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Lipase immobilization through the combination of bio-imprinting and crosslinked protein-coated microcrystal technology for biodiesel production Jing Gao, Luyan Yin, Kai Feng, Liya Zhou, Li Ma, Ying He, Lihui Wang, and Yanjun Jiang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b03273 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Industrial & Engineering Chemistry Research

1

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Lipase immobilization through the combination of

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bio-imprinting

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microcrystal technology for biodiesel production

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Jing Gaoa,b, Luyan Yina, Kai Fenga, Liya Zhoua,b, Li Maa,b, Ying Hea,b, Lihui Wanga,

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Yanjun Jianga,b *

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a School of Chemical Engineering and Technology, Hebei University of Technology,

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8 Guangrong Road, Hongqiao District, Tianjin 300130, PR China

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b Hebei Provincial Key Lab of Green Chemical Technology and High Efficient

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Energy Saving, Hebei University of Technology, 8 Guangrong Road, Hongqiao

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District, Tianjin 300130, PR China

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*Corresponding Author: E-mail: [email protected]; Tel: 86-22-60204945

and

cross-linked

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14

15

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ACS Paragon Plus Environment

protein-coated


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Abstract：：

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A novel immobilized lipase of Burkholderia cepacia lipase (BCL) was prepared for

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the first time through the combination of bio-imprinting and cross-linked

4

protein-coated microcrystal technology (CLPCMC). P-benzoquinone was used as the

5

cross-linker for preparing the bio-imprinted CLPCMC (denoted as IM-CLPCMC).

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The morphology and the properties of the IM-CLPCMC were investigated. The

7

IM-CLPCMC could remain ca. 84% of initial activity after incubated in

8

n-hexane/ethanol system at 80 °C for 4 h, and remain 98% of initial activity after

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stored in n-hexane/ethanol for 120 h. These results indicated that IM-CLPCMC

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exhibited improved thermal stability and long-term stability in organic solvents.

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Furthermore, IM-CLPCMC was used as biocatalysts for producing biodiesel from

12

Jatropha curcas L. oil. Under the optimal conditions, the maximum yield of biodiesel

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obtained via transesterification could reach 95.0%. After seven cycles, more than 54.7%

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of biodiesel yield can be retained. Such results demonstrated that IM-CLPCMC is a

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promising catalyst for biotechnological applications.

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Keywords ： Bio-imprinted, Cross-linked protein-coated microcrystal, Lipase,

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Biodiesel, Enzyme immobilization

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1.

Introduction

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It is well known that a finite amount of fossil fuels could only last for a few more

3

decades 1. Thus, developing renewable energy is extremely necessary. Among the

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alternative engine fuels to petroleum diesel, biodiesel has attracted much attention 2-6.

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Some chemical technologies have been industrialized for biodiesel production, while

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it can be found numerous inescapable disadvantages in these steps, for example, high

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energy consumption and waste pollution produced by catalysts such as H2SO4 or

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NaOH 7-10. Moreover, the follow-up processes such as the recovery of the glycerol, the

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separation of the inorganic salts and water from the biodiesel, and the treatments of

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alkaline or acidic wastewater are complex and need more energy 9-11.

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Fortunately, lipase-catalyzed transesterification technology can overcome the

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drawbacks of the chemical technologies due to the mild reaction conditions 11, easy

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separation of glycerol, and an ability to obtain high biodiesel yield 12-14. Until now, to

14

make the catalytic activity and stability of lipases better15, there are many

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immobilized strategies such as non-covalent adsorption 16, entrapment 17, covalent

16

bonding

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simplicity, amazing efficiency, and cost effectiveness, have been developed as a very

18

efficient method for lipase immobilization 12, 19-20. However, PCMC exhibited some

19

unsatisfactory catalytic properties due to the enzyme leakage and the partial

20

miscibility in the presence of water21. To overcome these limitations, cross-linked

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protein-coated microcrystal (CLPCMC) had been designed for lipase immobilization,

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, and cross-linking

12

. Protein-coated micro-crystal (PCMC), due to its

3



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where glutaraldehyde (GA) was cross-linking agent 2, 22-23. The activity and stability

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of the obtained CLPCMC was superior to PCMC in organic solvents without water.

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There were many available cross-linking agents, and GA was the widely used in

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various fields

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immobilization.26-27. For instance, Wang

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cross-linker to acquire robust and stable cross-linked enzyme aggregates (CLEAs) of

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lipase. The lipase CLEAs prepared by p-benzoquinone exhibited better stability than

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that used GA as cross-linker, because the existence of C-O and C-N bonds make

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CLEAs stable in acidic condition. Additionally, p-benzoquinone can dissolve in

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organic solvent, thus can avoid the addition of water in the cross-linking process and

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then avoid the re-dissolution of precipitated enzyme and microcrystals. Thus

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p-benzoquinone used in preparing CLPCMC was expected to obtain more stable

13

biocatalyst with high activity recovery. Nevertheless, no literature concerning

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CLPCMC with p-benzoquinone as the cross-linker has been published until now.

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Bio-imprinting is an efficient strategy to improve the enzymatic activity and stability

16

in anhydrous organic media 28. The method of bio-imprinting about enzymes was

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reported firstly by Russell and Klibanov.29. The activity of enzyme with 100-times

18

enhancement was obtained in their work. After that, many reports demonstrated that

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bio-imprinting of enzymes is a rapid, simple, and effective way to obtain extremely

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robust biocatalyst in organic medium30-32. However, there is no report that uses

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bio-imprinted enzymes in the preparation of CLPCMC.

24-25

. Some other cross-linking agents have also been used for enzyme 27

and coauthors used p-benzoquinone as

4


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Thus, in this work, the combination of CLPCMC and bio-imprinting was developed to

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form a novel biocatalyst of bio-imprinted cross-linked protein-coated microcrystals

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(IM-CLPCMC). Burkholderia cepacia lipase (BCL) was chosen as model enzyme

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and p-benzoquinone was chosen as the cross-linker. The preparation and the catalytic

5

performances of IM-CLPCMC were investigated. The IM-CLPCMC was used as

6

catalyst for producing biodiesel from Jatropha curcas L. oil and ethanol. The

7

synthesis conditions were optimized. The results indicated that IM-CLPCMC showed

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high catalytic performance in transesterification reactions.

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2.

Materials and Methods

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2.1. Materials

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Burkholderia cepacia lipase (BCL) and Novozyme 435 were purchased from

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Novozymes (Denmark). Jatropha curcas L. oil was purchased from local market.

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Fatty acid ethyl esters were purchased from Sigma-Aldrich (USA). Fatty acids were

14

purchased from heowns (China). Ethanol, acetone and other reagents were purchased

15

from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents

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were used as received without further purification.

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2.2. Bio-imprinting procedure

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50 mg of BCL was added in 1 mL of phosphate buffer (100 mM, pH 7.0). A certain

19

amount of fatty acid was dissolved in 0.25 mL of acetone and then added to the BCL

20

solution and kept at 4 oC with constant shaking (150 rpm) for 1 h. 5



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2.3. Preparation of IM-CLPCMC and CLPCMC

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1.5 mL of saturated solution of K2SO4 was added to the above mixture to obtain a

3

combined solution. Subsequently, the above solution was added drop-wise to 20 mL

4

of acetone with continuous stirring. The PCMC were separated by centrifugation

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(6000 g, 2 min), then added 10 mL of acetone and a certain amount of cross-linking

6

agent (p-benzoquinone). The mixture was shaken with a speed of 200 r/min at 25 oC

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for 2.5 h. The mixture was washed with acetone thrice to remove the imprint molecule

8

and unreacted p-benzoquinone. After vacuum dried for 2 h, IM-CLPCMC was

9

obtained.

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CLPCMC was prepared according to the above method by using non-imprinted

11

lipase.

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2.4. Assay for catalytic activity of the biocatalysts

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Esterification activities were determined by incubating 5 mL of an n-octyl alcohol

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(0.2 mL) solution and lauric acid (100 mg) in iso-octane at 40 °C with magnetic

15

stirring. Reaction was started with the addition of desired amount of biocatalysts

16

(IM-CLPCMC or CLPCMC) with equal protein content.

17

After reacting for 2h, the residual lauric acid was monitored by titration with NaOH

18

solution (20 mM). One unit of esterification activity (U) was defined as the amount of

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enzyme required to consume 1 µmol of lauric acid per hour under assay conditions.

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2.5. Operational stability of immobilized biocatalysts 6


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To investigate the thermostability, IM-CLPCMC or CLPCMC (100 mg) were

2

incubated in n-hexane/ethanol system (molar ratio 1:2, 5 mL) at different

3

temperatures for 4 h. The residual esterification activity was measured. And the

4

activity at 30 oC was defined as 100%. The stability was explained by relativity

5

activity (%). Then, long-term stability was measured by incubating 100 mg of

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IM-CLPCMC or CLPCMC in n-hexane/ethanol (5 mL) at 40 oC for a certain period.

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At certain time intervals, the residual esterification activity was detected. The initial

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activity was used as control and defined as 100%. The mechanical stability was

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measured by incubating IM-CLPCMC, CLPCMC and Novozyme 435 with same

10

activity under shaking condition (200 rpm) for some time. The residual activity was

11

measured by esterification. The results were explained by relative activity (%).

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2.6. Reusability of IM-CLPCMC in biodiesel production

13

The reaction parameters of biodiesel production were optimized (Supporting

14

Information) and the optimum conditions were as follows: molar ratio of Jatropha

15

curcas L. oil to n-hexane 1:2, temperature 40 oC, molar ratio of ethanol to oil 4:1, the

16

dosage of IM-CLPCMC 20% (w/w IM-CLPCMC to oil). Subsequently, the

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reusability study of IM-CLPCMC, CLPCMC and Novozyme 435 with the same

18

activity of 160 U was tested by repeated use for biodiesel production under the

19

optimum conditions. The biocatalysts were recovered by centrifugation, washed with

20

n-hexane three times. After air-dried, they were resuspended in a fresh aliquot of

21

substrates for the next catalytic cycle.

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2.7. GC analysis

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Biodiesel yields were analyzed on a shimadzu GC-2010 Plus, equipped with a flame

3

ionization detector and a fused silica capillary column (RTX-1, 30 m×0.25 mm,

4

Agilent Technologies). The column oven temperature was firstly kept at 160 oC for 2

5

mins. Subsequently, the temperature was heated to 220 oC at a rate of 15 oC/min. After

6

2 mins, The column oven temperature was increased to 280 oC at a rate of 8 oC/min

7

and then maintained for 1 min. The injector temperature was set at 240 oC and

8

detector temperature was at 280 oC. Nitrogen was chosen as the carrier gas at a

9

constant pressure of 65.3 kPa with linear velocity at 29.1 cm/sec. The amount of

10

biodiesel in the reaction mixtures was determined by comparing the retention times

11

and peak areas of standard ester peaks.

12

2.8. Characterization

13

The Nova Nano SEM450 field-emission microscope with an accelerating voltage of 1

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kV was used to obtain scanning electron microscopy (SEM) images. A Leica TCS

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SP5 optical microscope with excitation wavelength of 488 nm and emission

16

wavelength of 525 nm was used to obtain confocal laser scanning microscopy (CLSM)

17

micrographs. Lipases were labeled with fluorescein isothiocyanate (FITC) and then

18

used to prepare IM-CLPCMC for detecting.

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3.

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3.1. Optimizing the preparation conditions of IM-CLPCMC and CLPCMC

Results and Discussion

8


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Primary experiments were conducted to determine the amount of crosslinking agent

2

for the CLPCMC preparation, and the results were shown in Table 1. An obvious

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decrease of specific activity for the CLPCMC was observed with the increase of the

4

given concentrations of cross-linker solution from 5 mM to 20 mM. The reason might

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be that a high content of cross-linker could damage enzyme structure and deactivate

6

enzyme25. Subsequently, the mechanical stabilities of CLPCMC with different

7

cross-linker concentration were investigated with constant shaking speed of 200 rpm

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for certain time and the residual activity was measured at different time intervals. As

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shown in Fig. 1, CLPCMC prepared with 10 and 20 mM cross-linker could retain

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above 98% of its initial activity after 10 hours, while the rest one lost nearly 30% of

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its initial activity under the same conditions. The better performance can be explained

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by that the sufficient multi-point covalent linkages make CLPCMC as robust ‘solid

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biocatalyst’ to resist the external stlimuli27. Based on the above results, 10 mM of

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cross-linker was adopted to prepare CLPCMC and IM-CLPCMC in subsequent

15

experiments.

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The preparation of IM-CLPCMC was further studied and the procedure was

17

optimized by tuning the amount of fatty acid. Fig. 2 showed that the imprinting

18

process indeed enhance the activity of the biocatalysts. An evident increase of activity

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of IM-CLPCMC was observed with the increase of the given amount of n-decylic

20

acid from 0 to 0.05 mmol. While further increase of the amount of n-decylic acid to

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0.2 mmol, the relative activity remained almost constant. Thus, 0.05 mmol of fatty

22

acid was used for further experiments. To study the influence of fatty acids with the 9



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1

carbon chain length on the activity of the lipase, BCL was imprinted with fatty acids

2

to fabricate IM-CLPCMC. The specific activity of each biocatalyst was evaluated

3

through esterification and the results were shown in Table 2. In comparison with

4

non-imprinted BCL, the specific activities of IM-CLPCMC were significantly

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enhanced in range of 25.4%-53.8%, respectively. The molecular mechanism of the

6

bio-imprinting on improving enzyme’s performance was as follows. After the

7

combination of lipase’s active site with the imprint molecule in an aqueous solution,

8

small conformational changes was considered to happen. When the imprint molecule

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is washed away by an essentially anhydrous organic solvent, the lipase cannot return

10

its former conformation because of the rigid structure. This bio-imprinting process

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could make the three-dimensional structure of the lipase keep ‘frozen’ in a modified

12

form and then improve lipase’s catalytic performance in anhydrous or microaqueous

13

organic solvents30. Additionally, the activity enhancement was related to the carbon

14

chain length, which was consistent with the previous report 30. As can be seen in

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Table 2, BCL imprinted with n-hexylic acid (a C6-acid) had a very slight activity

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enhancement, but the activation effect of n-decylic acid (a C10-acid) was significant.

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The reasonable explanations of the activity enhancement of the IM-CLPCMC

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imprinted with n-decylic acid may be attributed to two reasons: one is the similarity to

19

the substrate of enzyme and the suitable solubility of n-decylic acid in the aqueous

20

phase. The other is the formation of lipase IM-CLPCMC with a activated

21

conformation that is favorable for catalysis 33. Therefore, n-decylic acid was chosen as

22

imprint molecule to fabricate IM-CLPCMC in the subsequent experiments. 10


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3.2 Operational stability of immobilized lipase

2

Thermostability: The thermal stabilities of IM-CLPCMC and CLPCMC were

3

described in Fig. 3. The activity of the two biocatalysts kept at 30 oC was defined as

4

100%. The two biocatalysts showed highest activities at 40 oC and the activities were

5

decreased with increasing the incubation temperature in n-hexane/ethanol system (Fig.

6

3a). Compared to IM-CLPCMC, a faster inactivation rate was observed for CLPCMC

7

at the tested temperatures. When temperature was changed to 80 oC, IM-CLPCMC in

8

n-hexane/ethanol remained 84% of initial activity, while only 63% of initial activity

9

was kept for CLPCMC after 4 h. Subsequently, CLPCMC lost about 55% of its

10

original activity after 8 h, while IM-CLPCMC could retain more than 65% of its

11

activity under the same condition (Fig. 3b). Thus, the halftime of CLPCMC and

12

IM-CLPCMC were nearly about 7 h and 11 h incubating in n-hexane/ethanol system

13

at 80 oC, respectively. The higher activity retention for IM-CLPCMC than that of

14

CLPCMC can be ascribed to the fact that molecular bio-imprinting was an effective

15

method to improve enzymatic activity and stability in organic media 33.

16

Organic solvent stability: Long-term stabilities of IM-CLPCMC and CLPCMC were

17

also investigated. The initial activities of IM-CLPCMC and CLPCMC were defined

18

as 100%. As shown in Fig. 4, after incubated in n-hexane/ethanol system for 120 h,

19

the IM-CLPCMC and CLPCMC retained nearly 98% and 90% of their initial

20

activities, respectively. All the results indicated that there were slight improvements of

21

thermal stabilities and long-term stabilities through imprinting process. It is

11



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1

interesting to note that hyper activation was observed for the immobilized lipase in

2

n-hexane/ethanol for a period time. The organic solvent system could facilitate the

3

conformational change of lipase hyper activation by keeping water layer on the

4

surface 34-35.

5

3.3 Characterization of IM-CLPCMC

6

SEM was used to investigate the physical characteristics of IM-CLPCMC. As shown

7

in Fig. 5a, the IM-CLPCMC particles were in the form of aggregate of crystals and

8

the size of the IM-CLPCMC was about 1~2.5 µm in diameter, which was smaller than

9

the previously reported PCMC-lipase 19. The surface of IM-CLPCMC was rough

10

indicating lipase molecule aggregation on the salt crystals. The overall surface of

11

IM-CLPCMC was different to the K2SO4 salt (Fig. 5b).

12

To further confirm the lipase was immobilized on the surface of the salt crystals,

13

lipase molecules were labeled with fluorescein isothiocyanate (FITC) before the

14

formation of IM-CLPCMC. Confocal image (Fig. S1) showed that the FITC-labeled

15

BCL molecules were distributed on the microcrystals homogeneously, indicating the

16

successful immobilization of BCL.

17

3.4. Reusability of IM-CLPCMC in biodiesel production

18

Jatropha curcas L. oil and ethanol was selected to synthesize biodiesel and the

19

reaction conditions were optimized (Fig. S2-S5). Under the optimal conditions, the

20

reusability of IM-CLPCMC was studied. As control experiments, CLPCMC and 12


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1

Novozyme 435 were used to synthesize biodiesel at the same conditions. Fig. 6

2

showed that the maximum yields of biodiesel (based on Jatropha curcas L. oil) were

3

96.4%, 95.0%, and 85.7% for the first run that catalyzed by Novozyme 435,

4

IM-CLPCMC and CLPCMC, respectively. The improved yield catalyzed by

5

IM-CLPCMC in comparison with CLPCMC may due to the activation that caused by

6

the bio-imprinting procedure 30. After seven cycles, the biodiesel yields that catalyzed

7

by Novozyme 435, IM-CLPCMC and CLPCMC dropped to 79.8%, 54.7%, and

8

47.2%, respectively. The results indicated that Novozyme 435 exhibited the best

9

catalytic activity and recycling stability. IM-CLPCMC showed enhanced catalytic

10

performance over time when it was compared with CLPCMC. The activity loss for

11

IM-CLPCMC and CLPCMC in cycles might be the mechanical inactivation caused

12

by shearing force, as well as adverse factors such as methanol deactivation and the

13

loss of lipases during centrifugation and wash procedures21, 36-37. In order to confirm

14

the reason, the mechanical stabilities of IM-CLPCMC, CLPCMC and Novezyme 435

15

were measured by continuous shaking (200 rpm) for 60 h in n-hexane/ethanol system

16

(Fig. 7). CLPCMC lost 33% of its initial activity after 60 h, while IM-CLPCMC

17

could retain 78% of its initial activity. Novozyme 435, a high enzyme loading located

18

in the acrylate-based polymer Lewatit VP OC 1600

19

activity. The results indicated that the mechanical stability of Novozyme 435 was

20

better than that of IM-CLPCMC. Thus, the mechanical stability of the IM-CLPCMC

21

still requires better understanding and further improvement.

38

, retained 86% of the original

13



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Page 14 of 34

1

Subsequently, the comparison of biodiesel production using IM-CLPCMC and several

2

other lipase systems in related studies for the transesterification is shown in Table 3.

3

Using the other immobilized BCL system, a 98% yield of biodiesel could be reached

4

with a process time of 48 h. While IM-CLPCMC gave a yield of 95% for biodiesel

5

production only in 8 h. Thus, the short reaction time and high biodiesel yield obtained

6

in this work could confirm the advantages of the IM-CLPCMC though the different

7

feedstocks and reaction conditions were adopted in the literatures. All the results

8

indicated

9

transesterification reactions through the combination of CLPCMC and bio-imprinting.

10

It should be noted that the lipase used for preparing IM-CLPCMC in this study was

11

Burkholderia cepacia lipase, which was different from Novozyme 435 (Candida

12

antarctica lipase). Compared to Novozyme 435, the preparation of IM-CLPCMC was

13

easy, fast and cheap 39. The low cost and high catalytic efficiency made IM-CLPCMC

14

a promising biocatalyst for enzymatic synthesis of biodiesel compared to Novozyme

15

435 and other immobilization methods.

16

4. Conclusions

17

The successful preparation of immobilized lipase through the combination of

18

bio-imprinting and cross-linked protein-coated microcrystal technology has been

19

demonstrated for the first time. The immobilized lipase IM-CLPCMC was used for

20

producing biodiesel from Jatropha curcas L. oil through transesterification. Various

21

parameters of biodiesel production were optimized, and the highest yield of biodiesel

that

IM-CLPCMC

showed

higher

catalytic

14


performance

in

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1

could reach 95.0%. The biodiesel yields catalyzed by IM-CLPCMC were

2

considerably enhanced compared to CLPCMC. Thus, bio-imprinted cross-linked

3

protein-coated microcrystals can be potential biocatalyst for industrial production of

4

biodiesel.

5

Supporting Information

6

CLSM of IM-CLPCMC, effect of the molar ratio of oil/n-hexane on biodiesel yield,

7

effect of temperature on the yield of biodiesel, effect of ethanol/ oil molar ratio on the

8

yield of biodiesel, effect of the dosage of IM-CLPCMC on yield of biodiesel. This

9

material is available free of charge via the Internet at http://pubs.acs.org.

10

Notes

11

The authors declare no competing financial interests.

12

Acknowledgements

13

This work was supported by the National Nature Science Foundation of China (Nos.

14

21276060, 21276062, 21106164 and 21576068), the Natural Science Foundation of

15

Tianjin (13JCYBJC18500), the Natural Science Foundation of Hebei Province

16

(B2015202082 and B2016202027), the Science and Technology Research Project for

17

Colleges and Universities in Hebei Province (YQ2013025) and Tianjin City High

18

School Science & Technology Fund Planning Project (20140513).

19

15



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10

21



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1

Figure captions

2

Figure 1 Effect of cross-linker concentration on stability of the CLPCMC

3

Figure 2 Effect of fatty acid amount on activity of the IM-CLPCMC

4

Figure 3 Thermal stabilities (a) and thermal stability at 80 oC (b) of IM-CLPCMC

5

and CLPCMC

6

Figure 4 Long-term stabilities of IM-CLPCMC and CLPCMC

7

Figure 5 SEM images of (a) IM-CLPCMC and (b) K2SO4

8

Figure 6 Reusability of biocatalysts in biodiesel production (The reaction conditions:

9

Jatropha curcas L. oil 500 mg, molar ratio of ethanol to oil 4:1, 20% of biocatalyst

10

dosage to oil content, molar ratio of oil to n-hexane 1:2, 40 oC)

11

Figure 7 Mechanical stabilities of IM-CLPCMC, CLPCMC and Novozyme 435

12

22


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1

2

Tables

Table 1 Effect of cross-linker concentration on activity of the CLPCMC

Cross-linker concentration(mM)

Specific activity(µmol/h•g CLPCMC)

5

1155±29

10

1038±32

20

941±26

3

4

23



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Page 24 of 34

1

Table 2 Influence of bio-imprinting with fatty acids on the activity of the

2

IM-CLPCMC

Fatty acids Specific

Non-imprinted n-hexylic

activity

n-caprylic acid

n-decylic acid

lauric acid

1538±11

1597±26

1509±17

IM-CLPCMC acid

U(µmol/h•g 1038±31

1302±26

IM-CLPCMC)

3

24


Page 25 of 34

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1

Table 3 Comparison of the biodiesel production conditions and performance of

2

IM-CLPCMC with other immobilized BCL systems reported in the literatures Immobilization methods

feedstocks

alcohols

molar ratio alcohol/oil

Temperatur e (oC)

Reaction time

Yield s

fixed-bed reactor

soybean oil

alcohol

3:1

50

46

95

40

Adsorption

babassu oil

ethanol

7:1

39

48

98

41

Adsorption

olive oil

alcohol

4:1

25

12

70

42

Adsorption

olive oil

ethanol

4:1

30

30

90

43

CLPCMC

waste cooking oil

alcohol

3:1

40

9

72

21

IM-CLPCMC

Jatropha curcas L oil

ethanol

4:1

40

8

95

3

25


References

this work


1

Figures

120

Relative activity(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

100 80

40 20

2 3 4

5mM 20mM 10mM

60

0

2

4

6

8

10

Time(h) Figure 1

26


Page 26 of 34

Page 27 of 34

1

120 105

Relative activity(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 75 60 45 30 0.00

0.10

0.15

0.20

Amount of fatty acid (mmol)

2 3 4

0.05

Figure 2

27



Relative Activity(%)

160

a

120

80

IM-CLPCMC CLPCMC

40 30

40

50

60

70

b

100 80 60 40

IM-CLPCMC CLPCMC

20 0

2 3 4

80

Temperature(°C)

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2

4

6

8

10

12

Time(h) Figure 3

28


Page 28 of 34

Page 29 of 34

150


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


120

90

60

IM-CLPCMC(n-hexane/ethanol) CLPCMC(n-hexane/ethanol)

30 0

8

16

24

48

72

120

Time(h)

1 2 3

4

Figure 4

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

2

3

Figure 5

4

30


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1

2 3

Figure 6

31



100


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

90 80 70

Novozyme 435 IM-CLPCMC CLPCMC

60 50 0

1 2 3

10

20

30

40

50

60

Time(h) Figure 7

32


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1

33



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