Asymmetric organic carbonate synthesis catalyzed by an enzyme with dimethyl carbonate: A fruitful sustainable alliance

Article abstract of DOI:10.1039/c3ra43698e

We have successfully developed an easy and efficient bioprocess for asymmetric organic carbonate synthesis by performing Novozym 435 mediated esterification of DMC and alcohols in this work. Under the optimized conditions (60 °C, molar ratio of alcohol to DMC 1:12), the highest yield of carbonate can reach 95.6%. An additional advantage of the new process is the fact that 90% of the original activity of the enzyme is retained after being recycled nine times. Consequently it has potential as a useful enzyme-catalyzed process for the industrial production of asymmetric organic carbonates. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3ra43698e

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Asymmetric organic carbonate synthesis catalyzed
by an enzyme with dimethyl carbonate: a fruitful
sustainable alliance†
Cite this: RSC Adv., 2014, 4, 7013
a
a
a
b
a
*
Yaoliang Zhou, Qiuyan Jin, Zhanyang Gao, Hongtao Guo, Haibo Zhang
and Xiaohai Zhoua
We have successfully developed an easy and efficient bioprocess for asymmetric organic carbonate
synthesis by performing Novozym 435 mediated esterification of DMC and alcohols in this work. Under
the optimized conditions (60 ꢀC, molar ratio of alcohol to DMC 1 : 12), the highest yield of carbonate can
reach 95.6%. An additional advantage of the new process is the fact that 90% of the original activity of
the enzyme is retained after being recycled nine times. Consequently it has potential as a useful
enzyme-catalyzed process for the industrial production of asymmetric organic carbonates.
Received 31st July 2013
Accepted 7th November 2013
DOI: 10.1039/c3ra43698e
www.rsc.org/advances
The new pathways involve carbonated building blocks like
DMC, which has been shown to be an efficient methoxy car-
1. Introduction
bonylating agent and can be formed by a clean synthetic
process. The synthesis of asymmetric organic carbonates
through the transesterication of DMC with alcohols has been
achieved by the use of solid base catalysts, for instance, MCM-
41-TBD (TBD, 157-triazabicyclo[4.4.0]dec-5-ene),8 Mg/La metal
oxide,9 CsF/a-Al2O3,10 (n-Bu2SnO)6 and nano-crystalline MgO.11
These heterogeneous catalytic systems are complicated,
requiring sophisticated and tedious procedures, high temper-
atures (>100 ꢀC) and rigorous pressure conditions. Recently
Matthieu Bandres et al.12 showed the efficiency of K2CO3, NaOH
Because of concerns regarding global sustainability, environ-
mentally benign chemicals and synthetic methods have become
urgently needed. Asymmetric and symmetric organic carbon-
ates,1 as essential intermediates,2 play an indispensable role in
the lithium battery industry and chemical manufacturing. For
instance, asymmetric organic carbonates, well-known solvents
with relatively high oxidation potentials, are actually a more
important component in lithium-ion battery electrolytes than
symmetric carbonates.3 Furthermore, organic carbonates can
be used as plasticizers, lubricants, protecting groups4 of alco-
hols and phenols, and mild carbonylation or methylation
agents in place of poisonous phosgene and dimethyl sulfate.
With the dramatically expanding applications of lithium
batteries, the importance of aryl and alkyl carbonates is
undoubted as reported by many patents5 and research articles,6
however, few carbonates are commercially available except for
propylene carbonate (PC), diethyl carbonate (DEC), ethylene
carbonate (EC) and dimethyl carbonate (DMC). Several methods
for the synthesis of alkyl carbonates from alcohols have thus
been exploited and reactants such as urea or its carbamate
derivatives, carbon monoxide or supercritical CO2, lead to low
yields. Moreover, the synthesis of these compounds still uses
highly toxic and harmful reagents,1,7 such as phosgene (COCl2),
dimethyl sulfate, pyridine and carbon monoxide.
ꢀ
or MeONa in such reactions with a high temperature of 90 C
(reux) which is still slightly high for DMC, but the result could
not be duplicated in our lab. Even the only successful TBD
catalysis published in 2012 still has some drawbacks, such as
causticity and inammability.13
Nowadays, as awareness of environmental protection is
gradually aroused, eliminating metal component involvement
in catalyst design and abating volatile organic solvent usage in
chemical syntheses is more promising for our future. Hence,
there is the need to identify “green” catalyst systems for trans-
esterication reactions which are “environment-friendly” and
can obtain high yields at ambient conditions. One of the most
promising strategies to achieve these goals is the application of
enzymes which exhibit a number of features that make their use
advantageous compared to conventional chemical catalysts.
Except for a high level of catalytic efficiency, enzymes generally
operate under mild temperature, pressure and pH conditions
with reaction rates of the order of those achieved by chemical
catalysts under more extreme conditions. Although enzymes
have been used mostly for aqueous phase reactions,14 non-
aqueous enzymology has potential applications in synthetic
chemistry.15 Advantages such as high solubility, the ability to
aColloid and Interface Sci. Lab, College of Chemistry and Molecular Sciences, Wuhan
University, Wuhan 430072, China. E-mail: haibozhang1980@gmail.com; Fax: +86-
027-87218534; Tel: +86-027-87218534
bShenzhen Leveking Bio-engineering Co. Ltd., Shenzhen 518000, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra43698e
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carry out new reactions impossible in water, greater stability,
and an easier synthesis procedure, mean that lipases are the
most frequently used enzymes for organic syntheses,16
including aminolysis, alcoholysis, amidation and perhydrolysis.
As a catalyst it has also been reported for the transesterication
of various rened vegetable oils in many literature reports. A
suitable choice for catalyzing transesterication is Candida
Antarctica lipase B (CaLB),17 while the commercially available
lipase for the immobilization of CaLB is Novozym 435.18 The
enzyme is adsorbed on a polymethylmethacylate carrier, mainly
due to its tolerance to organic solvents and reasonable thermal
stability, which has been shown to be a highly efficient
biocatalyst.
2.3. Experimental procedure
The enzymatic esterication was performed in a 50 mL ask on
a rotary shaker at 180 rpm. The entire reactor assembly was
immersed in a thermostatic water bath, which was maintained
at a desired temperature with an accuracy of ꢂ1 ꢀC.
A typical reaction mixture consisted of 0.0113 mol alcohol
and 0.136 mol DMC without solvent. The immobilized enzyme
was added to initiate the reaction. The mass ratio of enzyme/
ꢀ
DMC is 1/100. The reaction mixture was agitated at 60 C at a
speed of 180 rpm. Liquid samples were withdrawn periodically
from the reaction mixture and analyzed on
chromatograph.
a
gas
The lipase-catalyzed synthesis of glycerol carbonate from
glycerol (a renewable and cheap raw chemical) with dimethyl
carbonate has received increasing attention.19 However, these
catalytic processes require relatively harsh reaction conditions,
organic solvents and other additives like molecular sieves, then
moderate yields of the targeted product are obtained and yet
another intricate purication step by distillation is necessary. In
this paper, we extended the scope of the catalyst to the synthesis
of asymmetric organic carbonates. The main advantage of the
developed synthesis method is that the strategy follows an eco-
friendly and non-toxic route. The process efficiently synthesizes
asymmetric organic carbonates, with alcohols and DMC, and is
totally green since the reaction is catalyzed by Novozym 435, and
no other additives are involved except for an acyl donor and
solvent. This enzyme-catalyzed process has the potential for the
industrial production of asymmetric organic carbonates for
lithium-ion battery electrolytes. To the best of our knowledge, it
is the rst enzymatic example to synthesize asymmetric organic
carbonates.
2.4. Analysis
The analysis of the reaction mixture was carried out on a gas
chromatograph (sp-6890) equipped with a ame ionization
detector (FID) and a capillary column (SE-54, 30 m ꢃ 0.25 mm
ꢃ 0.25 mm). The column temperature was kept at 100 ꢀC for
ꢀ
ꢀ
1 min and then raised to 150 C for 10 min at a rate of 10 C
minꢁ1. The temperatures of the injector and detector were
ꢀ
maintained at 320 and 320 C, respectively. The products were
further identied using 1H NMR (Nuclear Magnetic Resonance).
The catalyst enzyme was recovered by centrifugation of the
resulting suspension and then washed using acetone. The
residue obtained was dried at 45 ꢀC under reduced pressure
overnight (at 1 Torr for 24 h) and was then used for the next
generation. The conversion of alcohol, the yield of alcohol and
the selectivity for asymmetric carbonate were calculated using
eqn (S1)–(S3),† where the number of moles was determined by
the Internal Standard Method from the chromatographic
analysis.
3. Results and discussion
2. Materials and methods
2.1. Enzyme
The effects of various parameters on the conversion and rate of
reaction were studied systematically. The reaction is shown by
Scheme 1.
Crude lipase from Penicillium expansum (PEL) (5000 U mgꢁ1
solid), Penicillium neutral expansum (PNEL) (10 000 U mgꢁ1
solid), Aspergillus niger (ANL) (120 000 U mgꢁ1 solid), Rhizopus
chinensis satio (RCSL) (10 000 U mgꢁ1 solid), Porcine pancreatic
(PPL) (10 000 U mgꢁ1 solid), neutral proteolytic enzyme
(20 000 U mgꢁ1 solid), acidic proteolytic enzyme (20 000 U
mgꢁ1 solid), alkaline proteolytic enzyme (20 000 U mgꢁ1 solid)
was kindly donated by Shenzhen Leveking Bio-engineering
Co. Ltd., China. These enzymes were produced by spraying
the concentrated supernatant from fermentation with the
addition of a certain amount of starch as a thickening agent.
3.1. The efficacy of various catalysts
Because it is cheap and renewable, fusel oil production is a
rapidly growing biomass industry, isoamyl alcohol, which is
distilled off fermented alcohol for use in beverages or biofuels,
has been investigated less compared to other alcohols, from C2
to C5, especially for carbonates. For this reason, isoamyl alcohol
was selected as a model substrate to carry out our elementary
experiments in the search for a highly efficient catalyst that
follows the principles of green chemistry, and the results are
listed in Table 1. As shown in Table 1, different lipases or
chemical catalysts were used to evaluate their efficacy under
similar conditions. Novozym 435 showed the highest catalytic
performance for the production of asymmetric organic
carbonates, while other lipases exhibited little activity and
almost no asymmetric organic carbonates could be found.
Immobilized (Novozyme 435) C. antarctica lipase
(EC 3.1.1.3) was donated by Novozymes (China) Investment
Co. Ltd.
B
2.2. Chemicals
All chemicals were purchased from Aladdin reagent company, NaOH, MeONa and CaCO3 didn't show high activity as repor-
which were of analytical grade and used without further ted.12 Based on these results, we selected Novozym 435 as the
purication.
desirable catalyst for the synthesis of asymmetric carbonates.
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Scheme 1 The enzyme-mediated esterification of alcohols with DMC.
Table
1 Screening the lipase sources for asymmetric organic
As shown in Fig. 1, the initial reaction rate increased with an
increase in enzyme loading, which is in contrast to the data at
2 h when the substrate concentration was much higher than the
enzyme concentration (as will be mentioned in Section 3.4).
Based on the Michaelis–Menten equation V ¼ Vmax[S]/(Km + [S])
(where [S] is the substrate concentration, V is the reaction rate,
Vmax is the maximum rate achieved by the system at maximum
(saturating) subtrate concentrations and Km is the Michaelis
constant i.e. the substrate concentration at which the reaction
rate is half of Vmax), when the substrate concentration is high
enough, i.e., [S] [ Km, then V z Vmax. At this time, if we
increase the concentration of the enzyme, the enzymatic reac-
tion rate will become proportional to the change in enzyme
concentration, i.e., V ¼ k3[ES] (ref. 20) (where [ES] is the enzyme
concentration). Because the dosage of 5–40 mg enzyme was
small and therefore the reaction was slow, we can regard the
data (2.3%, 5.1%, 10.0% and 18.1%) at the time of 2 hours as
proof of the existence of k3.
carbonate synthesisa
Entry
Cat.
Conv. (%)
1
2
3
4
5
6
7
8
No catalyst
Novozym 435
Parcine pancreas
Penicillium expansum
Penicillium neutral expannsum
Aspergillus niger
0
89
10
2
2
0
Rhizopus chinensis satio
Neutral proteolytic enzyme
Acidic proteolytic enzyme
Alkaline proteolytic enzyme
NaOH
0
0
0
0
20
38
52
9
10
11
12
13
MeONa
CaCO3
a
Conditions: DMC : isoamyl alcohol
¼
12 : 1; 1% (w/w) catalyst;
temperature ¼ 55 ꢀC; 48 h incubation time. For NaOH, MeONa,
CaCO3: temperature ¼ 90 ꢀC.
It was found that the rate of reaction and the overall
conversion exhibited a similar tendency to ascend and then
reach equilibrium with an increasing amount of catalyst from
5 mg to 120 mg. As the catalyst loading surpassed 120 mg the
rate of reaction and conversion did not increase any more at
72 h. We believe that the reaction reached equilibrium; under
these conditions it reached the highest conversion of up to 90%.
3.2. The effect of Novozym 435 loading
In the reaction process, the amount of lipase not only plays a
key role in the catalytic performance, but also demonstrates a
crucial economical factor for successful industrial application.
The effect of catalyst loading was studied from 5 to 180 mg
under similar conditions.
3.3. The effect of temperature
It is well known that the reaction temperature is a crucial
parameter in biocatalysis. The higher the reaction temperature,
the faster the reaction rate. However, the high temperature may
inactivate the enzyme. The temperature also has a big effect on
the thermodynamic equilibrium of a reversible reaction.
Therefore, the effect of temperature was studied in the range of
50–65 ꢀC. In Fig. 2, it is shown that the initial rate and the
conversion increased when the temperature was raised from 50
to 65 ꢀC. The nal conversions aer 6 h were 39.0%, 48.5%,
59.3% and 58.6% at 50, 55, 60 and 65 ꢀC respectively. It is well
known that Novozym 435 is thermally stable at 60 ꢀC and hence
there was no deactivation of the enzyme at 60 C.21 The asym-
ꢀ
metric organic carbonate yield reached the highest value of
93.4% and a further increase in temperature (up to 65 ꢀC) led to
a slight decrease in the yield, which supported the previous
nding that the reaction was intrinsically kinetically controlled.
The rise in temperature is responsible for the activation of
Novozym. Also the enthalpic contribution to the enzymatic rate
enhancement suggests that there are important electrostatic
and hydrogen-bonding interactions in the transition state of the
Fig.
1 Effect of catalyst loading. Reaction conditions – isoamyl
alcohol: 11.3 mmol, DMC: 0.136 mol, speed of agitation: 180 rpm,
temperature: 55 ꢀC, catalyst: 5–180 mg, time: 72 h, (-) 5 mg; (C)
10 mg; (:) 20 mg; (;) 40 mg; (*) 80 mg; (+) 100 mg; (A) 120 mg; (ꢃ)
160 mg; (,) 180 mg.
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Based on the data in Fig. 3, the initial conversion rate
increased along with an increase in the dosage of DMC before
the ratio of DMC to isoamyl alcohol exceeded 12 : 1. When the
DMC’s dosage surpassed 12 : 1, the increment of its concen-
tration wouldn't enhance the initial reaction rate, which
matched the Michaelis–Menten equation V ¼ Vmax[S]/(Km + [S]).
When the substrate concentration is very low, [S] ꢄ Km, the
reaction rate is proportional to the substrate concentration;
while if the substrate concentration is high enough, the reaction
rate will reach the limit Vmax and it won't change even though
the substrate concentration increases.
An increase in the isoamyl alcohol : DMC molar ratio from
1 : 2 to 1 : 12 generated an increasing DMC yield with an
improved product conversion from 60.6% to 93.4%. The law of
chemical kinetics tells us that the reaction equilibrium is
sharply impacted by the reactant concentration, and more
reactant can drive the reaction to move in the opposite direc-
tion. Therefore, adding more DMC should result in a higher
IMC (Isoamyl Methyl Carbonate) yield due to the reversible
nature of the transesterication step. On further increasing the
ratio of isoamyl alcohol to DMC from 1 : 14 to 1 : 25, a decrease
in the conversion to carbonates can be observed. It may be
attributed to the fact that excessive DMC decreases the valid
concentration of the lipase and the enzyme activity is lowered by
more and more DMC. Thus a suitable value (1 : 12) was deter-
mined and used in the subsequent investigation in consider-
ation of energy consumption.
Fig. 2 Effect of temperature. Reaction conditions – isoamyl alcohol:
11.3 mmol, DMC: 0.136 mol, catalyst: 0.12 g, speed of agitation:
180 rpm, temperature: 50–65 ꢀC, time: 48 h, (-) 50 ꢀC; (C) 55 ꢀC; (:)
60 ꢀC; (;) 65 ꢀC.
enzymatic reaction, which are responsible for the increased rate
with a raised temperature.
3.4. The effect of concentration of DMC
The catalytic performance is closely associated with the amount
of added reactive precursors in the reactions. In our present
study, the effect of the isoamyl alcohol/DMC molar ratio was
also investigated. As shown in Fig. 3, it is apparent that the
catalytic performance of lipase on the transesterication reac-
tion was signicantly affected by the isoamyl alcohol/DMC
molar ratio.
3.5. Reusability of the catalyst
To make the process more economical and feasible, it is
necessary to study the recyclability of the immobilized lipase
Novozym 435, which is regarded as the most important advan-
tage of an immobilised enzyme compared to a free enzyme. The
reusability was studied to ascertain its stability during the
reaction. To further examine the potential of this bioprocess for
industrial production, the catalyst was ltered aer each use
Fig. 3 Effect of concentration of DMC. Reaction conditions – isoamyl
alcohol: 11.3 mmol, DMC: 0.011–0.283 mol, speed of agitation:
180 rpm, temperature: 60 ꢀC, catalyst: 120 mg, time: 48 h, (-) 1 : 2; Fig. 4 Reusability of the catalyst. Reaction conditions – isoamyl
(C) 1 : 4; (:) 1 : 6; (;) 1 : 8; (,) 1 : 10; (A) 1 : 12; (|) 1 : 14; (-) 1 : 16; (*) alcohol: 11.3 mmol, DMC: 0.136 mol, speed of agitation: 180 rpm,
1 : 18; (ꢃ) 1 : 20; (+) 1 : 25.
temperature: 60 ꢀC, catalyst: 120 mg, time: 48 h.
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and washed with acetone and then dried at room temperature. Table 2, cyclohexanol has the biggest strict hindrance, so it has
As shown in Fig. 4, Novozym 435 shows good stability and the lowest conversion. Benzyl alcohol's hydroxide radical formed
retains more than 91% of its initial activity aer 9 consecutive the conjugated system with the benzene ring decreasing the strict
reuses. The loss of activity is believed to be due to the deacti- hindrance, and its methylene is mobilizable, so benzyl alcohol
vation of lipase or the loss of catalyst during handling as the has a higher conversion than cyclohexanol. It can be concluded
cycles are increased.
that the strict hindrance of the alkanols' hydroxide radical has an
important effect on the conversion.
3.6. Asymmetric organic carbonate synthesis
The alcohol used above was isoamyl alcohol and a number of
different alcohols were chosen to evaluate the scope of the
4. Conclusions
reaction. The results in Table 2 show that the esterication of In our present work, we rst used the enzyme to demonstrate a
DMC with different alcohols was very satisfactory.1
high-efficiency, low energy-consuming and easy-to-handle route
As can be seen, the length of the alkyl chains of the alcohols for the synthesis of asymmetric organic carbonates. The only by-
did not affect the conversion much. The alcohols with a branched product is methyl alcohol, which indicates this process has
chain, which has a huge steric hindrance, reacted with DMC and signicance in green chemistry. Besides, the catalyst Novozym
had lower conversion ratios. Among the alcohols mentioned in 435 is industrialized and easy to obtain and recycle.
Table 2 The synthesis of various asymmetric carbonates via Novozym 435 catalyzed transesterification of DMCa,b,c
Entry
Product
Time
Conv. (%)
Sel. (%)
Yield (%)
1
2
24 h
24 h
94.4
93.9
95.7
93.8
90.3
87.3
3
4
5
6
7
36 h
48 h
48 h
48 h
30 h
94.3
95.6
92.5
91.1
91.3
>99
94.3
91.9
92.5
86.9
90.2
96.1
>99
95.4
98.8
8
24 h
48 h
72 h
72 h
85.2
93.4
61.7
47.6
>99
>99
>99
>99
84.6
93.4
61.7
47.5
9
10
11
12
72 h
72 h
35.2
72.8
>99
>99
35.2
72.2
13
a
All reactions were carried out with 11.3 mmol alcohol, 136.1 mmol of the DMC and wEnzyme/wDMC ¼ 1.0% (120 mg) of Novozym 435 (where wEnzyme
is the mass of the enzyme and wDMC is the mass of the DMC). b The selectivity towards the asymmetric carbonate. c The conversion and selectivity
were calculated for crude reaction mixtures via GC (Gas Chromatography) with methyl benzoate as an internal standard.
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One of the products, methyl ethyl carbonate (yield 90.3%), is
an excellent lithium ion battery electrolyte solvent. It has been
mentioned in the literature that methyl pentyl carbonate (yield
94.3%) used for lithium ion battery electrolyte solvent can not
8 S. Carloni, D. E. D. Vos, P. A. Jacobs, R. Maggi, G. Sartori and
R. Sartorio, J. Catal., 2002, 205, 199–204.
9 B. Veldurthy and F. Figueras, Chem. Commun., 2004, 734–
735.
only increase the battery's capacity density and discharge 10 B. Veldurthy, J. M. Clacens and F. Figueras, Eur. J. Org.
capacity, but also extend the battery's lifetime. Furthermore, it Chem., 2005, 1972–1976.
can raise the performance at low temperature, effectively over- 11 M. L. Kantam, B. S. U. Pal and B. M. Choudary, Adv. Synth.
coming the defects of conventional lithium ion battery
electrolytes.22
Catal., 2007, 349, 1671–1675.
12 M. Bandres, P. d. Caro, S. T. Roux and M. E. Borredon, C. R.
Chim., 2011, 14, 636–646.
Although Novozym 435 has been commercialized and is
recyclable, its price is still higher than other enzymes, such as 13 H. Mutlu, J. Ruiz, S. C. Solleder and M. A. R. Meier, Green
the Porcine pancreatic lipase, which showed catalytic activity in Chem., 2012, 14, 1728–1735.
this reaction. Enzyme immobilization will be continued and 14 Z. Cabrera, G. F. Lorente, R. F. Lafuente, J. M. Palomo and
delved into aer this work.
J. M. Guisan, Process Biochem., 2009, 44, 226–231.
15 M. Fujii, M. Fukumura, Y. Hori, Y. Hirai, H. Akita,
K. Nakamura, K. Toriizuka and Y. Ida, Tetrahedron:
Asymmetry, 2006, 16, 2292–2298; M. Gruttadauria, P. Lo
Meo and R. Noto, Tetrahedron Lett., 2004, 45, 83–85;
E. Sundby, L. Perk, T. Anthonsen, A. J. Aasen and
T. Hansen, Tetrahedron, 2004, 60, 521–524.
Notes and references
1 A. A. Shaikh and S. Sivaram, Chem. Rev., 1996, 96, 951–976.
2 Y. Ono, Appl. Catal., A, 1997, 155, 133–166; J. P. Parrish,
R. N. Salvatore and K. W. Jung, Tetrahedron, 2000, 56,
8207–8237.
16 M. Reetz, Adv. Catal., 2006, 49, 1–69.
17 M. Arroyo and J. V. Sinisterra, J. Org. Chem., 1994, 59, 4410–
4417.
3 T. W. Greene, P. G. Wuts and J. Wiley, Protective groups in
organic synthesis, Wiley, New York, 1999.
18 Z.-Q. Duan, W. Du and D.-H. Liu, J. Mol. Catal. B: Enzym.,
2013, 89, 1–5.
4 S. Gryglewicz, F. Oko and G. Gryglewicz, Ind. Eng. Chem. Res.,
2003, 42, 5007–5010.
19 S. C. Kim, Y. H. Kim, H. Lee, D. Y. Yoon and B. K. Song,
J. Mol. Catal. B: Enzym., 2007, 49, 75–78.
20 I. H. Segel, Biochemical calculations, Wiley, New York,
1976.
21 A. Adnani, M. Basri, E. A. Malek and A. B. Salleh, Ind. Crops
Prod., 2010, 31, 350–356.
22 E. J. Plichta and W. K. Behl, J. Power Sources, 2000, 88, 192–
5 H. O. Osaka, H. G. Neyagawa and A. M. Takaishi, US Pat.,
5521027, 1996; H. Gan, S. Takeuchi and R. Rubino, US Pat.,
6759 170, 2004.
6 H. Buchold, J. Eberhadt, L. U. Wagner and H. J. Woelk, WO
Pat., 2005028415, 2005; M. Miyamoto and T. Tayama, JP
Pat., 2005126496, 2005; S. Yukio, T. Masahiro and
U. Makoto, JP Pat., 2004010491, 2004.
´
196; J. Vetter and P. Novak, J. Power Sources, 2003, 119, 338–
342.
7 A. F. Hegarty, Comprehensive Organic Chemistry, London,
1979.
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