COVALENT IMMOBILIZATION OF BOVINE PHOSPHOLIPASE A2

Journal of Food Biochemistry

29

(2005) 1–12.

All Rights Reserved.

©

Copyright 2005, Blackwell Publishing

1

COVALENT IMMOBILIZATION OF BOVINE PHOSPHOLIPASE A

2

SEUNG-HEE NAM and MARIE K. WALSH

1

Department of Nutrition and Food SciencesUtah State University

Logan, UT 84322-8700

Received for Publication October 16, 2003 Accepted for Publication February 2, 2004

ABSTRACT

Bovine phospholipase A

2

(PPLA

2

) was immobilized onto controlled poreglass (CPG) beads using four different immobilization methods. PPLA

2

wasimmobilized directly to CPG using glutaraldehyde without and with reductionby sodium borohydride (PP1 and PP2). Whey protein isolate was directlyimmobilized to CPG as a spacer followed by immobilization of PPLA

2

withoutand with reduction (PP3 and PP4). Immobilized enzyme samples were char-acterized with respect to total amount of protein immobilized, activity with afluorescent substrate and stability over 3 weeks. Among the methods, PP2 andPP4 showed the highest enzyme activity. All methods but PP2 showed asignificant decrease in enzyme activity over 3 weeks. Enzyme immobilized bytwo methods (PP2 and PP4) were compared with soluble enzyme for thehydrolysis of egg phospholipids. Soluble and immobilized enzyme (PP4)resulted in similar free fatty acid values.

INTRODUCTION

Immobilized enzymes are widely used in the food industry and offerseveral advantages over bulk or free enzyme preparations. Advantages includegreater productivity of the enzyme, automation and continuous processing,precise control of the extent of reaction, easy product recovery and the enzymedoes not contaminate the final product (Swaisgood and Horton 1989).

Examples of immobilized enzymes for food applications include glucoseisomerase, beta-galactosidase, proteases and lipases. Immobilized glucoseisomerase is used in the production of high fructose corn syrup because of thehigh costs associated with purification of this enzyme from yeast sources.Immobilized beta-galactosidase is employed for the hydrolysis of lactose insweetened dairy products (Heng and Glatz 1994). Proteases such as trypsin

Blackwell Science, LtdOxford, UKJFBCJournal of Food Biochemistry0145-8884Copyright 2005 by Food & Nutrition Press, Inc., Trumbull, Connecticut.2005291112Short Note

IMMOBILIZATION OF PHOSPHOLIPASE A

2

S.-H. NAM and

M.K. WALSH

1

Corresponding author. TEL: 1-435-797-2177; FAX: 1-435-797-2379; EMAIL: [email protected]

2 S.-H. NAM and M.K. WALSH

(Huang

et al

. 1999; Wang

et al

. 1999) and pepsin (Katchalski-Katzir 1993)are currently being used in an immobilized form for the limited hydrolysis ofmilk and wheat proteins. Improving the characteristics of complex fats can bedone with phospholipase A

2

(PPLA

2

), which selectively hydrolyzes the esterlinkage at the sn-2 position of phospholipids forming lyso-compounds. Thisbioconversion is important in food science because lysophospholipids arestrong bioemulsifiers, enhancing emulsion stability by improving solubility ofhydrophobic and/or hydrophilic compounds in oil and water phases (Madoery

et al

. 1995).The two methods currently used to immobilize phospholipase A

2

arenoncovalent adsorption and covalent cross-linking. Noncovalent adsorption isthe simplest and most economical method, but usually results in high enzymeleaching from the support matrix (Madoery

et al

. 1995, 1999). The result isa loss of enzyme productivity and contamination of the final product. Covalentimmobilization does not involve enzyme leaching from the matrix, but thismethod is more complex and expensive (Ferreira

et al

. 1993, 1994; Shen andCho 1995; Chen and Chen 1998).

Lipase, covalently immobilized using glutaraldehyde, retained a signifi-cant amount of activity with minimal leaching of the enzyme into the finalproduct (Rucka and Turkiewica 1989; Rucka

et al

. 1990; Cho and Rhee 1993).Immobilization techniques incorporating a linker molecule have previouslybeen more successful than direct coupling to the support, and treatment withsodium borohydride has been shown to increase stability in glutaraldehyde-immobilized proteases (Li and Walsh 2000).

Immobilization methods suitable for food applications are limited tothose employing techniques with GRAS (generally recognized as safe) status.Covalent attachment of PPLA

2

to solid supports has involved the use of toxicchemicals such as carbodiimide and hexane (Ferreira

et al

. 1993, 1994; Chenand Chen 1998). Here, we produced immobilized PPLA

2

matrices usingglutaraldehyde, which does have GRAS status (Cho and Rhee 1993).

The objectives of the present study were (1) to develop a covalentlyimmobilized PPLA

2

using glutaraldehyde with or without a linker protein(whey protein isolate) and a reducing agent (sodium borohydride) and (2) tocompare soluble with immobilized enzyme activity using a fluorescent sub-strate and liquid eggs.

MATERIALS AND METHODS

Materials and Chemicals

PPLA

2

(Bovine Pancreas, EC 3.1.1.4) was obtained from M.G. Wald-baum (Gaylor, MN). Other reagents included 3-aminopropyltriethoxylsilane


2

3

(Acros, Geel, Belgium); glutaraldehyde, 25%, Grade II, and methyl sulfoxide(Sigma Chem. Co., St. Louis, MO); sodium borohydride (Fisher Scientific,Pittsburgh, PA); and whey protein isolate (WPI) containing 90% protein(Glanbia, Twin Falls, ID). Support material was controlled pore glass (CPG),180–840 microns, 2191 Å average pore diameter (CPG, Inc., Lincoln Park,NJ). Fluorescent substrate specific for PPLA

2

activity [2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) hexanoyl-1-hexadecanoly sn-glycero-3-phos-phocholine (NBD C

6

-HPC)] was purchased from Molecular Probes (Eugene,OR).

Enzyme Immobilization

Acid-cleaned CPG-beads were silanized with 3-aminopropyltriethoxysi-lane according to Walsh and Swaisgood (1993) and activated using 2% (v/v)glutaraldehyde in 50 mM sodium phosphate buffer, pH 6.5 for 1 h at roomtemperature. Excess reagent was washed off with distilled water as describedby Li and Walsh (2000). PPLA

2

was coupled to the activated beads by directcovalent attachment or using cross-linker proteins (WPI) as described by Liand Walsh (2000). Aliquots of both samples were further reacted with sodiumborohydride to compare the influence of reducing on enzyme activities(Fig. 1).

For direct immobilization, 10 mL enzyme solution (3 mg protein/mL)was added to 1 g beads and allowed to react at room temperature for 4 h onan orbital shaker (PP1). Enzyme-coupled beads were reduced with 6.25 mgsodium borohydride in 10 mL water for 30 min (PP2). Beads were washedwith 10 vol of 1 M NaCl in 50 mM sodium phosphate buffer, pH 6.5 to removefree enzyme (Li and Walsh 2000). For immobilization involving a cross-linker,a solution of 100 mg WPI in 25 mL 50 mM sodium phosphate buffer, pH 6.5,was mixed with the glutaraldehyde activated beads for 1 h at room tempera-ture. Excess protein was removed with distilled water and the WPI-coatedbeads were reduced by the addition of 10 mL water containing 6.25 mgsodium borohydride. The reduced, WPI-coated beads were activated with 2%glutaraldehyde as described above. After completely washing off excess glu-taradehyde with 10 vol of 50 mM sodium phosphate buffer, pH 6.5, 10 mL ofenzyme solution (3 mg protein/mL) was added to 1 g beads and allowed toreact at room temperature for 4 h (PP3). Sodium borohydride (6.25 mg) in10 mL of 50 mM phosphate buffer, pH 6.5, was added slowly to the enzyme-coupled beads before washing with 10 vol of 1 M NaCl in 50 mM sodiumphosphate buffer, pH 6.5, to remove free enzyme (PP4).

The beads were assayed for immobilized protein after immobilizationwas complete. Total amount of protein immobilized onto the CPG was deter-mined directly by a dye-binding assay as described in Bond

et al

. (1992).


Volumes of 50

m

L (PP1, PP2) and 20

m

L (PP3, PP4) immobilized beads wereassayed for total protein and reported as mg protein/g dry beads.

After completion of enzyme activity studies, noncovalently bound pro-tein was desorbed from 50

m

L (PP1, PP2) and 20

m

L (PP3, PP4) immobilizedmatrices using 1% SDS and assayed using the Bio-Rad Protein Assay Kit.

FIG. 1. FOUR DIFFERENT IMMOBILIZATION METHODSCPG, controlled pore glass; GA, glutaraldehyde; WPI, whey protein isolate; NaBH4, sodium

borohydride; NaN3, sodium azide.

React w/ 2% GA

Add enzyme(30 mg/g beads)

4 g CPG beads

Degas and react w/ 2% GA

Divide into 4 equal portions

PP1 PP2 PP3 PP4


Add WPI protein (100 mg/g beads)

Reduce w/ NaBH4

(6.25 mg/g beads)Reduce w/ NaBH4

(6.25 mg/g beads)

Add WPI protein (100 mg/g beads)

Reduce w/ NaBH4

(6.25 mg/g beads)

Wash in 1M NaCl & Store in reaction Buffer w/ 0.02% NaN3


Reduce w/ NaBH4

(6.25 mg/g beads)

React w/ 2% GA



2

5

Proteins desorbed from the matrices are classified as noncovalently boundenzyme.

Enzyme Activity

Immobilized and soluble enzyme activities were determined using afluorescent substrate (NBD C

6

-HPC) or liquid egg yolks. The fluorescentsubstrate (NBD C

6

-HPC) in 50 mM HEPES buffer (pH 7) was added to UV-pass fluorescent cuvettes containing phospholipase-immobilized glass beadsin 10 mM Tris buffer, pH 8, 100 mM KCl, 4 mM CaCl

2

(with modificationsdescribed by Moreau 1989). The absorbance maximum was 466 nm with anemission of 533 nm. The final concentration of substrate was 10

m

M. Thechange in fluorescence was measured every 5 min for 60 min and the slopeof this plot resulted in an activity unit of

D

RFU/min. For each aliquot of beads(50

m

L of PP1, PP2 and 20

m

L of PP3, PP4) assayed for activity, the dryweight of the beads was also determined to obtain an activity value per grambeads. Activity is expressed as relative fluorescence units

D

RFU/min/g beads.After each 5 min activity measurement, the cuvettes containing the beads wereinverted twice prior to the next measurement. Soluble and immobilizedenzymes were stored for 3 weeks in 10 m

M

Tris (pH 8), 100 KCl, 4 m

M

CaCl

2

buffer containing 0.02% NaN

3

at 4C.Enzyme activity with liquid egg yolks was determined using Easy Eggs,

pasteurized liquid egg product (M.G. Waldbaum, Gaylor, MN). Immobilizedor soluble enzyme was added to 5 g of liquid eggs in 150 mL, Teflon-lined,screw-capped tube and incubated at room temperature with shaking (40 rpm)using a single-platform shaker (Reliable Scientific, Inc., Hernando, MS) for30 min or 120 min. PPLA

2

activity was obtained by manual titration ofreleased fatty acids as described by Helrich (1990) with modifications. Eggfat was first extracted using anhydrous MgSO

4

(5 g) and ethyl ether (60 mL)at 30C for 30 min with occasional shaking. After evaporating ether undervacuum, samples were dried for 1 h at 105C. A phenolphthalein and toluene-methanol mixture (30 mL, 2:1, v/v) was added dropwise to samples. Manualtitration was accomplished potentiometrically with 0.02 N methanolic-potas-sium hydroxide. Activity values are given in free fatty acid (FFA) (mL KOH/g sample).

Immobilized Enzyme Stability

To monitor the stability of the immobilized enzyme preparations, aliquotsof the matrices were assayed once a week for 3 weeks with the fluorescentassay. Beads were washed in 50 mM Tris (pH 8) prior to enzyme analysis.This gives the stability of the enzyme under controlled conditions and pro-vides an indication of stability over storage.


Statistical Analysis

A 4

¥

4 factorial design was used to determine the influence of theimmobilization method (PP1, PP2, PP3 and PP4) and time (week 0, 1, 2 and3) on the immobilized enzyme activity. A 3

¥

2 factorial design was used todetermine the influence of enzyme type (soluble, PP2 and PP4) and incubationtime (30 and 120 min) on the enzyme activity on liquid eggs. Samples wereprepared and assayed in triplicate for each factorial experiment. The data wereanalyzed by

MANOVA

(STATISTICA/Mac program; Stat Soft Inc., Tulsa, OK)and results reported to be statistically significant have

P

£

0.05.

RESULTS AND DISCUSSION

Enzyme Immobilization

Four different methods were performed to determine the influence of alinker protein (WPI) and a reducing agent (sodium borohydride) on the immo-bilization efficiency and activity of PPLA

2.

The four immobilization methodswere (1) direct enzyme immobilization (PP1), direct enzyme immobilizationplus reduction (PP2), enzyme immobilization using a linker protein (PP3) andenzyme immobilization using a linker protein plus reduction (PP4). High ionicstrength solution (1 M NaCl) was applied to remove noncovalently attachedenzyme after immobilization. CPG beads were used as the support matrix inthis study because this material possesses a greater surface area per unitvolume than nonporous beads, thus allowing preparation of an immobilizedbiocatalyst with high enzyme activity (Li and Walsh 2000).

We used 10 mM Tris buffer, pH 8.0, for the enzyme reaction as thehighest reported enzyme activity was achieved between pH 7.4 and 8.0(Thuren

et al

. 1987, 1988). It was also reported that the pH optimum is similarfor both soluble (pH 8.3) and immobilized enzyme (pH 8.2) and the immobi-lized enzyme rate is less sensitive to pH changes (Thuren

et al

. 1988). In thisstudy, 4 mM CaCl

2

was added into enzyme–substrate reaction because cal-cium is essential for achieving the maximum activity for both free and immo-bilized PPLA

2

(Kim

et al

. 2001). Madoery

et al

. (1995) also found that 5 mMCaCl

2

in the reaction leads to a 61 to 100% increase in PPLA

2

activity. Thespecific affinity of Ca

2

+

for soluble or immobilized PPLA

2

is in the mM rangeand the values are about 10

2

-

10

3

times higher than the specific affinity of Ca

2

+

for phospholipid (Madoery

et al

. 1999).

Immobilization Efficiency and Stability

Immobilization efficiency and stability for four immobilization methodsare shown in Table 1. For immobilization efficiency, the total amount of


2

7

immobilized protein and the total activity per gram beads were measured atweeks 0, 1, 2 and 3. The total amount of protein immobilized varies primarilywith respect to the use of a linker protein. PP1 and PP2 did not employ alinker protein and show a lower total amount of immobilized protein than PP3and PP4 (Table 1).

Enzyme activity was determined using a fluorometric assay and reportedas

D

RFU/min/g beads. The main effects of immobilization method(

P

= 0.001), time (P = 0.003), and their interaction (P = 0.009) were signifi-cantly different. The average activity for each immobilization method wasdifferent from each other although the average activities of PP2 and PP4approached nonsignificance (P = 0.04). These two methods incorporated areducing agent that reduces the labile imino bonds, resulting in a strongcovalent bond between the enzyme and the matrix. This reducing step mayalso reduce disulfide bonds present in enzymes, resulting in lower activity,although this was not observed with this enzyme (Fleer et al. 1981; Jeyaseelanet al. 2000).

Soluble enzyme activity was 4.98 DRFU/min per milligram protein withno change over a 3-week period. PP2 showed a relatively high activity (2.22DRFU/min/g beads) compared with PP1 (1.52 DRFU/min/g beads). This maybe a result of the amount of enzyme immobilized because immobilized phos-pholipase specific activity decreased sharply with increasing protein loading(Chen and Chen 1998). Enzyme molecules with less steric restrictions maylead to a more favorable spatial arrangement for the enzyme-substrate com-plex formation (Ferreira et al. 1993; Madoery et al. 1995).

Our immobilization method showed about 50% activity retention com-pared with soluble enzyme, which is higher than the previously reportedvalues of 20% (Ferreira et al. 1994) and 20–50% (Ferreira et al. 1993; Chenand Chen 1998), but lower than those of other studies showing 60–70% (Shenand Cho 1995) and 82% activity (Madoery et al. 1995) compared with solubleenzyme.

Higher immobilization efficiency has been obtained with noncovalentadsorption (Madoery et al. 1995, 1999) compared with covalent cross-linking(Ferreira et al. 1993, 1994; Chen and Chen 1998) and with snake venomPPLA2 than pancreatic PPLA2. Covalent cross-linking methods can lead tomore conformational changes than noncovalent adsorption because of themultiple and complex reactions.

We obtained a low coupling yield for which there are at least two possiblereasons. First, the amount of enzyme offered for immobilization to the solidsupport was in excess or second, the immobilization process led to undesirablemodifications of catalytically important residues including lysine, asparticacid and glutamic acid residues in PPLA2 (Chen and Chen 1998). PPLA2

coupled through e-amino groups showed a decrease in the coupling yield


TAB

LE

1.

IMM

OB

ILIZ

AT

ION

EFF

ICIE

NC

Y A

ND

STA

BIL

ITY

*

Enz

yme

imm

obili

zatio

nm

etho

ds‡

Tota

l pr

otei

n†(m

g/g

bead

s)To

tal

activ

ity (

DRFU

/min

/g b

eads

¥ 1

000)

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Ave

rage

PP1

1.53

± 0

.00

1.74

a ± 0

.04

1.54

b ± 0

.18

1.55

b ± 0

.10

1.25

c ± 0

.26

1.52

A ±

0.2

3PP

2 (r

educ

ing)

0.77

± 0

.03

2.20

a ± 0

.15

2.33

a ± 0

.02

2.15

a ± 0

.04

2.22

a ± 0

.11

2.22

B ±

0.1

1PP

3 (l

inke

r)3.

40 ±

0.0

11.

47a ±

0.2

91.

46a ±

0.1

01.

18b ±

0.0

61.

26b ±

0.1

91.

34C ±

0.2

0PP

4 (l

inke

r, re

duci

ng)

3.76

± 0

.12

2.11

a ± 0

.14

2.19

a ± 0

.20

1.74

b ± 0

.21

1.94

c ± 0

.10

2.00

D ±

0.2

3

*Im

mob

ilize

d ph

osph

olip

ase

A2

(PPL

A2)

enz

yme

activ

ity w

as d

eter

min

ed u

sing

a fl

uore

scen

t su

bstr

ate

(NB

D C

6-H

PC).

For

the

im

mob

ilize

d PP

LA

2, th

ebe

ads

wer

e as

saye

d ev

ery

5 m

in f

or 6

0 m

in.

The

dry

wei

ght

of t

he b

eads

was

det

erm

ined

aft

er a

ctiv

ity m

easu

rem

ents

. A

ctiv

ity i

s ex

pres

sed

as r

elat

ive

fluor

esce

nce

units

(D

RFU

)/m

in/g

bea

ds.

†To

tal

amou

nt o

f pr

otei

n im

mob

ilize

d on

to t

he c

ontr

olle

d po

re g

lass

(C

PG b

eads

) w

as d

eter

min

ed d

irec

tly b

y a

dye-

bind

ing

assa

y as

des

crib

ed i

n B

ond

et a

l. (1

992)

.‡

Four

dif

fere

nt m

etho

ds w

ere

used

to

cova

lent

ly i

mm

obili

ze P

PA2

onto

CPG

bea

ds. M

etho

ds w

ere

dire

ct o

r in

corp

orat

ing

a lin

ker

prot

ein

(WPI

) w

ith o

rw

ithou

t re

duci

ng w

ith s

odiu

m b

oroh

ydri

de.

a,b,

cM

eans

with

in t

he r

ows

for

wee

ks f

ollo

wed

by

no c

omm

on s

uper

scri

pt d

iffe

r P

£ 0

.05.

A,B

,C,D

Mea

ns a

mon

g av

erag

e en

zym

e ac

tivity

val

ues

for

imm

obili

zatio

n m

etho

d fo

llow

ed b

y no

com

mon

sup

ersc

ript

dif

fer

P £

0.0

5.

IMMOBILIZATION OF PHOSPHOLIPASE A2 9

because of the affected lysine residues, which played an important role in thecatalysis and/or maintaining enzyme structure (Shen and Cho 1995). Interest-ingly, our immobilization result is inconsistent with those of previous studies,which showed that when the Œ-amino groups are used to couple to a support,the coupling yield is high but the activity of immobilized enzyme towardsubstrates decreases drastically. We show a low coupling yield with a 50%activity level based on bead protein content. Other studies have shown thatthe coupling of lipase carboxylic groups to the solid support improves theactivity retention up to 50% of original activity with low coupling yield (Fleeret al. 1981; Ferreira et al. 1993; Shen and Cho 1995).

For immobilization stability, PP1, PP3 and PP4 showed decreasedenzyme activity over the 3-week testing. The amount of noncovalently boundprotein was determined by treating the matrices with 1% SDS after completionof the activity studies. No noncovalently bound protein was detected, whichmay be a result of the detection limit (minimum 2.5 mg/mL) of the proteinassay

Enzyme Activity on Liquid Eggs

Activity of soluble versus immobilized enzyme is shown in Table 2.Immobilized enzyme preparations that showed the highest activity (PP2 andPP4) were used in this assay. Each enzyme format (soluble and immobilized)was tested at room temperature at two different times, 30 and 120 min.Approximately the same units of enzyme activity (600 units based on thefluorometric assay) were used in each format.

TABLE 2.ENZYME ACTIVITY ON LIQUID EGGS*

Samples Free fatty acids (FFA)†(mL KOH/g sample) 30 min

120 min

No enzyme 14.76a ± 0.89 14.86a ± 1.19Soluble enzyme* 25.85b ± 2.72 42.22c ± 2.43Immobilized enzyme*

PP2 (reducing) 21.40d ± 1.39 36.05e ± 0.65PP4 (reducing, linker) 25.36b ± 1.31 41.86c ± 2.44

* Approximately 600 units of enzyme (soluble or immobilized) were added to 5 g of liquid eggs andincubated for 30 min or 120 min at room temperature.† The FFA value after incubation was determined with the manual titration method using methanolic-potassium hydroxide. Activity values are given in FFA (mL KOH/g sample). Values are the means ofthree experiments ± the standard deviation.a, b, c, d, e Fatty acid values with no common superscript differ P £ 0.05.


As shown in Table 2, the FFA value of no enzyme remained constant ateach time point. For both the soluble and immobilized enzymes, the FFA valueincreased with an increase in time. Soluble and immobilized enzyme (PP4)exhibited similar activities; therefore, the immobilized enzyme preparationswere at least as active as the soluble. Enzyme activity on liquid eggs wassignificantly affected by enzyme type (P = 0) and incubation time (P = 0) butnot affected by their interaction (P = 0.09).

CONCLUSIONS

Significant differences were observed among the four immobilizationmethods with respect to total activity. These differences most likely resultedfrom the use of a reducing agent to form a more stable imino bond betweenthe enzyme and the matrix. The use of a cross-linker, in this case WPI, didnot lead to higher enzyme activity using the fluorescent substrate. Theinfluence of the reducing agent did lead to a more stable and highly activematrix.

Activity of each enzyme format (immobilized and soluble) on liquid eggyolks was similar, demonstrating that immobilization of PPLA2 does lead toa matrix that can be used on liquid eggs.

ACKNOWLEDGMENTS

This research was supported by the Western Dairy Center and UtahAgricultural Experiment Station, Utah State University, Logan, UT 84322-4810. Approved as journal paper number 7562.

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COVALENT IMMOBILIZATION OF BOVINE PHOSPHOLIPASE A2