Highly efficient solvent-free synthesis of 1,3-diacylglycerols by lipase immobilised on nano-sized magnetite particles

Article abstract of DOI:10.1016/j.foodchem.2013.07.132

Recently, 1,3-DAGs (1,3-diacylglycerols) have attracted considerable attention as healthy components of food, oil and pharmaceutical intermediates. Generally, 1,3-DAG is prepared by lipase-mediated catalysis in a solvent free system. However, the system's high reaction temperature (required to reach the reactants' melting point), high substrate concentration and high viscosity severely reduce the lipase's activity, selectivity and recycling efficiency. In this report, MjL (Mucor javanicus lipase) was found to have the best performance in the solvent-free synthesis of 1,3-DAGs of several common commercial lipases. By covalent binding to amino-group-activated NSM (nano-sized magnetite) particles and cross-linking to form an enzyme aggregate coat, MjL's specific activity increased 10-fold, and was able to be reused for 10 cycles with 90% residual activity at 55 °C. 1,3-DAGs of lauric, myristic, palmitic, stearic, oleic and linoleic acid were prepared using the resulting immobilised enzyme, all with yields greater than 90%, and the reaction time was also greatly reduced.

Full text of DOI:10.1016/j.foodchem.2013.07.132

Food Chemistry 143 (2014) 319–324
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Highly efficient solvent-free synthesis of 1,3-diacylglycerols by lipase
immobilised on nano-sized magnetite particles
⇑
⇑
Xiao Meng, Gang Xu, Qin-Li Zhou, Jian-Ping Wu , Li-Rong Yang
Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, 1,3-DAGs (1,3-diacylglycerols) have attracted considerable attention as healthy components of
food, oil and pharmaceutical intermediates. Generally, 1,3-DAG is prepared by lipase-mediated catalysis
in a solvent free system. However, the system’s high reaction temperature (required to reach the reac-
tants’ melting point), high substrate concentration and high viscosity severely reduce the lipase’s activity,
selectivity and recycling efficiency. In this report, MjL (Mucor javanicus lipase) was found to have the best
performance in the solvent-free synthesis of 1,3-DAGs of several common commercial lipases. By cova-
lent binding to amino-group-activated NSM (nano-sized magnetite) particles and cross-linking to form
an enzyme aggregate coat, MjL’s specific activity increased 10-fold, and was able to be reused for 10
cycles with 90% residual activity at 55 °C. 1,3-DAGs of lauric, myristic, palmitic, stearic, oleic and linoleic
acid were prepared using the resulting immobilised enzyme, all with yields greater than 90%, and the
reaction time was also greatly reduced.
Received 23 March 2013
Received in revised form 8 June 2013
Accepted 29 July 2013
Available online 7 August 2013
Keywords:
1,3-Diacylglycerol
Mucor javanicus lipase
Regioselective
Immobilisation
Magnetite
Nano-sized
High viscosity
Solvent-free
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction
glycerolysis and partial alcoholysis tend to produce more MAG
(monoacylglycerol) and 1,2-DAG than 1,3-DAG (Fureby et al.,
1, 3-DAGs (1,3-diacylglycerols) are known to be minor compo-
nent of edible fats (D’Alonzo, Kozarek & Wade, 1982; Fureby, Tian,
Adlercreutz, & Mattiasson, 1997). Recent studies have claimed that
consumption of rich 1,3-DAG oil has beneficial effects on suppress-
ing the accumulation of body fat and preventing increased body
weight (Lo, Tan, Long, Yusoff, & Lai, 2008; Meng, Zou, Shi, Duan,
& Mao, 2004; Morita & Soni, 2009; Reyes et al., 2008; Yanai
et al., 2008). Additionally, 1,3-DAGs have attracted growing atten-
tion as intermediates for the synthesis of various compounds with
pharmaceutical applications. When esterified with 1,3-DAG at the
2-position hydroxyl group, drugs, such as Niflumic Acid (a channel
blocker), are often better absorbed and have fewer side effects than
in the original form (Berger & Schneider, 1993; Mantelli, Speiser, &
Hauser, 1985).
Due to their minor content in their natural form, the synthesis
of 1,3-DAGs becomes important. 1,3-DAGs can be prepared both
chemically and enzymatically through glycerolysis, partial alcohol-
ysis and esterification. The enzyme mediated processes, usually
carried out with lipase as a catalyst, have been found to be superior
to conventional chemical methods due to their mild reaction con-
ditions, higher yield and procedural convenience. Nevertheless,
1997; Garcia, Yang, & Parkin, 1996; Kristensen, Xu, & Mu, 2005a;
Kristensen, Xu, & Mu, 2005b). Therefore, the ideal procedure to ob-
tain a high yield of 1,3-DAG is through the esterification of glycerol
and FFA (free fatty acid).
Unfortunately, glycerol is poorly soluble in most of the common
hydrophobic organic solvents that lipase favours, such as n-hexane
and toluene. Moreover, in solvent systems, side reactions such as
acyl migration may occur and result in a reduction of the 1,3-
DAG purity. It has been reported that in t-butanol, both glycerol
and oleic acid are soluble, and their esterification catalysed by Nov-
ozym 435 (immobilised Candida antarctica lipase B produced by
Novozymes A/S) achieved a reasonable yield of 1,3-DAG (Duan,
Du, & Liu, 2010), but t-butanol could not dissolve most other FFAs,
especially saturated long-chain ones. Worse still, most common
commercial lipases other than Novozym 435 have notably low cat-
alytic activity in t-butanol.
From the above discussion, the ideal method for 1,3-DAG prep-
aration is clearly the solvent-free esterification of glycerol and
FFAs. However, a solvent-free glycerol/FFA system has high sub-
strate concentration and high viscosity, and requires a relatively
high reaction temperature (to reach the FFAs’ melting point to keep
the reaction liquid), which severely impact the lipase’s catalytic
activity, selectivity and recycling efficiency. Therefore, until now,
to the best of our knowledge, related studies have only solved
⇑
Corresponding authors. Tel./fax: +86 0571 87952363.
E-mail addresses: wjp@zju.edu.cn (J.-P. Wu), lryang@zju.edu.cn (L.-R. Yang).
0308-8146/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.07.132

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X. Meng et al. / Food Chemistry 143 (2014) 319–324
problems, such as operation conditions or reaction equilibrium
(Berger, Laumen, & Schneider, 1992; Rosu, Yasui, Iwasaki, & Ya-
mane, 1999; Watanabe et al., 2003), whilst research on the key
problem, the improvement of the heat resistance, activity and
operational stability in the system of solvent-free glycerol/FFA
esterification of the biocatalyst, has never been reported. In this
work, common commercially available lipases were screened,
and MjL (Mucor javanicus lipase) was selected because it performed
better. To enhance its performance further, MjL was covalently
bound to surface activated NSM (nano-sized magnetite) particles
and later cross-linked to form a CLEA (cross-linked enzyme aggre-
gate) coating structure. The resulting nano-sized magnetite immo-
bilised lipase was used as catalyst for preparing 1,3-DAGs of lauric,
tein in the supernatant was determined by the Bradford method
(Bradford, 1976) using BSA (bovine serum albumin) as a standard.
To investigate influence of pH’s on immobilisation, PBS solu-
tions at pH 5, 6, 7, 8 and 9 were used. To investigate protein con-
centration’s influence, a series of MjL solution were used, whose
concentration ranged between 0 and 16 (mg protein)mLꢀ1 were
used.
2.4. Esterification of the fatty acids
Esterification of free fatty acid and glycerol was used to prepare
1,3-DAG. The general equation of the reaction is as follows:
myristic, palmitic, stearic, oleic and linoleic acid (C12:0, C14:0, C16:0
,
C
18:0, C18:1 and C18:2), all with good yield (>90%) with shortened
RCOOH þ CH2OHCHOHCH2OH
! RCOOCH2CHOHCH2OCORð1; 3 ꢀ DAGÞ
reaction time (from 24 h to 8 h or less). The immobilised MjL was
able to be reused for 10 cycles at 55 °C with only a 10% loss of
activity. Thus, a highly efficient, widely usable and low-cost system
for the synthesis of 1,3-DAG has been established.
þ RCOOCH2CHOCORCH2OHð1; 2 ꢀ DAGÞ þ H2O
ð1Þ
where R – represents the carbon chain of fatty acids. The detail
reaction condition was: free fatty acid (100 mmol, approximately
20.0, 22.8, 25.6, 28.4, 28.2 and 28.0 g for lauric, myristic, palmitic,
stearic, oleic and linoleic acids, respectively), glycerol (50 mmol,
approximately 4.60 g), MjL (in native or immobilised form, 1 g),
and molecular sieves (4 Å, 10 g, for water removal) were adequately
mixed in a 50 mL conical flask at atmospheric pressure, and reacted
at 55 °C for lauric, oleic and linoleic acid, and 60, 65 and 70 °C for
myristic, palmitic and stearic acid (to reach the three fatty acids’
melting point), respectively, stirring at 200 rpm for 6–12 h. At
0.5 h of the reaction (during this time, conversion rate of FFA was
2. Materials and methods
2.1. Materials
GA (glutaraldehyde, 25% water solution), APTES ((3-aminopro-
pyl)triethoxysilane), glycerol, FFAs (free fatty acids: lauric, myris-
tic, palmitic, stearic, oleic and linoleic acid, all purity >98%) and
Candida rugosa lipase (aka CRL Type VII) were purchased from Sig-
ma-Aldrich Co.; lipases from Aspergillus niger, Pseudomonas fluores-
cens, M. javanicus, Rhizopus niveus, Burkholderia cepacia (aka Lipase
A, AK, M, N and PS, respectively) were purchased from Amano En-
zyme Inc.; native and immobilised lipase from Rhizomucor miehei
(aka Lipozyme RM and Lipozyme RM IM), together with immobi-
lised Candida antarctica lipase B (Novozym 435) were purchased
from Novozymes A/S. All other reagents referred in this article
were of analytical grade.
beneath 5%), 50 lL of the reactant was sampled to determine the
activity and selectivity.
2.5. Assay of lipase’s activity and selectivity
Esterification of glycerol and oleic acid (that is the case when
R- = CH3(CH2)7CH = CH(CH2)6CH2– in Eq. (1) was used as a model
reaction to assay lipase activity and selectivity. Activity of esterifi-
cation was defined as the initial consumption rate of oleic acid
2.2. Preparation of surface modified NSM
(
l
mol) per minute (when conversion was under 5%), that is, 1
Fe3O4 particles with a diameter of 10–20 nm were prepared by
coprecipitation (Molday, 1984), as shown in Fig. 1a-1, the NSM was
modified with APTES according to Ma’s work (Ma et al., 2003).
Afterwards, as shown in Fig. 1a-2, 1 g of the APTES modified
NSM, 2 mL of GA (25% aqueous solution) and 16 ml of PBS (phos-
phate buffer solution, 25 mmol Lꢀ1, pH = 7) were mixed and stirred
at 200 rpm and 25 °C for 2 h. The resulting solid was magnetically
UE = 1 (
l
mol oleic acid) minꢀ1. The specific activity of esterification
was based on the total protein content, that is, (specific activ-
ity) = (activity of esterification)/(total protein in the lipase powder
or loaded on the immobilisation carrier). Activity recovery = (total
esterification activity of the immobilised lipase)/(total esterifica-
tion activity of the lipase powder used for immobilisation)ꢁ100%.
The protein content of the lipase solution was determined accord-
ing to the Bradford method (Bradford, 1976), in which BSA was
used as protein standard. The amount of bound protein of immobi-
lised enzyme was indirectly determined by comparing the differ-
ence between the total amount of protein used and the amount
of protein in the washing solution.
separated by
a
strong magnet (N52 Nd-Fe-B magnet,
60 ꢁ 60 ꢁ 40 mm, Yantai Metal Material Company, Shanghai, Chi-
na), washed several times with PBS, and vacuum frozen for 12 h.
Finally, a dry powder of surface activated NSM was obtained.
2.3. Lipase immobilisation
In analogy to the ee value used for the description of enantio-
meric excess, a similar value for the regioisomeric excess was de-
fined: re = [(1,3-DAG)%ꢀ(1,2-DAG)%]/[(1,3-DAG)% + (1,2-DAG)%].
The larger the re value is, the more 1,3-selective the lipase is. FFA
and glycerides concentrations were determined by GC. The sample
was analysed using a HP 1890 gas chromatograph (Hewlett-Pack-
ard Co. CA, US) equipped with a FID detector and a DB-17ht capil-
lary column (30 m ꢁ 0.25 mm, Agilent Technologies Inc., CA, US).
Hydrogen was used as a carrier gas. Both the injector and detector
temperatures were 350 °C. The temperature program was as fol-
lows: initial temperature of 80 °C, then heating to 340 °C at
10 °C minꢀ1. The final temperature was 340 °C and was held for
15 min.
As shown in Fig. 1a-3, 1 g of activated NSM as prepared above
and 1000 mg of MjL crude powder (containing about 320 mg pro-
tein) was resolved in PBS (4 °C, 20 mL). The mixture was stirred
at 200 rpm at 4 °C for 12 h, at which point 0–2.4 mL of GA (25%
weight/weight) was added. This mixture was stirred for another
12 h to prepare CLEA as shown in Fig. 1b. Finally the insoluble solid
was magnetically separated from the mixture, washed several
times with PBS (pH = 7) to remove the unbound protein, and
freeze-dried for 12 h. The resulting immobilised lipase was kept
at 4 °C before use. The amount of lipase protein bound onto the
carrier was calculated by a mass balance. The amount of lipase pro-

X. Meng et al. / Food Chemistry 143 (2014) 319–324
321
Fig. 1. Process of immobilisation. (a) Activation of NSM and binding the lipase onto the carrier. (a-1) The nano-sized Fe3O4 particle was modified by APTES; (a-2) GA was
attached to the particle via the APTES branch; (a-3) lipase was immobilised onto the activated particle by covalent bonding of its surface amino group and the carrier’s
aldehyde group. (b) Cross linking and enzyme aggregate coating. (b-1) surface bonding; (b-2) cross-linking; (b-3) enzyme aggregate coating. In this figure, a circle with a
notch denotes an enzyme molecule, and the tiny dots with sticks around the molecule denotes amino groups.
2.6. Assay of the lipase operational stability
Table 1
Lipase screening.
The esterification activity of the lipase at 55 °C was determined
in batched reactions, with the operation parameters stated in Sec-
tion 2.4. After every cycle had finished, the old reactant and molec-
ular sieves were removed (for native enzyme, the reaction mixture
was centrifuged at 12,000 rpm for 10 min; for the immobilised en-
zyme, a strong magnet was applied for 10 min to completely pre-
cipitate the magnetic immobilised lipases), and fresh ones were
added for the next batch.
Enzyme
Source
Activity of
esterification
re
UE (g solid)ꢀ1
2.7
6.4
13.0
44.0
2.4
29.5
14.6
(%)
67
95
95
75
95
75
95
95
75
CRL Type VII
Lipase A
Lipase AK
Lipase M
Lipase N
Lipase PS
Lipozyme RM
Candida rugosa
Aspergillus niger
Pseudomonas fluorescens
Mucor javanicus
Rhizopus niveus
Burkholderia cepacia
Rhizomucor miehei
2.7. Assay of lipase thermal stability
Lipozyme RM IM Rhizomucor miehei (immobilised) 61.2
Novozym 435
Candia antarctica (fraction B,
92.4
One gram of native or immobilised MjL powder was dispersed
in 20 g oleic acid by stirring at 200 rpm at a 55, 65 and 75 °C for
12 h, afterwards, the solid was recycled by centrifugation or mag-
netic separation. The activity of the heat-treated enzyme was then
determined according to the method described in Section 2.5.
immobilised)
pases is 37 °C, whilst their enzymatic activity was assayed at 55 °C.
This temperature was too high for these lipases and inactivated
them. Moreover, in the solvent-free system, the substrate concen-
tration was very high, resulting in substrate inhibition. Therefore,
MjL was selected as the catalyst for 1,3-DAG preparation. It should
be noted that although the immobilised lipases had good activity
and at least acceptable selectivity, in the high-viscosity system
containing strong hydrophilic substrate, glycerol, the lipase protein
would desorbed from their carrier, causing the problem of mass
loss during reuse. Worse still, similar to the native lipases, these
immobilised lipases can be recycled from the reactant only by
high-speed centrifugation (12,000 rpm as stated in Section 2.6),
which is not a convenient way for separation. Besides, these
commercial immobilised lipases lacked potential of further
3. Results and discussion
3.1. Screening of commercial lipases
Esterification activity and regioisomer excess of common com-
mercial lipases in native or immobilised form were investigated for
use in 1,3-DAG solvent-free synthesis. As shown in Table 1, two
immobilised lipases (Lipozyme RM IM and Novozym 435) had
the highest activity. Amongst the native enzymes, M. javanicus li-
pase (MjL) had the best activity and not very low selectivity
(re = 75%). The lipases with good re values (90% or more) had very
low activity. This was because the optimal temperature for most li-

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X. Meng et al. / Food Chemistry 143 (2014) 319–324
enhancement via special immobilisation techniques such as cross-
linked enzyme aggregate and employing nano-sized magnetite
carrier.
for MjL (Ishihara, Okuyama, Ikezawa, & Tejima, 1975). As in
Fig. 2a, the protein binding rate at acidic or alkaline pH was greater
than that at neutral pH, as an aldehyde group of the carrier and
amino group of the protein tended to react in the presence of acid
or base. However, the specific activity at those pH values were the
lowest, potentially because many MjL molecules were deactivated
during the 12-hour-long immobilisation process at far-from-opti-
mum pHs.
A series of different concentrations of MjL were used for immo-
bilisation. The results are depicted as an adsorption isothermal
curve (Fig. 2b). From the figure, it can be observed that as the pro-
tein concentration reached approximately 16 mg mLꢀ1, covalent
3.2. Immobilisation of MjL
pH strongly affected the results of assays of enzyme immobili-
sation based on covalent binding. Native MjL powder was dissolved
in PBS (25 mmol Lꢀ1) at different pHs and mixed with the surface-
activated NSM for 12 h. After that process, the resulting powder
was separated magnetically and washed with PBS (25 mmol Lꢀ1
,
pH = 7.0) because neutral pH was reported to be the optimum pH
Fig. 2. Investigation of the immobilisation conditions. (a) pH influence. (b) Binding equilibrium curve of immobilisation. In this figure, the title of the horizontal axis,
[Protein], stands for the total protein concentration of the lipase used for immobilisation. (c) Effect of GA cross-linking: In this figure, the horizontal axis represents the ratio of
GA concentration vs. MjL protein concentration; both concentrations are in mg mLꢀ1. Meaning of the symbols: —d— bound protein, - -s- -adsorption rate, —j— activity,
- -h- - specific activity.

X. Meng et al. / Food Chemistry 143 (2014) 319–324
323
Fig. 3. Stability of the lipase. (a) Operational stability (at 55 °C) of the immobilised MjL and other commercial lipases: - -s- - native MjL (Lipase M), —d— immobilised MjL, —
j— immobilised CaL-B (Novozym 435), —N— immobilised Rml (Lipozyme RM IM). (b) Thermal stability of MjL in immobilised or native form: —d— Immobilised MjL (55 °C),
—j— immobilised MjL (65 °C), —N— immobilised MjL (75 °C), - -s- - native MjL (55 °C), - -h- - native MjL (65 °C), - -4- - native MjL (75 °C).
binding was saturated. It should be noted that in Fig. 3b, as bound
protein increased, the specific activity did not decrease accord-
ingly, indicating that the immobilisation method employed was
able to avoid mass transfer limitations, which is one of the great
advantages of nano-sized particles.
As Fig. 1b indicates, enzyme cross-linking and the aggregate
coating method was proposed to attach more molecules and aggre-
gates in the solution onto ‘seed’ enzyme molecules that had al-
ready been covalently attached onto the support. In our study, to
further improve the activity and stability of the immobilised MjL,
we attempted to prepare the cross-linked enzyme-aggregate deriv-
ative and investigated the characteristics of the immobilised prep-
aration. The experimental results (Fig. 2c) confirmed that as the
amount of cross-linker (glutaraldehyde) increased, the amount of
bound protein, together with esterification activity of the immobi-
lised MjL, increased also.
3.3. Stability of the immobilised MjL
As shown in Fig. 3a, the immobilised MjL retained 90% residual
activity after 10 reaction cycles, whilst native MjL (Amano Lipase
M) retained only 25% for 5 batches. The critical improvement of
the stability of the lipase might be the result of the following:
cross-linked enzyme molecules were tightly bound to each other;
covalent binding minimised the amount of enzyme released from
the carrier; and, the higher recycling efficiency of the magnetic
separation reduced the mass loss of the catalyst.
The esterification activity of the native and immobilised MjL
after several hours of heat treatment at 55, 65 and 75 °C in oleic
acid were determined (Fig. 3b). At 55 °C, the immobilised MjL
was able to keep 100% activity for 36 h compared with the full
deactivation of native MjL in less than 12 h. As the temperature
rose to 65 and 75 °C, even the immobilised MjL began to lose activ-
ity. Regardless, the thermal stability of the lipase was critically en-
hanced, enhancing the potential for DAG synthesis at high
temperatures.
Through immobilisation onto nano-sized magnetite particle in
optimum conditions (MjL (22 (mg protein) mLꢀ1), carrier
(1 g mLꢀ1), stirring 200 rpm for 12 h at 4 °C in PBS (25 mmol Lꢀ1
,
pH = 5), 12 h cross-linking with GA (40 mg mLꢀ1) at 4 °C and
200 rpm stirring), the MjL specific activity of esterification in-
creased 10-fold (from 0.133 to 1.42 UE (mg protein)ꢀ1), and re in-
creased from 75% to 90%. The activity recovery (esterification of
oleic acid) of the immobilisation was 285%.
3.4. Preparation of 1,3-DAGs using immobilised MjL
Using the immobilised MjL as the catalyst, 1,3-DAGs of lauric,
myristic, palmitic, stearic, oleic and linoleic acid were synthesised.
Table 2
Preparation of 1,3-DAGs catalysed by the immobilised MjL.
Free fatty acids
Reaction temperature
Reaction time
(h)
Conversiona
(%)
re
Glyceride compositionb
TAG
DAG
MAG
(°C)
(%)
(%mol)
(%mol)
(%mol)
By native MjLc
Lauric acid
Oleic acid
(C12:0
(C18:1
(C18:2
)
)
)
55
55
55
24
24
24
92
90
90
73
75
75
0.0
0.0
0.0
88.7
86.4
88.0
11.3
13.6
12.0
Linoleic acid
By Immobilised MjL
Lauric acid
Myristic acid
Palmitic acid
Stearic acid
(C12:0
(C14:0
(C16:0
(C18:0
(C18:1
(C18:2
)
)
)
)
)
)
55
60
65
70
55
55
6
9
12
12
12
12
97
96
94
92
90
91
90
90
91
91
92
92
0.0
0.0
0.0
0.0
0.0
0.0
99.0
98.9
98.0
95.0
93.3
94.4
1.0
1.1
2.0
5.0
6.7
5.6
Oleic acid
Linoleic acid
a
Conversion was calculated according consumption of fatty acid in the reactant.
TAG = triacylglycerol, DAG = diacylglycerol, MAG = monoacylglycerol. The ratio of tri-, di-, and mono-acylglycerols was calculated based on the molar percentage of total
b
glyceride. No triacylglycerol was detected in the analysis of the glyceride composition of the esterification product. This finding could be observed because in the reactant,
molar ratio of glycerol vs. FFA was set to 1:2, and TAG could not be generated in this condition.
c
There was no data on the esterification of myristic, palmitic and stearic acid catalysed by native MjL because the reaction temperature required by these FFAs was
sufficiently high that the native lipase would be inactivated during the reaction.

324
X. Meng et al. / Food Chemistry 143 (2014) 319–324
Berger, M., Schneider, M. P. (1993). Regioisomerically pure mono and
&
As shown in Table 2, all products’ FFA conversion, DAG percentage
from total glyceride and re value were greater than 90%. In con-
trast, the re of 1,3-DAG prepared with native MjL was only 75%.
The reaction time was reduced from over 24 h to less than 12 h.
These results indicated that the selectivity of the immobilised li-
pase was quite reasonable, and the very reaction conditions (the
glycerol/FFA solvent-free system) were quite suitable for highly
efficient 1,3-DAG synthesis.
diacylglycerols as synthetic building blocks. Fat Science Technology, 95,
169–175.
Bradford, M. M. (1976).
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microgram quantities of protein utilizing the principle of protein–dye binding.
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D’Alonzo, R. P., Kozarek, W. J., & Wade, R. L. (1982). Glyceride composition of
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preparation via esterification in t-butanol system. Process Biochemistry, 45,
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4. Conclusions
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213–222.
In this work, common commercial lipases were screened, and
M. javanicus lipase was found to have most acceptable perfor-
mance. Surface-activated nano-sized magnetite particle was em-
ployed as carrier for the immobilisation of MjL via covalent-
bonding. Further treatment by glutaraldehyde was employed to
form a cross-linked enzyme aggregate. The resulting nano-sized
magnetite immobilised lipase exhibited critical improvement in
esterification activity, regioselectivity and thermal/operational sta-
bility in a high-viscosity and high-temperature system for 1,3-DAG
preparation. Good yields of 1,3-DAGs of lauric, myristic, palmitic,
stearic, oleic and linoleic acid synthesised by the immobilised li-
pase were achieved with re > 90%, and the reaction time was signif-
icantly reduced. It can be concluded that using the immobilised
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The authors thank the National Basic Research Program of Chi-
na (973 Program, No. 2011CB710800), Hi-Tech Research and
Development Program of China (863 Program, 2011AA02A209),
National Natural Science Foundation of China (No. 20936002),
and the Science and Technology Planning Project of Zhejiang Prov-
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