solubility tests were made, and the results revealed that
-
1
concentrations of maximum 25 nmol ml of quercetin were
◦
soluble in the buffer at 95 C. The highest concentration of
quercetin species used in the remaining work was thus limited to
-
1
1
7 nmol ml , to ensure full solubility of the produced quercetin.
Enzymatic hydrolysis of the different quercetin glucosides
To obtain quantitative data, a number of enzyme:substrate ratios
for the wt TnBgl1A and mutated variant, TnBgl1A_N221S, were
tested, using the substrates, Q-3 and Q-4¢, to find optimal ratios
for determination of kinetic parameters. The detection method
used requires sampling followed by quantification; hence the
challenge was to find ratios where the initial reaction is neither
too fast nor too slow. Indeed, the obtained results showed that
different enzyme:substrate ratios were necessary for different en-
zyme/substrate combinations: for Q-3, 1 : 250-1250 and 1 : 2500-
12500 for wt TnBgl1A and TnBgl1A_N221S, respectively, and
for Q-4¢, 1 : 2500-12500 and 1 : 5000-250000 for wt TnBgl1A
and TnBgl1A_N221S, respectively. The higher concentration
required of the wt was in accordance with the higher turnover
-
1
and specific activity reported for TnBgl1A_N221S (1700 s at
◦
-1
◦
8
0 C) as compared to the wt (485 s at 80 C) for hydrolysis of
18
pNPG.
In Table 1 all kinetic data are summarised. As shown in Fig. 1,
the glucose on Q-4¢ is situated at a position resulting in an
extended molecule, while for Q-3 the glucose is in the middle
of the flavonoid backbone resulting in a different overall shape,
which may be sterically more difficult to accommodate in the
active site. This is reflected in the higher K
m
of Q-3 than for
Fig. 2 Catalysis by TnBgl1A with (a) Q-3, (b) Q-4¢ and (c) Q-3,4¢.
Q-4¢. Furthermore, the K (Michaelis–Menten constant) value
m
2
0 pmol of TnBgl1A and 200 nmol of substrate were used. Relative
standard deviation (RSD) of the concentration values at the different
sampling time points ranges between 0.3 and 20.6% (n = 3).
of TnBgl1A_N221S is lower for both Q-3 and Q-4¢ compared to
the wt (Table 1), which indicates that the mutation has increased
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 159–168 | 161

View Article Online
Table 1 Kinetic studies of enzymes TnBgl1A and TnBglA_N221S, and the substrates Q-3 and Q-4¢ (n = 3). K
m
– Michaelis–Menten constant, Vmax
–
maximal conversion rate, kcat – turnover number and kcat/K
m
– catalytic efficiency.
max/mmol min mg-1
-
1
k
cat/s-1
(kcat/K
m
)/s-1 M-1
Enzyme
Substrate
K
m
/mM
V
TnBgl1A
Q-3
Q-4¢
Q-3
Q-4¢
0.129 ± 0.059
0.059 ± 0.026
0.046 ± 0.020
0.022 ± 0.015
8.3 ± 2.1
8.3 ±1.4
8.1 ±1.3
7.1 ± 1.1
7.2
7.2
7.1
6.2
55
122
154
281
TnBgl1A_N221S
the affinity for both quercetin substrates. The effect is, however,
especially pronounced for Q-3. The smaller size of the serine
residue at position 221 may have reduced the steric hindrance
of this molecule, leading to a better fit in the binding site
resulting in higher catalytic efficiency. The maximal conversion
rate (Vmax) and the turnover number (kcat) were in the same range
for all enzyme and quercetin-substrate combinations, but as a
Table 2 Irreversible thermal deactivation of TnBgl1A and
TnBgl1A_N221S. k is the deactivation constant and t1/2 is the
half-life of the enzymes
TnBgl1A
k/h-1
TnBgl1A_N221S
◦
k (h-1)
T/ C
t
1/2/h
t1/2/h
8
90
9
5
0.018
0.027
0.73
20
11
0.46
0.029
0.040
1.51
7.0
6.4
0.23
consequence of the change in K
m
the catalytic efficiency (kcat/K
m
)
5
was higher for the mutant (see Table 1).
Despite the improved affinity for Q-3 using TnBgl1A_N221S,
◦
experiments with this variant at 95 C showed a hydrolysis
a slightly higher deactivation rate, but both the wt and mutant
◦
pattern with decreased deglucosylation rate at increasing in-
cubation times (Fig. 3, 0–30 min). A second addition of the
enzyme after 30 min restarted the reaction (Fig. 3, 30–65 min).
To understand if this was a result of deactivation or product
inhibition, thermal deactivation experiments of the enzyme in
the absence of substrate, as well as incubations of the enzyme in
the presence of products, were made (see below).
were judged to be stable at temperatures up to 90 C (t of the
1
/2
mutant was approximately 6 h).
◦
A larger difference was seen at 95 C, where the deactivation
constant (k) for the mutant was doubled, and its half-life
reduced to 13 min (0.23 h, Table 2). Thermal transitions
by differential scanning calorimetry have shown the apparent
m
transition temperature (T ) from the folded to unfolded state at
◦
◦
18
1
02.5 C (wt) and 100.5 C (mutant), respectively. However,
inspection of the chromatograms showed that the starting point
◦
of the transition occurred around 95 C (data not shown),
explaining the reduced stability at this temperature. This sets a
limitation in the time frame applicable for the hydrolysis reaction
◦
if a temperature of 95 C is used, and should only be applied
if the overall reaction time is short (≤10 min). This relatively
high temperature was used in the kinetic study and the total
◦
reaction time was less than 5 min. A temperature of 95 C was
also used in the hydrolysis of the onion extract (with a total
reaction time of 5 min) to minimise the temperature difference
between extraction and hydrolysis temperatures in the process.
However, in longer studies of the deglucosylation, deactiva-
Fig. 3 A second addition of 20 pmol TnBgl1A_N221S 35 min after the
first addition of 20 pmol TnBgl1A_N221S. RSD of the concentration
values at the different sampling time points ranges between 0.4 and
◦
tion will be significant at temperatures above 90 C. This means
that the slow deglucosylation rate of Q-3 seen within the 30 min
interval for the wt cannot be a consequence of deactivation, as
1
6.9% (n = 3).
◦
this was run at 90 C (Fig. 2), while in the reaction performed
◦
with the mutated variant at 95 C, deactivation should be
Thermal stability of the b-glucosidases TnBgl1A and
TnBgl1A_N221S
significant (Fig. 3).
Activation/inhibition of the enzyme
The occurrence of irreversible thermal deactivation at 85, 90 and
◦
95 C was investigated to establish the tolerance of the enzymes
An experiment was set up to evaluate if the mutated enzyme was
product-inhibited. In this experiment, glucose and quercetin,
respectively, were added to TnBgl1A_N221S 18 min prior to
adding a hydrolysable substrate (in this case Q-4¢). Product
inhibition acts on the enzyme, and the presence of substrate can
hence be used to detect if inhibition has occurred after the pre-
incubation. To avoid thermal deactivation, the temperature was
to prolonged exposure to these temperatures (Table 2). Almost
no enzymatic activity was lost for wt TnBgl1A during the first
◦
3
0 min of incubation at 85 and 90 C. Prolonged incubations up
to 24 h fitted a first-order deactivation rate during the complete
incubation period, resulting in a deactivation constant (k) of
1
◦
0
.027 h- (at 90 C). The half-life of the enzyme (t1/2) could thus
◦
◦
◦
be calculated to exceed 10 h at 90 C. A further 5 C increase
set to 90 C. The results in Fig. 4 show that most of the Q-4¢ was
in temperature led to significant activity loss, giving a t1/2 of
hydrolysed after 10 min with either quercetin or the control (just
2
8 min (0.46 h, Table 2). The mutant TnBgl1A_N221S showed
the buffer). Interestingly, incubations in the presence of glucose
1
62 | Green Chem., 2010, 12, 159–168
This journal is © The Royal Society of Chemistry 2010

View Article Online
Fig. 4 The effect of incubation of TnBgl1A_N221S with glucose,
quercetin and buffer (control) on the hydrolysis of Q-4¢. RSD of
the concentration values at the different sampling time points ranges
between 0.3 and 6.1% (n = 3).
led to complete Q-4¢ hydrolysis after just 5 min. The conclusion
is that there is no inhibition effect by either quercetin or glucose
on the enzyme, but rather that glucose is an activator of the
enzyme. An activating effect of glucose on the b-glucosidases
from the glycoside hydrolase family 1 (GH1) is in accordance
2
0-22
with results reported by other researchers,
been reported for wt TnBgl1A.
and has previously
18,21
This work shows that the
activating effect of glucose also works for the mutated variant
of the enzyme.
The activating effect of glucose may also explain the improved
conversion of Q-3 to Q seen after the second addition of
TnBgl1A_N221S (Fig. 3), where glucose released after the first
enzyme addition is present in the reaction mixture. Currently,
one can only speculate that glucose may prevent non-productive
binding of the Q-3 or Q-4¢ substrate, for example by blocking a
high affinity sub-site. To obtain proof of this suggestion, detailed
studies on the binding affinity of the subsites of TnBgl1A will
be necessary.
Application to an onion sample
Whole yellow onion was chopped and both the skin and
the pulp was mixed and extracted using subcritical water at
◦
1
20 C and 50 bar as solvent. The result of the HPLC-UV
analysis of the extract was that seven flavonoids were identified
in the yellow onion extract – Q-4¢, Q-3, Q-3,4¢, quercetin,
isorhamnetin-4¢-glucoside (I-4¢), isorhamnetin-3,4¢-diglucoside
(
I-3,4¢) and isorhamnetin. Quercetin and the three quercetin
glucosides were identified by comparing the retention time
and elution order with standards, and isorhamnetin, I-4¢ and
3
I-3,4¢ were identified based on the results by Turner et al. One
of the peaks labelled “unknown” is most likely kaempferol,
3
and this assumption is based on a previous study. Q-4¢ and
Q-3,4¢ are the most abundant flavonoids identified in the yellow
onion extract and this corresponds well to other publications of
3
,6
flavonoid content in yellow onion. Three different amounts of
Fig. 5 HPLC-UV analysis of a yellow onion extract before and
after enzyme treatment. Concentration change of Q-4¢, Q-3, Q-3,4¢
and Q during enzymatic reaction (a), RSD of concentration at the
different sampling time points ranges between 0.8 and 10.6%, within
the calibration curve range (n = 3). HPLC-UV chromatogram of yellow
onion extract before addition of enzyme (b) and 5 min after addition
of TnBgl1A_N221S (c). Peak labels; 1 (mix of Q-3,4¢ and I-3,4¢), 2
TnBgl1A_N221S were tested (20 pmol, 100 pmol and 3 nmol)
data not shown) resulting in the following method optimised for
.2 ml of onion extract corresponding to 0.4 g of onion (50 mg
dw); 3 nmol of TnBgl1A_N221S, reaction temperature of 95 C,
pH 5.0 and 5 min reaction time. Chromatograms of yellow
onion extracts before and after enzymatic treatment at 95 C can
be seen in Fig. 5b and 5c. In Fig. 5a the amounts of quercetin and
(
1
◦
◦
(
Q-3), 3 (Q-4¢), 4 (I-4¢), 5 (morin (IS)), 6 (Q), 7-8 (unknowns) and 9
(
isorhamnetin).
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 159–168 | 163

View Article Online
Table 3 Primary energy consumption and emission of greenhouse gases relating to the manufacture of the compounds used in the extraction
methods per 10 g of quercetin. Ranges of values are in brackets
Primary energy (MJ kg-
1
GWP (kg CO
2
-eq kg-1
Compounds
solvent/catalyst)
solvent/catalyst)
Primary energy/MJ
2
GWP (kg CO -eq)
a
Methanol
1.5
27
110
17
1.6
9
46 (24–83)
86 (46–160)
0.33 (0.22–0.66)
550 (270–960)
5.1 (2.7–9.4)
0.027 (0.018–0.054)
b
HCl
c
Enzyme
a
23
b
24
c
25
Furnander. Boustead. Nielsen et al.
Table 4 Primary energy consumption and emissions of greenhouse gases relating to the heating needed in the extraction methods per 10 g of
quercetin. Ranges of values are in brackets
Extraction method
Heating/MJ
Extraction/kJ
Total energy/MJ
2
GWP (kg CO -eq)
Traditional method
New method
13 (9.3–25)
8.4 (4.2–15)
70 (60–80)
11 (8–13)
13 (9.4–25)
8.4 (4.2–15)
1.3 (0.94–2.5)
0.85 (0.42–1.5)
quercetin glucosides have been plotted as a function of
sampling time points. Almost all the Q-4¢ and Q-3,4¢ have
been hydrolysed after 1 min, while Q-3 concentration has
increased, and after 5 min only trace amounts of glucosides are
detected (Fig. 5c). Within the few minutes it takes to conduct
the reaction of the yellow onion extract, TnBgl1A_N221S
◦
maintains its activity, even though the reaction is done at 95 C.
The quercetin aglycone concentration increased at the same
rate as the concentration of glucosides decreased. The increase
in Q-3 concentration (after 1 min) shows that the presence of
the glucosylation at this position does not limit the activity of
the enzyme, but that accommodation of the substrate favours
hydrolysis of glucose bound at the 4¢-position. By comparing
the peaks in Fig. 5b and 5c it is seen that after 5 min of reaction
no I-4¢ or I-3,4¢ peaks are detected, but instead an isorhamnetin
peak can be found. This indicates that TnBgl1A_N221S
also catalyses the hydrolysis of isorhamnetin glucosides to
isorhamnetin. However, to confirm if it the enzyme catalyses the
reaction, a study using isorhamnetin species standards should
be done.
Environmental performance
The results from the environmental impact assessment are shown
in Table 3 and 4. The traditional method with organic solvents
leads to a significantly higher contribution to the GWP than the
extraction with subcritical water. This is mainly due to the use of
fossil methanol as a solvent, but also due to the larger amount
of solvents needed per gramme of onion for the traditional
method. Although clear differences in energy consumption can
be seen for the two methods, energy for heating gives relatively
small contributions to the overall environmental impact. The
major input of primary energy is in the manufacture of HCl and
methanol.
2
Fig. 6 Total contribution to the GWP expressed as kg CO -equivalents
per 10 g of quercetin, for the two extraction methods (a), and as total
primary energy consumption, expressed as MJ per 10 g of quercetin, for
the two extraction methods (b).
that subcritical water extraction is more favourable. These “hot-
spots” are the effect of the use of fossil-fuel-derived methanol
on GWP, and in addition the effect of the use of HCl on the
primary energy consumption.
The environmental impact is summarised in Fig. 6. There are
several uncertainities involved in the environmental assessment
performed, such as the linear scaling-up of the laboratory
methods. Furthermore, depending on the primary fuel used
for producing heat (in this case natural gas), the contribution
to the GWP will differ. However, despite these uncertainties,
there are some “hot-spots” identified, supporting the conclusion
Experimental
Materials
Methanol and formic acid were purchased from Merck
(Darmstadt, Germany). Quercetin dihydrate, quercetin
1
64 | Green Chem., 2010, 12, 159–168
This journal is © The Royal Society of Chemistry 2010

View Article Online
3-b-D-glucoside (Q-3), citric acid monohydrate, disodium
to the amount of enzyme that will release 1 mmol p-nitrophenol
hydrogen phosphate and pNPG were purchased from Sigma-
Aldrich (Steinheim, Germany). Quercetin 4¢-O-b-glucopyrano-
side (Q-4¢) and quercetin 3,4¢-di-O-b-glucopyranoside (Q-3,4¢)
were purchased from Polyphenols Laboratories AB (Sandnes,
Norway). Ultrapure water (MilliQ) was used at all times.
per minute under the described conditions.
Enzymatic hydrolysis of quercetin standards
In an initial activity screening, 200 nmol sample of Q-3, Q-4¢
and Q-3,4¢ dissolved in methanol (in triplicate) were evaporated,
1.0 ml of 100 mM citrate–phosphate buffer, pH 5.0, was added
Enzyme production and purification
◦
and the vials were heated at 90 C until substrate dissolved. A
The gene encoding wt TnBgl1A was cloned in pET22b(+), under
50 ml fraction was collected and added to 450 ml of mobile phase
3
control of the T7lac-promoter, as described by Turner et al.
composed of methanol–water (50 : 50) and 0.13 M (0.5 vol%)
The TnBgl1A_N221S was obtained by site-directed mutagenesis
formic acid (0 min fraction in Fig. 2). The reaction took place
18
using megaprimer PCR. The fragment encoding the mutated
TnBgl1A was cloned in pET22b(+) (Novagen, Madison, WI,
USA) under control of the T7lac-promoter, and transformed to
E. coli Bl21(DE3) for production.
◦
at 90 C and the reaction was started by adding 20 pmol
of wt TnBgl1A. 50 ml fractions were collected and added to
450 ml of mobile phase 1–60 min after addition of enzyme.
Samples were analysed by HPLC-UV. Collection of fractions
and enzyme addition was done with a 50 ml GC-syringe. The
fraction-collection procedure and enzyme addition were similar
for all experiments described below, unless otherwise noted.
Both enzymes were produced in 2.5 l batch cultivations,
in strain Bl21(DE3) after induction with isopropyl-1-thio-b-D-
galactopyranoside (IPTG) (2 h), as described by Abou-Hachem
26
et al. The cells were harvested by centrifugation (10000g,
◦
1
0 min, 4 C). Cell pellets were resuspended in buffer (20 mM
citrate–phosphate pH 5.6 for a single step heat treatment
Thermal stability of the quercetin standards
◦
(
80 C, 30 min, used only for the irreversible deactivation
studies)/or binding buffer (20 mM Tris-HCl, 20 mM imidazole,
Samples (150 nmol) of Q-3, Q-4¢ and Q-3,4¢ dissolved in
methanol were evaporated, 1.5 ml of 100 mM citrate–phosphate
buffer was added (triplicate samples for each glucoside) and
0
.75 M NaCl, pH 7.5) for a two-step procedure involving
◦
heat treatment (70 C, 30 min) and immobilized metal ion
affinity chromatography). The cell pellets were disintegrated by
sonication (60 W cm , 0.5 cycle, for 5 ¥ 3 min) with a UP400S
sonicator (Dr. Hielscher, Stahnsdorf, Germany) while cooling
in an ice–water mixture. The sonicated extracts were centrifuged
◦
the vials were heated at 95 C until substrate was dissolved.
-
2
Fractions were collected 10, 30 and 60 min after the first fraction
collection. Fractions were collected and diluted as above. The
samples were analysed by HPLC-UV.
◦
at 25000g (20 min, 4 C), and the resulting supernatants stored
◦
at 4 C until purified.
After the heat treatment, the samples were again centrifuged
30 min at 25000g and 4 C) for removal of denatured aggregated
Enzyme kinetics of the hydrolysis of Q-3 and Q-4¢
◦
(
To determine the kinetics of the hydrolysis reactions, 50–
proteins. The supernatants obtained after heat treatment at
2
50 nmol samples of Q-3 and Q-4¢ dissolved in methanol were
◦
8
0 C were used directly to study irreversible deactivation, while
evaporated and re-dissolved as described above, and the volume
of buffer used was 1.5 ml. Fractions were collected at 0, 1, 3, and
◦
those treated at 70 C were filtered (0.4 mm pore size) and further
purified by immobilised metal-ion affinity chromatography
5
2
min after enzyme addition. The enzyme amounts used were
00 pmol of wt TnBgl1A and 20 pmol of TnBgl1A_N221S to
27
(
IMAC).
Q-3 and 20 pmol of wt TnBgl1A and 10 pmol of
TnBgl1A_N221S to Q-4¢. Samples were analysed by HPLC-UV.
The enzyme activity using Q-3 and Q-4¢ is expressed in Units
Enzyme assays
The total protein concentrations were estimated using the bicin-
choninic acid method (Sigma-Aldrich, Steinheim, Germany)
or the Bradford protein assay (BioRad, Hercules, CA, USA)
according to the manufacturers’ instructions. Bovine serum
albumin (BSA) was used as standard (in concentrations from
(
U). One U corresponds to the amount of enzyme required
to release 1 mmol quercetin per minute under the described
conditions. Obtained data was analysed using Enzpack (Biosoft,
UK), using a time-range allowing fit to the Michaelis–Menten
curve. Kinetic constants were calculated using the Wilkinson
non-linear regression method, supplied in the program.
-
1
0
.2 to 1.0 mg ml ). The absorbance was measured at 562 nm
(
bicinchoninic acid method) and 595 nm (Bradford method)
using an Ultrospec 1000 (Pharmacia, Uppsala, Sweden). To
estimate the purity of the enzyme, SDS-PAGE according to
Laemmli was used.
Repeated enzyme additions at the applied conditions
28
For thermal inactivation tests, the b-glucosidase activity was
A sample of Q-3 (triplicate of 150 nmol) in methanol was
evaporated and re-dissolved as above at 95 C. Fractions were
◦
measured as the release of pNPG after 5 min incubations at
◦
8
5 C. To 960 ml preheated substrate (2.92 mM pNPG dissolved
collected at 0, 5, 10, 20 and 30 min after addition of 20 pmol
TnBgl1A_N221S. 35 min after the first enzyme addition, the
same amount of TnBgl1A_N221S was added and fractions were
collected 40, 45, 55 and 65 min after the first enzyme addition.
Samples were analysed by HPLC-UV.
in citrate–phosphate buffer pH 5.6), 40 ml of enzyme solution
was added, and the assay was performed as described in Turner
29
et al. Absorbance was measured at 405 nm using Ultrospec
000 (Pharmacia). One Unit (U) for this substrate corresponds
1
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 159–168 | 165

View Article Online
◦
Thermal stability of the enzymes in absence of substrate
for 5 min. Extraction temperature was 120 C and extraction
time 15 min (three extraction cycles of 5 min each). Purging
between extractions was performed with nitrogen. The extract
was collected in 100 ml clear glass vials, cooled down to room
temperature and diluted with degassed water to a total volume
of 50 ml.
Thermal stability of the enzymes was measured as irreversible
-
1
deactivation. The heat-treated wt (TnBgl1A, 1.5 mg ml , 29 mM)
-
1
and mutant (TnBgl1A_N221S, 2.0 mg ml , 38 mM) glucosidase
were aliquoted into microcentrifuge tubes. These samples were
◦
incubated at 85, 90 and 95 C, at time intervals ranging from 0 to
2
4 h. During the first 30 min, duplicate samples were withdrawn
Enzymatic hydrolysis of onion extract
every 5 min, then samples were taken after 1, 2, 3, 4, 5, 18 and
2
0
4 h, and were chilled on ice (at least 30 min), centrifuged (13
00g, 30 min, 4 C) and stored at 4 C until activity analysis was
A 1.2 ml aliquot of onion extract, prepared by subcritical water
◦
◦
extraction, was mixed with 300 ml of 100 mM citrate–phosphate
performed. The inactivation kinetics of the different constructs
was analysed assuming a first-order reaction rate. For a constant
temperature:
◦
buffer, pH 5.0, and heated at 95 C until the sample components
dissolved. Fractions were collected at 0, 1, 3, 5 and 10 min after
addition of 3 nmol of TnBgl1A_N221S. Samples were analysed
by HPLC-UV.
-
kt
A/A
0
= e
(1)
(2)
HPLC analysis
lnA- lnA
0
= -kt
HPLC-UV analysis was performed using the chromatographic
system UltiMate 3000 from Dionex (Germering, Germany). An
Agilent Zorbax SB-C18 column (100 ¥ 2.1 mm, 3.5 mm) was
used for isocratic separation with a methanol–water (50 : 50) and
formic acid (0.13 M, 0.5 vol%) mobile phase at a flow rate of
-
1
where A
0
is the initial activity expressed in U ml , A is the
activity at time t, expressed as above, t (h) is the time, and k is
-
1
the inactivation rate constant (h ). The linear regression line of
the natural logarithm of the residual activity versus incubation
time were obtained using Excel (Microsoft Office 2003). The
half-life (t1/2) at the respective temperature was obtained at A =
-
1
0
.15 ml min . The injection volume was 10 ml and detection was
accomplished at 350 nm. Quantification of quercetin and its
glucosides was performed using a five-point calibration curve
of a quercetin dihydrate, Q-3, Q-4¢ and Q-3,4¢ standards at
0.5A0.
Product inhibition studies
-
1
concentrations between 0.5 and 25 mg ml . Each vial taken
to analysis had a total volume of 500 ml.
Quercetin samples (triplicates of 100 nmol each) in methanol
were evaporated and re-dissolved in 750 ml of 100 mM citrate–
◦
phosphate buffer pH 5.0, and the vials were heated at 90 C until
Environmental impact assessment
the quercetin had dissolved. Glucose (triplicates of 100 nmol)
dissolvedin750 ml 100mMcitrate–phosphate buffer, pH5.0, and
controls containing 750 ml of 100 mM citrate–phosphate buffer
The global warming potential (GWP) and primary energy
consumption for the new and a traditional extraction method
16,30
◦
were assessed from a gate-to-gate perspective.
The calcu-
pH 5.0, (triplicate) were also heated at 90 C, and treated in the
lations are based on a functional unit of 10 g of quercetin.
The new, subcritical water extraction method is described by
same way as the quercetin samples. TnBgl1A_N221S (10 pmol)
◦
was added to the vials and the samples were heated at 90 C.
3
Turner et al. (see Subcritical water extraction, above), while
Then, 18 min after addition of enzyme, pre-heated 100 nmol
of Q-4¢ dissolved in 750 ml of 100 mM citrate–phosphate buffer,
pH 5.0, was added to the vials. Fractions were collected as above,
the enzymatic reaction method is described in this study (see
Enzymatic hydrolysis of onion extract, above). The traditional
method using methanol extraction and HCl-catalysed hydrolysis
1
, 3, 5, 10, 20 and 30 min after addition of Q-4¢. The first point
◦
7
(
80 C for 2 h) is described by Hertog et al. and Nuutila
in Fig. 4 is the 1 min fraction. Samples were analysed by HPLC-
UV.
8
et al. Extractions were scaled up linearly to produce 10 g of
quercetin. The dry matter content in onion is around 13.5%
-
1
and the quercetin content in onion is, on average, 13 mg g dry
Subcritical water extraction
-
1
3
matter, varying between 7 and 25 mg g (see Table 5).
Energy demand is calculated for heating and heat losses
1
5–18 g of onion samples, in triplicate, were weighed into 33 ml
31
stainless steel extraction cells containing a cellulose filter at the
during extraction according to Welty et al. Extraction is set to
be carried out in vessels of stainless steel insulated with mineral
wool. Production of extraction equipment and generation of
pressure are not considered in this study. A natural gas burner
with 60% efficiency is set to be used for heating. Emissions due to
ꢀ
R
bottom. Extractions were performed on a Dionex ASE -200
subcritical water extraction system (Dionex, Sunnyvale, CA,
USA) using water as the only solvent as described by Turner
3
et al. The pressure was set to 50 bar and the initial heating lasted
◦
◦
Table 5 Data for the extraction methods both producing 10 g of quercetin. Traditional method: 80 C for 2 h. New method: extraction at 120 C
◦
for 15 min and biocatalysis at 95 C for 5 min. Ranges of values are in brackets
3
Extraction method
Onion/kg
Amount of solvent/kg
Amount of catalyst/kg
Total volume (dm )
Traditional method
New method
5.7 (3.0–10.6)
5.7 (3.0–10.6)
Methanol: 31 (16–56)
Water: 13 (6.5–23)
HCl: 3.2 (1.7–5.9)
Enzyme: 0.003 (0.002–0.006)
87 (41–150)
21 (9.4–33)
1
66 | Green Chem., 2010, 12, 159–168
This journal is © The Royal Society of Chemistry 2010

View Article Online
production and combustion of natural gas are from Uppenberg
et al. The environmental impact of water is assumed to be
insignificant.
wt
Wild type
Transition temperature
Glycoside hydrolase family 1
32
T
m
GH1
Conclusions
Acknowledgements
In this paper, we have developed analytical methods to study the
kinetics of a thermostable b-glucosidase (wt TnBgl1A), and a
mutant of the same enzyme (TnBgl1A_N221S) towards Q-3 and
Q-4¢. We have also developed an off-line biocatalysis method, in
which the hydrolysis of all quercetin glucosides present in yellow
The Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning (FORMAS, 2006-1346); the
Swedish Research Council (VR, 2006-4084 and 2006-6048);
the Swedish International Development Cooperation Agency
(
SIDA, SWE-2005-473); the Swedish Foundation for Strategic
◦
onion extract is completed within 5 min at 95 C using only
Research (SSF, 2005 : 0073/13) and the Foundation for Strategic
Environmental Research (MISTRA, Greenchem) are greatly
acknowledged.
3
nmol of enzyme per 1.3 mmol of quercetin species. This study
shows that TnBgl1A_N221S has higher activity towards both
Q-3 and Q-4¢ compared to the wt TnBgl1A, i.e. a mutation
of residue 221 at the +2 binding site of the enzyme does
significantly affect substrate affinity, but this effect is higher
for the Q-3 substrate. Another conclusion that can be drawn
References
1
A. Schieber, F. C. Stintzing and R. Carle, Trends Food Sci. Technol.,
2001, 12, 401–413.
◦
is that deactivation is significant at 95 C, and to avoid this,
◦
the temperatures should be kept below 95 C, if reactions are
2 M. G. L. Hertog, P. C. H. Hollman and M. B. Katan, J. Agric. Food
Chem., 1992, 40, 2379–2383.
more than a few minutes long. To increase the hydrolysis rate
of quercetin glucosides, addition of glucose can be considered.
Future aspects include immobilisation of TnBgl1A_N221S onto
a support material allowing recycling of the enzyme, and the
use of an on-line flow system of hot water extraction and
enzymatic hydrolysis of quercetin and quercetin glucosides
from yellow onion waste and other potential by-products and
waste. The immobilisation of the enzymes may also improve the
thermostability of the enzymes.
From an environmental point of view, extraction with subcrit-
ical water followed by an enzymatic hydrolysis is to be preferred
regarding primary energy consumption and global warming
potential. Shorter extraction time and smaller volumes makes
the process more energy-efficient, although a higher temperature
and pressure have to be generated. Furthermore, avoiding the
use of organic solvents from fossil origin, such as methanol,
makes the subcritical water extraction more resource efficient
and favourable from a greenhouse gas perspective. Handling of
large quantities of toxic chemicals, such as HCl and methanol,
is also avoided.
3
C. Turner, P. Turner, G. Jacobson, K. Almgren, M. Waldeback, P.
Sj o¨ berg, E. N. Karlsson and K. E. Markides, Green Chem., 2006, 8,
949–959.
4
5
A. Murakami, H. Ashida and J. Terao, Cancer Lett., 2008, 269, 315–
3
25.
K. Ono, T. Hamaguchi, H. Naiki and M. Yamada, Biochim. Biophys.
Acta, Mol. Basis Dis., 2006, 1762, 575–586.
6 K. R. Price and M. J. C. Rhodes, J. Sci. Food Agric., 1997, 74, 331–
39.
3
7
M. G. L. Hertog, P. C. H. Hollman and D. P. Venema, J. Agric. Food
Chem., 1992, 40, 1591–1598.
8 A. M. Nuutila, K. Kammiovirta and K. M. Oksman-Caldentey,
Food Chem., 2002, 76, 519–525.
S. H. Hakkinen, S. O. Karenlampi, I. M. Heinonen, H. M. Mykkanen
9
and A. R. Torronen, J. Sci. Food Agric., 1998, 77, 543–551.
0 J. B. Harborne, Phytochemistry, 1965, 4, 107–120.
1
11 Y. K. Do, J. M. Kim, S. M. Chang, J. H. Hwang and W. S. Kim, in
International Conference on Biorefinery, Elsevier Science Bv, Beijing,
JAPAN, 2007, pp. 173-178.
1
2 F. Siewek and R. Galensa, J. Chromatogr., A, 1984, 294, 385–
389.
1
3 N. Lambert, P. A. Kroon, C. B. Faulds, G. W. Plumb, W. R.
McLauchlan, A. J. Day and G. Williamson, Biochim. Biophys. Acta,
Protein Struct. Mol. Enzymol., 1999, 1435, 110–116.
4 E. Ibanez, A. Kubatova, F. J. Senorans, S. Cavero, G. Reglero and
S. B. Hawthorne, J. Agric. Food Chem., 2003, 51, 375–382.
5 K. Hartonen, K. Inkala, M. Kangas and M. L. Riekkola, J. Chro-
matogr., A, 1997, 785, 219–226.
1
1
1
Abbreviations
6 A. Azapagic, Handbook of Green Chemistry and Technology,
Q
Quercetin
Blackwell Science, Oxford, 2002, 62-85.
Q-3
Q-4¢
Quercetin-3-glucoside
Quercetin-4¢-glucoside
Quercetin-3,4¢-diglucoside
p-Nitrophenyl-b-D-glucopyranoside
p-Nitrophenol
Bovine serum albumin
Global warming potential
Life cycle assessment
Isorhamnetin-4¢-glucoside
Isorhamnetin-3,4¢-diglucoside
Isopropyl-1-thio-b-D-galactopyranoside
Dry weight
2,2-Diphenyl-1-picrylhydrazyl
Relative standard deviation
Immobilised metal-ion affinity chromatography
17 I. S. Organization, Enviromental management - Life cycle assesment -
Requirements and guidelines, ISO 14044, 2006.
8 S. Khan, T. Pozzo, C. Paul, S. Lindahl, C. Turner, and E. Nordberg
Karlsson, 2009, unpublished work.
1
Q-3,4¢
pNPG
pNP
BSA
GWP
LCA
I-4¢
I-3,4¢
IPTG
dw
19 J. G. Berrin, W. R. McLauchlan, P. Needs, G. Williamson, A.
Puigserver, P. A. Kroon and N. Juge, Eur. J. Biochem., 2002, 269,
2
49–258.
2
0 R. M. Wright, M. D. Yablonsky, Z. P. Shalita, A. K. Goyal and D. E.
Eveleigh, Appl. Environ. Microbiol., 1992, 58, 3455–3465.
21 D. A. Yernool, J. K. McCarthy, D. E. Eveleigh and J. D. Bok,
J. Bacteriol., 2000, 182, 5172–5179.
2
2 J. A. Perezpons, X. Rebordosa and E. Querol, Biochim. Biophys.
Acta, Protein Struct. Mol. Enzymol., 1995, 1251, 145–153.
23 A˚ . Furnander, Life Cycle Assessment of Dimethyl Ether as a
Motor Fuel, Chalmers University of Technology, Gothenburg,
1
996.
DPPH
RSD
IMAC
2
4 I. Boustead, Eco-Profiles of the European Plastic Industry - Hydrogen
Chloride, PlasticEurope - Association of Plastics Manufacturers,
2005.
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 159–168 | 167

View Article Online
2
2
2
5 P. H. Nielsen, K. M. Oxenboll and H. Wenzel, Int. J. Life Cycle
Assess., 2007, 12, 432–438.
30 A. E. V. Petersson, L. M. Gustafsson, M. Nordblad, P. Borjesson,
B. Mattiasson and P. Adlercreutz, Green Chem., 2005, 7, 837–
843.
31 J. R. Welty, C. H. Wicks, R. E. Wilson and G. Rorrer, Fundamentals
of momentum, Heat and Mass Transfer, John Wiley & Sons, Inc.,
2001, 201-218.
32 S. Uppenberg, M. Almemark, M. Brandel, L.-G. Lindfors, H.-O.
Marcus, H. Stripple, A. Wachtmeister and L. Zetterberg,
Milj o¨ faktabok f o¨ r Br a¨ nslen Del 2, IVL Swedish Enviromental
Research Institute Ltd., 2001.
6 M. Abou-Hachem, F. Olsson and E. N. Karlsson, Extremophiles,
2
003, 7, 483–491.
7 P. Turner, D. Svensson, P. Adlercreutz and E. N. Karlsson, J. Biotech-
nol., 2007, 130, 67–74.
2
2
8 U. K. Laemmli, Nature, 1970, 227, 680–685.
9 P. Turner, A. Pramhed, E. Kanders, M. Hedstrom, E. N. Karlsson and
D. T. Logan, Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun.,
2
007, 63, 802–806.
1
68 | Green Chem., 2010, 12, 159–168
This journal is © The Royal Society of Chemistry 2010