-\ fa § @- I] CROSSLINKED |3- GLUCDSIDASE IN MESOCEI.I.UI.AB SILICA FCAMS: CHARACTERIZATION AND CATALYTIC ACTIVITY STUDIES 7.1 Introduction 7.2. Immobilization nf[3 — glucosidase 7.3 Cross - Linked Enzyme Aggregates lCLEAs1 7.4 Immobilization and activity measurements 7.5 Results and discussion 7.6 Biochemical characterization of free and [ZLEA-GL 7.7 Conclusions jfigfucosidase can 5e used 6) tfie food industry to increase t/ie 5z'oa'vai[ci6:'[it_y of tlie isqffiwones in tlie tiumcm intestine, and 5)’ tlie Eeverage industry to improve tfie aromatic composition qfjuices and wines. I t is welt Known tfiat gt:ycos1dic compounds are useftd products in pfzarmaceuticafl food, cosmetic and fine cfiemicaf industnes. In addition, since (iota tlie iinmooifization procedure and sofid support add to tfie cost iftlie iuso[u5[e enzyme’, tfiere is a great interest in clieap sutistrates on wfiicfi flgfucosidase can 5e immooifized 6y a simpfe and economic process. To overcome tfie s/iortcomings as a reszdt of adsorption and covafent Einding 0]‘ e-nzymes on solid supports, a simple and /iigliév eflrectioe metfiod of crossliiifiing en:;_y1nes'oza muftipoint attacfzment was adopted in t/iis context wfzicfi invofoes simple adsorption of enzyme foffivwed 6)» enzyme cr(iss[i:z!{;':zg using g[utaratdefi_yde. ‘Hie pure supports as welt as tfie crossfinlied supports were cfiaracterized 5)’ 3\/itro(qen adsorption studies, FUR ‘T§'/®’T§ and IPWMQ jt[[ tfie studies coufirmedt/ze incorporation Qferu-:_yme as weft as afdefiyde moieties into t/ie support. ‘Hie optimaf immofiilization conditions (enzyme finding, immo6i[ization time and amount qfg[utara.[a’efz_ye) were eagamined jllso, tlie operation stafiitities of immooitized enzymes w/ien used repeatedél (temperature, p91; storage, etc. ) and tlie Kinetic properties were studied and compared witfi t/iose 0)‘ free enzyme. ant; is cjpracticaf impoitanceforfu-rtfzer appt'ication.s'. We reszdts are promising for afuture tec/inofogicafappfication tfze i-nimo5z'[i:eder1:y:ne in 'wine-mafiing.
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fa§
@I]CROSSLINKED |3- GLUCDSIDASE IN MESOCEI.I.UI.AB
SILICA FCAMS: CHARACTERIZATION AND
CATALYTIC ACTIVITY STUDIES
7.1 Introduction
7.2. Immobilization nf[3 — glucosidase
7.3 Cross - Linked Enzyme Aggregates lCLEAs1
7.4 Immobilization and activity measurements
('onte2z!s'
7.5 Results and discussion
7.6 Biochemical characterization of free and [ZLEA-GL
7.7 Conclusions
jfigfucosidase can 5e used 6) tfie food industry to increase t/ie 5z'oa'vai[ci6:'[it_y of tlieisqffiwones in tlie tiumcm intestine, and 5)’ tlie Eeverage industry to improve tfie aromaticcomposition qfjuices and wines. I t is welt Known tfiat gt:ycos1dic compounds are useftdproducts in pfzarmaceuticafl food, cosmetic and fine cfiemicaf industnes. In addition, since(iota tlie iinmooifization procedure and sofid support add to tfie cost iftlie iuso[u5[e enzyme’,
tfiere is a great interest in clieap sutistrates on wfiicfi flgfucosidase can 5e immooifized 6y a
simpfe and economic process. To overcome tfie s/iortcomings as a reszdt of adsorption and
covafent Einding 0]‘ e-nzymes on solid supports, a simple and /iigliév eflrectioe metfiod ofcrossliiifiing en:;_y1nes'oza muftipoint attacfzment was adopted in t/iis context wfzicfi invofoes
simple adsorption of enzyme foffivwed 6)» enzyme cr(iss[i:z!{;':zg using g[utaratdefi_yde. ‘Hie
pure supports as welt as tfie crossfinlied supports were cfiaracterized 5)’ 3\/itro(qen adsorption
as weft as afdefiyde moieties into t/ie support. ‘Hie optimaf immofiilization conditions(enzyme finding, immo6i[ization time and amount qfg[utara.[a’efz_ye) were eagamined jllso,
tlie operation stafiitities of immooitized enzymes w/ien used repeatedél (temperature, p91;storage, etc. ) and tlie Kinetic properties were studied and compared witfi t/iose 0)‘ freeenzyme. ant; is cjpracticaf impoitanceforfu-rtfzer appt'ication.s'. We reszdts are promising
for afuture tec/inofogicafappfication tfze i-nimo5z'[i:eder1:y:ne in 'wine-mafiing.
Chapter-7 g "_ j7.1 Introduction
Flavor compound synthesis by biotechnological processes plays nowadays
an increasing role in the food industry. This is the result of scientific advances in
biological processes, making use of microorganisms or enzymes as an alternative
to chemical synthesis, combined with recent developments in analytical techniques
such as high-performance liquid chromatography (HPLC), gas chromatography
(GC), infrared (TR) or mass spectrometry (MS). Research in this area for new
products and bioprocesses is also enhanced by a growing market and increasing
public concem for the wholesomeness and chemical safety of food ingredients.
I3-glucosidase
Glycoside hydrolases (also called glycosidases) catalyze the hydrolysis of
the glycosidic linkage to generate two smaller sugars. They are extremely common
enzymes with roles in nature including degradation of biomass such as cellulose
and hemicellulose. Glycoside hydrolases are classified into EC 3.2.1 as enzymes
catalyzing the hydrolysis of O- or S-glycosides.
‘-.,,_...O J08 + H29 ---»--¢- \"""‘”‘O: ‘OH + HOR
B-Glucosidase (BG, EC 3.2.1.21) is one of the most interesting glycosidases,
especially for hydrolysis of glycoconjugated precursors, in musts and wines, and
the release of active aromatic compounds. B-Glucosidase (E.C.3.2.l.2l) comes
from many sources including bacteria, animals, and ‘plants, which exhibits wide
substrate specificity and is capable of cleaving B—glucosidic linkages of conjugated
glucosides and disaccharides [1]- It can hydrolyze cellooligosaccharides and
cellobiose into glucose and is capable of hydrolyzing anthocyanins that are the
main coloring agents found in the foods of vegetable origin [2]. The main interest
in this enzyme is related to its, potential applications in food processing industry
(for example, the production of wines and fruit juices) for improving organoleptic
Fig 7.4 shows the TG/DTG profiles obtained for immobilized B-glucosidase
on silica and that after crosslinking with glutaraldehyde- The typical TG and DTG
cun/es ofM-GL demonstrate the significant weight loss between 200-400°C, which
is attributed to the enzyme decomposition._¢
80
_ -— ——— —_—_. -ii |- ~ ——' -— 1 0ll Ci‘ .
auwu
ss %
zwauag
|l aw Ill.; ‘ I_.A "g\ 1 _' _
%
It-I5
e ght 0
iqfi a
*" A-\ ‘ ' I. ‘ lg—~ 01¢I 0 3
We ght
0.1%
W
0.1%
9 “' 0F O '0'. mu00 .,"' 0 no ”°*‘"l it 0l 0 ' " ‘Jl’ if’ 1-H‘ i l ‘ l ‘ li‘ I ‘ l ' *1“ ' l ' l l , t H Y t ‘-__”,r' ' |7"ifi-Ir’,-e--*‘—‘-e-"_*f - “'0 100 200 300 400 s00 600 700 000 900 5 1&0 460 600 300 who
Temperature(°C) Tempe;-atu;-e (°C)(3) (b)Fig 7.4 TG/DTG curves of (a) MAI (b) CLEA-GL
After crosslinking (C LEA-GL) there is only a single weight loss extending
from 100-650°C which is attributed to the decomposition of the organic groups of
glutaraldchyde as well as the cnzyme moieties which showed that the thermal
The "c NMR of CLEA-GL (Fig 7.7) exhibited peaks due to the enzyme
groups at 10, 23, 71 and 101 ppm with decreased intensity. The effective binding
of aldehyde was evident from the peak at 184 ppm.
t
n
‘ >
I F - T - I F". - 1 ~I—- 1 -W-w—'v—i 1 ‘I ‘1200 150 100 50 0Fig 7.7 "c NMR ofCl;F.A-GI,
7.6 Biochemical characterization of free and CLEA—GL
7.6.1 Stability and activity of various immobilizates
The mesopores of MCF supports were used as a template for the
preparation of CLEAs, which can enhance the enzyme loading by utilizing all the
pore volume, pore diameter and surface area and possibly prevent the enzyme
leaching. It is anticipated that the above characteristics would place a good
resistance against leaching [3-GL from MCF’s.
Figure 7.8 shows the stability of free GL (B-glucosidase), adsorbed GL and
CLEA—GL in aqueous buffer (l On1M sodium acetate, pH 4.8) at room temperature.
At each time point, the residual activity of GL was measured by the hydrolysis of
p-ntrophenyl B-D glucopyranoside in an aqueous buffer ( lOml\/I sodium acetate, pH
4.8), and the relative activity was calculated from the ratio of residual activity to
the initial activity. The enzyme stability of various immobilizates was investigated
by incubating each sample at room temperature and under harsh shaking condition.
229
Time (h)
-0- Free GL —I—ADS-GL—-A— CA-GL -0- CLEA-GL
Fig 7.8 Stability of CLEA-GL, Adsorbed GL, covalently bound-GL andfree GL in a shaking condition at 30°C. CLEA+GLs were preparedwith 2mg/ml and 0.1% glutaraldehyde concentration.
In harsh-shaking condition, the activity of free GL rapidly dropped, and
no measurable activity remained after 24 h incubation. ADS-GL in MCF and
CA-GL marginally enhanced the enzyme stability, but the relative activities
after 24h incubation were 50% and 68%, respectively. CA-GL showed a
fairly-good stabilization of enzyme activity by retaining 65% of initial
enzyme activity after 48h incubation. However, CLEA~GL in MCF showed
negligible activity decrease after 48h incubation in the same condition.
CLEA-GL also showed an impressive stabilization of enzyme activity when
compared to other immobilization approaches. This impressive stability under
rigorous conditions demonstrates that the mesocellular pore structure with
interconnected windows was successful in preventing enzyme aggregates in
the main mesocellular pores (16 nm) from being leached out. Most of the
B-glucosidase in the mesoporous channels are crosslinked with CLEAS in
mesocellular pores and do not leach out from MCF’s. The successful enzyme
stabilization via the CLEA approach can be explained by the effective
prevention of enzyme leaching as well as the improvement of intrinsic
enzyme stability via multipoint covalent linkages between enzyme molecules.
230
Crosslinfieat ji - Qfutfosizfase in Slfesocelfufarsificafoams . . . . . . ...jlctivity studies
Table 7.2 Enzyme loadings and activities of various immobilizatesI ,_.'-. -.
-..-»--...>.....
i
7 Free GLI‘Ans-01.? 150 t s7 5.2 774 i
i
i
-:-i
i
i
CA-GLI 150 132 8.5 i 77.4LEA-GLI i
~.---»-.-----__-.-__¢a~...-.4_q_4.____,.,_.,,,-.
, ;(Pg'inr1mg r (I12 in img (pmovimnrz 1 §y~'eide({y°)ri~ silica) silica) mg e~nzyme)_§1 .- - 12.3 ,1 | ,___ l "- - --------------.--.| ||,.......5....................._......................._..... -- .. . -...,..-->.-i..-|..|.................... .................._...............__...._...._ _
The enzyme activity depends on the protonation of the amino acids at the
reactive site and that enzyme activity is inhibited by complexation of the reactive
sites by the heavy metal cations. Activation by Ca2+, Cop, Mn2+, Mgzl, may be
explained by stabilization of the enzyme structure [60]. Divalent cations such as
Ca2+ and Mg” at 1mM concentrations in the reaction media almost showed not
much influenced on activity of the immobilized B-glucosidase while at 50 inM,
there was a significant decrease when compared to free enzyme (5OmM) which
may be due to the destabilization effect of metal salts on enzyme structure after
immobilization. There was not much effect on free enzyme with higher
concentration of salts used except in the case of CuCl2, AgNO3 and HgCl2 where
there was complete inactivation.
The mechanisms of the effect of these metal ions on the enzyme activity are not
known. (1-glucosidases and B—glucosidase could catalyze the synthesis of glucosides in
lower yields without metal ion which indicated that metal ions are not essential for
enzymatic synthesis of glucosides in the study carried out by Wang et al.[6l]. KCI and
MgS()4 had little effect on the two B-giucosidases from almond and apple seal at 5mM
concentration while CaCl2, MnCl;, CoCl,; and ZnSO4 inhibited the enzymes to some
extent while heavy metal salts, such as CuSO4 and AgNO3, severely inhibited the
activity [62]. The effects of various cations and reagents on B-glucosidase from
Aspergillus Niger activity were investigated by Riou et al. and it was found that there
significant inactivation with Ag’, Hgzl, Cuzl, Zn2l, and Fell“ and slight stimulation by2+ 2*‘ 21Mn ' while C o ' and Ca had no activity dependence [63].
7.6.8 Activation by alcohols
The effect of alcohols on the activity of beta glucosidase activity is shown in
Table 7.5. Methanol and ethanol increased the activity of beta glucosidase by
1.1 fold and 1.3 fold while propanol and butanol increased the activity by 1.6 and
2 fold (O.5M). However ethanol, propanol and butanol inhibited the activity of the
enzyme at above 1-2 M.
241
Cfiapter-7
Table 7.5 Effect of various alcohols on the activity of free B-glucosidaseand CLEA-GL
The time changes of product concentration (p-nitrophenol) at different substrate
concentrations, S0 (p-nitrophenyl [3-D glucopymnoside, PNPG) for free [3-glucosidase and
CLEA-GL are shown in Fig. 7.14 and Fig 7.15. The slope of the straight line is the initial
rate of reaction V (mM/min). The relation between the rate of reaction and substrate
concentration can be described by the Michaelis-Menten equation [55, 68].
V _ vniaxsKM+5
where Vmx is the maximum rate of reaction and KM is the Michaelis constant (mM).
Figure 8.16 shows Lineweaver-Burk plots of data obtained in experiments where
the rates of the reactions catalysed by native and crosslinked B-glucosidases were
monitored at varying pNPG concentrations.
l l K = l I*. = "'.—- "l" —ll’, l/nisnt l"'n1::t1 S
The values of l/mm. and KM by free and immobilized [3-glucosidase at 25°C
are listed in Table 7.6. The reaction follows Michaelis-Menten kinetics over the
range of substrate concentrations studied. The Km values of the crosslinked enzyme
(8.2mM) obtained were higher than those of the free enzyme (5.9mM), suggesting
that the crosslinked network limited the permeation rate of substrate and product.
This means that internal diffusional effects are mainly responsible for the increase
of Michaelis constants- The reduction in affinity of the enzyme may be due to the
conformational changes on immobilization.
Table 7.6 The maximum rate of reaction (V,,,,,.) and the Michaeli’s constant(Km) using free and immobilized B-glucosidase(Sr,=0.25-2.5mM PNPG)
T l.samp1¢ip , a%.;¢i§V;..;(mM/min). T K...(mM) R2 QFreeenzyme 0.0135 7 775.9 7 0.9989CLEA 0.0120 8.2 0.9970
243
_C_liap_ter-7 _0.6
)
9U1itm .t
mM
i\i
Product concentrat on.° P P-K N w-ml __
. i
Pas
i
l ..- A* A- Ai A . 0J A 2 . : - I0.01 1 I ‘ I -~ F ' -" | -—t |' 0 20 40 60 80 100Reaction time (min)
Fig 7.14 Time profiles of catalytic reaction of free [3-glucosidase underdifferent substrate concentrations at 25°C and pH 4.8 (acetatebuffer).S0(mM)- (I)—0.25, (O)-0.5, (A)-I, (*)-2.5
On the other hand, the V,,,a_‘ of the immobilised enzyme (0.0l2O mM/min)
was smaller than those of the native enzyme (0.0l35ml\/l/min). The decrease in
specific activity and increase in Km value due to mass transfer and diffusional
limitations has been reported by several authors [69, 70, 71].
0.5 as K a 5-4% I *
mM
P-bu
__1__.
la-V at
Product concentrat onO O'-» Lo6 3IO P
8 T I .
_l
l
bi~41“ I *
.°B)t p
A_ ‘k ‘ O5
I~
P
O
O
I
I
0.0 -r a ~- - -~60 90 t 120 150 180Reaction time (min)
Fig 7.15 Time profiles of catalytic reaction of CLEA-GL under differentsubstrate concentrations at 25°C and pH 4.8 (acetate buffer).So(mM)- (I)-0.25, (I)-0.5, (A)-1, (*)-2.5
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249
Cfiapter-7 p H p WW[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[Z3]
[24]
[25]
[26]
1.27]
L28]
.29]
I39]
3111
[32]
[33]
[34]
[35]
250
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