255

Artifi cial Cells, Nanomedicine, and Biotechnology, 2013; 41: 255–258

Copyright © 2013 Informa Healthcare USA, Inc.

ISSN: 2169-1401 print / 2169-141X online

DOI: 10.3109/10731199.2012.731413

Giant vesicles as encapsulating matrix for stabilizing alcohol oxidase and as container for coupled enzymatic reactions

Sabitoj S. Virk 1,2 , Vishwa J. Baruah 1 & Pranab Goswami 1

1 Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India, and 2 Present address:

Tissue Modulation Laboratory, Department of Bioengineering, DSO (Kent Ridge) Building, NUS Tissue Engineering

Programme (NUSTEP), Singapore

Introduction

Th e ability of liposomes to mimic the behavior of natural cell

membranes has been exploited to develop liposome – based

constructs for various applications (Walde and Ichikawa

2001). Th e liposomal encapsulated enzyme system protects

the enzyme from external agents, such as proteases and

also prevents the enzyme from denaturation (Nasseau et al.

2001). Th e enzyme-encapsulated liposomes can be used

as a functional biocatalytic container in organic synthesis.

Various liposome-encapsulated enzymes have been pre-

pared and characterized for drug delivery, biocatalytic, and

synthetic biology applications (Walde and Ichikawa 2001,

Walde et al. 2009, Stano et al. 2011). Diff erent methods,

including rapid evaporation method, for the preparation

of enzyme containing lipid vesicles have been reviewed by

Walde and Ichikawa (Walde and Ichikawa 2001). We report

here the multilameller giant vesicles (GVs) prepared by rapid

evaporation method for encapsulating a broad substrate

specifi c alcohol oxidase (AOx) isolated from Aspergillus

terreus 6324 (Kumar and Goswami 2006). In general, the AOx

is a complex enzyme protein consisting of many subunits as

reported mostly from the fungal sources (Ozimek et al. 2005,

van Dijk et al. 2002). Th e enzyme is highly sensitive to ionic

strength and temperature (Kumar and Goswami 2008) and

hence stabilization of AOx is a prerequisite for its applica-

tion in biocatalytic process. We utilized the giant liposome

system not only to stabilize the enzyme but also to use it as

container to carry out a coupled reaction for detection of

alcohol in the sample.

Experimental

Chemicals L- α -Phosphatidylcholine ( � 99% TLC), Stearylamine

( � 99.0% GC), Cholesterol ( � 99%), Green fl uorescence pro-

tein (GFP), Peroxidase ( ~ 200 units/mg), ABTS [2,2 � - azino-

di-(3-ethylbenzthiazoline-6-sulphonic acid)], were obtained

from Sigma-Aldrich, India. DMSO (dimethylsulfoxide), BSA,

and alcohol substrates were obtained from Merck, India.

Isolation of AOx Th e AOx from Aspergillus terreus MTCC 6324 was isolated

following the reported procedure (Kumar and Goswami

2008). In brief, the harvested cells in 50 mM Tris-HCl buf-

fer, pH 8.0 (THB) was disrupted at 4 ° C. Th e AOx present in

20,000 � g supernatant of the disrupted cells homogenate

was precipitated by ammonium sulfate (35% w/v saturation)

and desalted against THB. Th e membrane bound AOx was

solubilized by CHAPS (0.5% w/v). Th e supernatant obtained

at 114000 � g was lyophilized to obtain powdered AOx

containing 130 U/mg-protein.

Preparation of composite enzyme/GFP containing liposomes Giant vesicles (GVs) were prepared by rapid evapora-

tion method (Moscho et al. 1996). A lipid composition of

Stearylamine/L- α -Phosphatidyl Choline/Cholesterol (1:4:5

mole ratio) was dissolved in 1 ml of chloroform in a 50 ml

round bottom fl ask. Th e aqueous phase containing suitable

concentration of AOx, or AOx, ABTS and HRP (composite

Abstract

Encapsulation of a multimeric alcohol oxidase (AOx) in giant

vesicles (GVs) was prepared by rapid evaporation method. The

specifi c activity (477 U/mg-protein) of the GV encapsulated AOx

was 3.5 fold higher than the free AOx. The half-life ( ~ 59.4 hours)

of the encapsulated AOx was 20-fold higher than the free enzyme

at 30 ° C. Further, the GV was used to encapsulate AOx and HRP

together as coupled reaction catalyst for chromogen-based optical

detection of alcohols. The results confi rmed potential application

of the GV as micro-dimensional container for enhancing stability,

activity and biosensing applications of AOx.

Keywords: ABTS , alcohol , alcohol oxidase , coupled reaction , giant

vesicles

Correspondence: Sabitoj Singh Virk, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. Tel: ( � 65)

93767932. E-mail: sabitoj@alumni.iitg.ernet.in

(Received 16 July 2012 ; revised 9 September 2012 ; accepted 10 September 2012)

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256 S. S. Virk et al.

enzyme) for coupled reaction or GFP in the respective buff er

was then added along the fl ask walls. For GFP encapsulation

THB were adjusted to 7.0. Th e organic solvent from the mix-

ture was removed by vacuum evaporator at 30 ° C. An opal-

escent fl uid containing GV obtained after the evaporation

was subjected to 15,000 � g for 45 min at 4 ° C to separate

un-capsulated enzyme/proteins from the liposomes. Th e

supernatant was removed and 5 mL THB was added to the

pellet to re-suspend the liposomes. Th is washing process

was repeated to ensure the prepared liposomes free from

un-encapsulated proteins, which was further verifi ed by

analyzing protein concentration/enzyme activity in each

extracting supernatant concomitantly. Th e encapsulated

enzyme/protein liposome preparation was fi nally sus-

pended in a 3 mL THB for storing at 4 ° C. Th e entrapment

percent (EP) was calculated as follows:

EP � (TE-FE)/TE � 100%.

Where, TE � Total enzyme, FE � Free enzyme

Th e AOx activity was measured using the spectropho-

tometric assay method (Kemp et al. 1988) using ABTS and

n -heptanol as substrate. Th e HRP-coupled assay was used

to monitor the production of H 2 O 2 at 405 nm for ABTS radi-

cal at 30 ° C ( ε 405 � 18,400 μ M � 1 cm � 1 ). Th e stoichiometric

equivalence of 1 mM ABTS with 0.5 mM oxidized substrate

was considered to measure the product. One unit (IU) of

enzyme was defi ned as the amount of enzyme activity pro-

ducing 1 μ mol of H 2 0 2 per min under the assay conditions.

Th e activity of the AOx in GV was measured by mixing an

aliquot of 0.5 mL (2500 GVs/ml) of AOx-GV with 5 mL of THB

containing HRP (1 mg/ml or 7 U/ml), ABTS (0.7 mg/ml) and

n-Heptanol (10 mM). Liposome encapsulated with BSA was

used as control for the experiments. Protein estimation was

done following Bradford method.

Results and discussions

Initially, GFP was encapsulated in the GVs and the image of the

GFP-GVs was captured by a camera (Nikon E8400) attached to

a microscope (Nikon E-200) under bright fi eld mode ( Figure

1A ) and also fl uorescence emission mode using a laser light of

488 nm ( Figure 1B ). Th e entrapment of GFP was clearly visible

from the specifi c fl uorescence emitted by the GVs. Th e major-

ity of the vesicles were spherical with sizes distributed largely

within 1 – 100 μ m (dia). Th e GVs count [suspension chamber

1 cm length � 1 cm breath � 3 mm high) was 5 GVs/ μ l at an

initial lipid concentration of 1.071 mg/ml-CHCl 3 . Th e number

and sizes of the GFP encapsulated GVs were constant for at

least three weeks when stored at 4 ° C. No aggregation of the

GVs was also observed during the period.

In our attempt to encapsulate AOx in GVs, an EP of 99.8

was achieved at the AOx protein of 0.84 mg/ml. Th e specifi c

activity of 477 U/mg-protein of AOx in the AOx-GV with sub-

strate n-heptanol was 3.5 fold higher than the specifi c activ-

ity of the free AOx (130 U/mg-protein). Th e reason for the

increased specifi c activity is attributed to the supporting role

of the lipoidic vesicle bilayer that provides the natural envi-

ronment to the enzyme for enhanced catalysis. Notably, AOx

is a membrane – bound enzyme that loses substantial activity

upon its dissociation from the microsomal membrane. Th e

effi ciency of liposomal encapsulation of proteins is generally

low ranging from ca. 5% to 20% for the enzymes of diff erent

sizes (Hwang et al. 2012). Th e exceptionally high level of

Figure 1. Th e image of GFP-GVs captured through a 40 × objective in a (A) bright fi eld mode and B: Fluorescence mode. C: Enzs-GV upon addition of n-heptanol. D: Image of multilamellar vesicle containing Enzs-GV. In 1C, 1D Scale Bar: 40 μ m. Each camera pixel represents 0.2 μ m in the images 1A, 1B.

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Giant vesicles as container for alcohol oxidase 257

entrapment of AOx in the GVs achieved through this inves-

tigation is thus a signifi cant breakthrough in encapsulation

of protein in liposomes. Th e high level of entrapment in the

present case is attributed to the lipoidal nature of the AOx

(Kumar and Goswami 2008) that facilitates avid binding of

the protein with the liposome matrix. Polymeric membrane

vesicles known as artifi cial cell, for the bio-encapsulating

enzymes have also been reported (Chang 2007). However,

studies on interaction between polymeric membranes and

the enzyme Alcohol oxidase need to be explored to under-

stand the stability of this enzyme within the polymeric

vesicles. Our attempt is to stabilize a massive multimeric

enzyme in a membrane that mimics native microsomal

membrane, hence an environment that would be the closest

mimic to the biological cell membrane i.e. a simple bilayer

lipid membrane was used. Th e half life (t1/2) of GV encap-

sulated AOx (calculated from 0.693/k where ‘ k ’ is the fi rst

order rate constant) was ~ 59.4 hours, whereas for the free

enzyme it was ~ 3 hours at 30 ° C and pH 8.0. While monitor-

ing the half-life of the GV encapsulated AOx the GVs count

(through microscopic observation) of the preparation was

compared with the original value and confi rmed that there

Figure 2. Spectrophotometric monitoring of the Enzs-GV catalyzed oxidation of n-heptanol. Th e Enzs-GV concentrations used were A � 100 μ l, B � 200 μ l, C � 400 μ l, D � 600 μ l, where the count of GV was 5/ μ l in THB. Th e concentrations of n-heptanol for the entire sample were fi xed (100 μ l) and the stock was prepared in DMSO.

Figure 3. Visible spectra for the AOx catalyzed oxidation of diff erent substrate alcohols (each 50 μ l in DMSO) added externally to Enzs-GV. A, methanol; B, butan-1ol; C, n-heptanol; D, R-(2)-octanol; E, 1- undecanol; F, 2-dodecanol.

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258 S. S. Virk et al.

stability of the AOx upon its encapsulation in the GVs was

also demonstrated. Further, the constructed GVs were used

as a container for AOx-HRP-coupled reaction to probe the

presence of alcohol in the samples using optical detec-

tion method. Th e fi ndings have opened up a new avenue

to explore GVs as a micro-dimensional container for high

molecular weight enzyme and coupled enzymes for stability

and biocatalytic reactions.

Acknowledgements

DBT, India provided JRF to V. Barua. Th e help rendered by

Seraj Ahmad, Mitun Chakraborty, Soma Sekhar Reddy,

Ankana Kakoti for purifi cation of AOx is acknowledged.

Declaration of interest

Th e authors report no confl icts of interest. Th e authors alone

are responsible for the content and writing of the paper.

References

Chang TMS . 2007 . Artifi cial cells: biotechnology, nanomedicine, regenerative medicine, blood substitutes, bioencapsulation, cell/stem cell therapy . In: Lim SC, Ed. Regenerative medicine, artifi cial cells and nanomedicine , vol. 1 . Hackensack, N.J.: World Scientifi c Publishers , pp. 299 – 332 .

Frimer AA . 1985 . Th e Semipermeability of biological membranes: an undergraduate laboratory experiment . J Chem Educ . 62 : 89 – 90 .

Hwang SY , Kim HK , Choo J , Seong GH , Hien TB , Lee EK . 2012 . Eff ects of operating parameters on the effi ciency of liposomal encapsula-tion of enzymes . Colloids Surf B Biointerfaces. 94 : 296 – 303 .

Johannes C , Majcherczyk A . 2000 . Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems . Appl Environ Microbiol . 66 : 524 – 528 .

Kemp GD , Dickinson FM , Ratledge C . 1988 . Inducible long chain alco-hol oxidase from alkane-grown Candida tropicalis . Appl Microbiol Biotechnol. 29 : 370 – 374 .

Kumar AK , Goswami P . 2006 . Functional characterization of alcohol oxidases from Aspergillus terreus MTCC 6324 . Appl Microbiol Bio-technol . 72 : 906 – 911 .

Kumar AK , Goswami P . 2008 . Purifi cation and properties of a novel broad substrate specifi c alcohol oxidase from Aspergillus terreus MTCC 6234 . BBA Protein Proteome. 1784 : 1552 – 1559 .

Moscho A , Orwar O , Chiu DT , Modi BP , Zare RN . 1996 . Rapid prepa-ration of giant unilamellar vesicles . Proc Natl Acad Sci USA. 93 : 11443 – 11447 .

Nasseau M , Boublik Y , Meier W , Winterhalter M , Fournier D . 2001 . Substrate-permeable encapsulation of enzymes maintains eff ective activity, stabilizes against denaturation, and protects against prote-olytic degradation . Biotech Bioeng. 75 : 615 – 618 .

Ozimek P , Veenhuis M , van der Klei IJ . 2005 . Alcohol oxidase: a complex peroxisomal, oligomeric fl avoprotein . FEMS Yeast Res. 5 : 975 – 983 .

Sol í s-Oba M , Ugalde-Sald í var VM , Gonz á lez I , Viniegra-Gonz á lez GJ . 2005 . An electrochemical – spectrophotometrical study of the oxidized forms of the mediator 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) produced by immobilized laccase . J Electroanal Chem. 579 : 59 – 66 .

Stano P , Carrara P , Kuruma Y , Pereira De Souza T , Luisi PL . 2011 . Com-partmentalized reactions as a case of soft-matter biotechnology: synthesis of proteins and nucleic acids inside lipid vesicles . J Mat Chem. 21 : 18887 – 18902 .

van Dijk R , Lahtchev KL , Kram AM , van der Klei IJ , Veenhuis M . 2002 . Isolation of mutants defective in the assembly of octameric alcohol oxidase of Hansenula polymorpha . FEMS Yeast Res. 1 : 257 – 263 .

Walde P , Ichikawa S , Yoshimoto M . 2009 . Th e fabrication and appli-cations of enzyme-containing vesicles . In: Ariga K, Nalwa HS, Eds. Bottom-Up Nanofabrication , vol. 2 . Los Angeles: American Scien-tifi c Publishers , pp. 199 – 221 .

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was no signifi cant degradation of the GVs, which further

indicates the high stability of the GVs used for the analysis.

Encapsulation of AOx within the vesicles led to localization

of the enzyme in hydrophobic bilayers that may protect and

stabilize the enzyme from rapid denaturation.

Th e Enzs-GVs were examined for their AOx catalytic

activity using n-heptanol as the sole substrate. On addition

of substrate n -heptanol the internal phase of the GVs turned

green with an intense bluish-green layer at the boundary of

the vesicle as shown in Figure 1C . Th e formation of the green

color is attributed to the production of ABTS. � radical as a

result of the AOx catalytic oxidation of the substrate alcohol

sequestered through the GV membrane. No such bound-

ary could be observed in control GV samples. Formation

of multilamellar Enzs-GVs was also observed as shown in

Figure 1D . From the results obtained it can be inferred that

AOx gets entrapped in the hydrophobic bilayer of the vesicle

and the bilayer is permeable to the long chain alcohol sub-

strate. Th e ABTS cation radical generally shows an absorp-

tion spectrum with fi ve maxima, at 214, 394, 414, 646 and 728

nm (Sol í s-Oba et al. 2005, Johannes and Majcherczyk 2000).

With increasing Enzs-GV concentration in the reaction

mixture containing n-heptanol, the absorbance of the ABTS-

specifi c peak at λ 340 nm was decreased with a concomitant

increase in the ABTS radical-specifi c absorption at λ 394,

414, 646, 728 nm ( Figure 2 ). An almost linear dependence

of concentration of Enzs-GV to [ABTS. + ] could be observed

from the inset of Figure 2 . Using ε 414 � 36000 M � 1 .cm � 1 for

ABTS . + , the sensitivity of the response was found to be 6.82

nM ABTS. + per Enzs-GV count. Th e fi ndings demonstrated

the occurrence of AOx-HRP coupled redox reaction inside

the GVs and hence provide evidence on the retention of HRP

and ABTS in the liposome matrix. ABTS cation radical is

impermeable to bilayers and so is ABTS (Frimer 1985).

AOx is reported to oxidize a broad range of alcohol sub-

strates with varying K m values (Kumar and Goswami 2008).

We tested various substrate namely, methanol, butan-1-ol,

n-heptanol, R-(2)-octanol, 2- dodecanol, and 1-undecanol

for the catalytic activity of the Enzs-GV. Spectrophotomet-

ric scans have shown the production of ABTS. + , thus imply

the activity realized with all the substrates used for the

assay. However, exceptionally high activity of Enzs-GV was

detected when n-heptanol ( curve C in Figure 3 ) was used as

substrate. Very low but signifi cant activity of the Enzs- GV

was detected for small chain (A, B) and long chain alco-

hols (D,E,F) as shown in the exploded spectra in the inset

of the Figure 3 . Th e activities of Enzs-GVs obtained for the

rest of the alcohol substrates were less by atleast 14-fold,

than the ones obtained for n-heptanol. Th e low activity of

AOx from A. terreus for the short chain alcohol substrates

is known (Kumar and Goswami 2006); however, the reason

for low activity of Enzs-GV for other long chain alcohols

namely, R-(2)-octanol, 2-dodecanol, and 1-undecanol is

not known.

Conclusion

Th e GV as an encapsulating matrix for the massive mul-

timeric AOx protein has been demonstrated. Enhanced

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