Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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442 ADÁNYI ET AL.

FAD

FADH

Glucose oxidase

reductive half-reaction

oxidative half-reaction

Figure 1. The reaction scheme for the oxidation of -D-glucose catalysed by glucose oxidase

P. purpurogenum, P. variabile, P. chrysogenum and A. fumaricus are also good

candidates for industrial applications (Crueger and Crueger, 1990; Petruccioli

et al., 1993; Leiter et al., 2004). The A. niger and P. amagasakiense enzymes

are homodimeric glycoproteins of 160 kDa molecular mass with one molecule of

non-covalently bound FAD cofactor per subunit (Fig. 2; Wohlfahrt et al., 1999).

Dissociation of subunits is only possible under denaturing conditions and is accom-

panied by the loss of the coenzyme FAD (Gouda et al., 2003). A. niger GOx has

10–16% mannose-type carbohydrate content, the carbohydrate moieties being N -

or O-glycosidically linked to the protein (Takegawa et al., 1989). The two GOxs

share 81% sequence similarity and exhibit similar glucose oxidation kinetics.

GOxs function by a Ping-Pong Bi-Bi mechanism which is divided into a reductive

half-reaction and an oxidative half-reaction (Fig. 1). In the reductive half-reaction

the substrate reduces FAD by hydride transfer forming dihydro-FAD FADH



and the oxidized product. Numerous sugars and derivatives of -D-glucose are

the substrates of GOx but the enzyme shows far greater activity with -D-glucose

(Pazur and Kleppe, 1964). In the oxidative half reaction, the reduced flavin is

regenerated by O

that undergoes reduction to H

. Besides O

, both one-electron

acceptors (such as transition metal complexes, ferrocene derivatives, and nitroxide

radicals) and two-electron acceptors (benzoquinone derivatives) are good substrates

for the reduced enzyme (Chan and Bruice, 1977; Bourdillon et al., 1993; Ryabov

et al., 1999).

One-electron acceptors are generally used as mediators between GOx and

the metal or graphite electrode. The largest mediator groups are derivatives of

ferrocene that are small enough to penetrate to the active centre of GOx and

exchange electrons (Alvarez-Icaza et al., 1995; Forrow et al., 2002). When the

mediator cannot penetrate to the active site due to its bulkiness, a long-range

electron transfer event takes place. Theoretical studies have predicted the length

and path for electron transfer from the flavin cofactor to the surface of the protein

(Alvarez-Icaza et al., 1995).

PRODUCING AND DECOMPOSING ENZYMES 443

Figure 2. The homodimeric structure of glucose oxidase from Penicillium amagasakiense with non-

covalently bound FAD represented by MOLSCRIPT (Kraulis, 1991)

2.2. Galactose Oxidases

Galactose oxidase (D-galactose:oxygen 6-oxidoreductase, GAO, EC 1.1.3.9.) is a

Type II (non-blue) copper protein (Giordarno et al., 1974) with a molecular mass of

69 kDa. It catalyses the oxidation of a wide range of primary alcohols and polysac-

charides (e.g. D-galactose) to the corresponding aldehydes, coupling this reaction to

the reduction of O

to H

 RCH

OH+O

→RCHO+H

(Bretting and Jacobs,

1987). The substrate specificity of GAO is very broad, ranging from small alcohols

to polysaccharides but the enzyme is highly stereospecific and does not oxidise

either D-glucose or L-galactose (Klibanov et al., 1982; Goudsmit et al., 1984). The

physiological function of GAO is unclear.

Different fungal species: Fusarium dendroides (Dactylium dendroides),

Gibberella fujikuroi and G. zeae as well as the basidiomycete Polyporus circinatus,

can be used for industrial-scale GAO production. GAO genes can also be expressed

efficiently in either Pichia pastoris (Whittaker and Whittaker, 2000) or Aspergillus

oryzae (Xu et al., 2000). GAO contains a single copper ion in addition to a protein

base redox site (Whittaker and Whittaker, 1988, 1990) (Fig. 3), which comprises a

444 ADÁNYI ET AL.

Tyr495

Tyr272

Cys228

His581

His496

RCH

Tyr495

Tyr272

Cys228

–H

abstraction

Tyr495

Tyr272

Cys228

H atom abstraction

Tyr495

Tyr272

Cys228

e- transfer

Tyr495

Tyr272

Cys228

–H2O2

Figure 3. Proposed mechanism for D-galactose oxidation by galactose oxidase according to Whittaker

(1988). R represents the pyranose ring associated with D-galactose

tyrosine residue (Tyr272) covalently linked to a cysteine (Cys228) through thioether

bond, forming a cysteinyl-tyrosine (C-Y) “built-in” cofactor (Ito et al., 1991). The

enzyme undergoes self-processing in the presence of oxygen and copper to generate

this covalent cross-link, which is required for enzyme activity (Xie and van der

Donk, 2001). The cross-linked tyrosine serves as an axial ligand to the copper and

is oxidized to the radical form in the active state of the protein. The Cu(II)/Tyr

cofactor on GAO carries out the two-electron oxidation of primary alcohols to the

corresponding aldehydes via a radical mechanism.

The enzyme uses a Ping-Pong mechanism with respect to the substrate and

. Tyr495 functions as a base for abstracting a proton from the bound substrate

followed by hydrogen atom abstraction by the Cys-Tyr radical generating an

PRODUCING AND DECOMPOSING ENZYMES 445

alkoxide radical and diamagnetic Cys-Tyr (Whittaker and Whittaker, 1990). From

the reactive alkoxide radical one electron transfers to Cu

reducing it to Cu

and

forming the aldehyde product. O

converts Cu

back to Cu

(Fig. 3).

2.3. Cholesterol Oxidases

Cholesterol oxidases (cholesterol:oxygen oxidoreductase, ChOx, EC 1.1.3.6.) are

bifunctional flavoenzymes that catalyse two reactions at a single active site. The

first is the oxidation of cholesterol to cholest-5-ene-3-one and the isomerisation of

the labile cholest-5-ene-3-one intermediate to cholest-4-ene-3-one product (Fig. 4).

Although the enzymes exhibit a wide-range of steroid specificities, the 3-hydroxyl

group is essential for their activity (Inouye et al., 1982).

ChOxs are produced by bacteria including species from the Rhodococcus,

Streptomyces and Nocardia genera. However, several Brevibacterium, Proactino-

myces, Pseudomonas and Cellulomonas species also possess ChOx activity suitable

for industrial production (Goodhue and Risley, 1978; Watanabe et al., 1986;

MacLachlan et al., 2000). Recombinant Escherichia coli strains expressing bacterial

ChOx genes have been reported (Sakka et al., 1994). Several fungi from the Basid-

iomycete taxon (e.g. Lentinus edodes, Oudemansiella radicate, Coprinus comatus

and Auricularia polytricha) are also promising candidates for future industrial ChOx

production (Matsui et al., 1982). In non-pathogenic bacteria, e.g. Streptomyces,

ChOx is secreted and is a component of the metabolic pathway for utilizing choles-

terol as a carbon source (Fukuda et al., 1973; Cheetman et al., 1982). Pathogenic

bacteria, e.g. Rhodococcus equi and slow-growing Mycobacterium spp., require

ChOx for invasion of the host macrophage and cholesterol regulates the expression

of this enzyme (Fernanandez-Garayzabal et al., 1996; Navas et al., 2001). ChOx is

a water soluble, interfacial enzyme that binds transiently to the membrane surface

during catalysis (Sampson et al., 1998).

ChOxs utilise a Ping-Pong mechanism with respect to the steroid substrate and

. In the reductive half-reaction the cholesterol reduces FAD by hydride transfer

from the 3C of cholesterol to N5 of the flavin moiety yielding cholest-5-ene-3-one

cholesterol

Isomerization

Oxidation

FAD

FADH + H

cholest-5-ene-3-one cholest-4-ene-3-one

Figure 4. Cholesterol oxidation and isomerization catalysed by cholesterol oxidase

446 ADÁNYI ET AL.

and 1,5-dihydroflavin (FADH

 (Medina et al., 1997). In the oxidative half-reaction

the reduced flavin (FADH

 is converted back to the active, oxidized form (FAD)

by O

with the generation of H

2.4. Catalases

Catalases (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, CAT, EC

1.11.1.6.), more correctly hydroperoxidases, catalyse the degradation of H

O and O

 2H

→2H

O+O

Considering the possibility of industrial enzyme production, a variety of different

micro-organisms including bacteria (e.g. Micrococcus, Bacillus, Microscilla, Alcali-

genes spp.), moulds (e.g. Aspergillus, Penicillium, Thermomyces, Thermoascus,

Acremonium spp.) and yeasts (e.g. Saccharomyces, Candida, Mycotorula spp.)

are known as good CAT producers. CATs from animal sources (e.g. bovine

liver) are generally cheap, therefore the production of microbial CATs will only

be economical when better producer (preferably recombinant) strains and cheap

technology can be used or CATs with special properties (e.g working at high or low

temperatures or at alkaline or acidic pH) are produced (Fusho and Yajima, 1995;

Kou et al., 1998; Takeuchi and Isobe, 1999).

In general, three distinct types of proteins, which do not share either sequence or

structural homologies, exhibit significant CAT activity. The class I CATs, which

are the most widely spread in nature, consists of monofunctional haem-containing

enzymes which can be divided into two subclasses having large > 75KDa or small

< 60 kDa subunits (Klotz et al., 1997). The second, less widespread class includes

bifunctional haem-containing CAT-peroxidases, which are closely related to plant

peroxidases. The third class comprises members of the non-haem or Mn-containing

CATs (Wu et al., 2004).

Monofunctional haem-containing CATs all possess a two-stage mechanism for

the degradation of H

. The first, oxidative, step in the catalyses is the mono-

oxygen transfer from H

to the iron centre with the release of water (Fig. 5).

Under physiological conditions the resting state of haem CATs contains iron (III).

In the oxidation, one molecule of H

oxidizes the haem to an oxy-ferryl species,

in which one electron is removed from the iron and one electron is removed from

the coordinated porphyrin ring to generate a porphyrin radical cation (Fig. 5). In

the second stage, another H

molecule is utilised to regenerate the enzyme by

reducing the porphyrin radical coordinated oxy-ferryl species to the resting haem

Enz(Por

–Fe

= O) + H

Enz(Por–Fe

III

) + H

O + O

Enz(Por–Fe

III

) + H

Enz(Por

–Fe

= O) + H

(1)

(2)

Figure 5. The two-stage process of H

disproportionation catalysed by catalases

PRODUCING AND DECOMPOSING ENZYMES 447

releasing H

O and O

(Matsunaga and Shiro, 2004). The haem reactivity is enhanced

by the axial phenolate ligand of Tyr.

CATs do not follow classical Michaelis-Menten kinetics except across a certain

range of substrate concentrations (Switala and Loewen, 2002). Most small subunit

enzymes are inactivated by H

at concentrations above 300–500 mM and never

reach the maximum rate extrapolated from the Michaelis–Menten curve determined

at lower substrate concentrations. Large subunit enzymes show a greater resistance

to H

damage even at concentrations above 3 M and the observed rates exceed

the theoretical Michaelis-Menten maximum rate (Switala and Loewen, 2002).

Most of the monofunctional haem-containing CATs are uniquely stable and resist

proteolysis due to their very rigid structure that resists unfolding (Fig. 6). Enzymes

with larger subunit such as HPII CAT from E. coli, show enhanced thermal stability,

the activity of the enzyme starting to drop at temperatures above 80



C (Switala

et al., 1999).

Figure 6. The tetrameric structure of catalase from beef liver. Each domain contains haem and NADP

cofactor represented with ribbon diagram (Kraulis, 1991)

448 ADÁNYI ET AL.

3. BIOSENSORS BASED ON OXIDASES AND CATALASE

3.1. Biosensors Based on Glucose Oxidase

Currently, GOx is the most important model enzyme in both basic and applied

biosensor research because its enzymological properties are well understood and

it is very cheap in comparison to other oxidases. Different design approaches

have been considered and employed in the construction of GOx-based biosensors,

including needle type sensors, sensors for in vivo monitoring, flow injection analysis

(FIA) applications, combinations of glucose biosensors with microdialysis sampling

techniques and disposable sensors (Wilson and Gifford, 2005).

Electrochemical glucose biosensors prepared by immobilising GOx on the

surface of carbon film electrodes using glutaraldehyde and bovine serum albumin

(BSA) with or without Nafion (a sulfonated tetrafluorethylene copolymer)

have been reported by Florescu and Brett (2005). An ISFET (Ion Sensitive

Field Effect Transistor) biosensor containing GOx and horseradish peroxidase

(HRP) co-immobilized with BSA using glutaraldehyde and covered by poly(4-

vinylpyridine-co-styrene) polymeric film could also be used for glucose deter-

mination (Volotovsky and Kim, 1998). Monolayer enzyme electrodes have been

made by covalently or electrostatically binding the recognition molecule onto

electrodes modified with conducting polymer films (e.g. self-assembled monolayers,

Langmuir–Blodgett films; Malhotra and Singhal, 2003). A flow-through electro-

chemical micro-cell can be used as an on-line detector in microdialysis-based assays.

The micro-detector modified by enzyme-based chemistry can be directly connected

to the outlet of the microdialysis probe (Gáspár et al., 2004).

Redox couples, or mediators, are able to shuttle electrons between the redox

centre of the enzyme and the electrode. The electron transfer mediator must be

chemically stable in both the reduced and oxidised forms, have a relatively low and

stable redox potential, and should be easy to immobilize at the electrode surface

(Harwood and Pouton, 1996). Carbon paste electrodes or graphite–Teflon rigid

composite biosensors offer the possibility of co-immobilization of several enzymes

via simple physical inclusion in the bulk of the electrode matrix by non-covalent

linkages in the presence of a mediator like ferrocene (Guzman-Vázquez de Prada

et al., 2004). Yu et al. (2003) constructed a biosensor by immobilizing GOx with

titanium isopropoxide forming GOx-titania sol-gel film, which retains the native

structure and activity of the entrapped enzymes. Carbon film resistor electrodes

can be modified with Prussian Blue (PB, ferric hexacyanoferrate) and then covered

with a layer of covalently immobilized enzyme. These enzyme electrodes can be

used to detect glucose via the oxidation of H

at +50mV vs. Ag/AgCl in the

low micromolar range, while also avoiding or reducing electrochemical interference

(Ricci and Palleschi, 2005). Electrodes produced with the ‘screen printing’ thick-

film technique can be chemically modified with PB prior to enzyme immobilisation

(Newman and Turner, 2005).

The use of metallized carbon electrodes resulted in remarkably selective amper-

ometric biosensors because these transducers eliminated major electroactive

PRODUCING AND DECOMPOSING ENZYMES 449

interference. Platinization has been used to increase electrode surface area

and thus electrode sensitivity, immobilizing GOx by the electropolymer-

ization of o-phenylenediamine (Reyes De Corcuera et al., 2005). Glucose

nanosensors based on the co-electrodeposition of an osmium redox polymer/enzyme

composite on low-noise carbon fibre nanoelectrodes have been constructed

(Fei et al., 2005). Chromium and manganese half-sandwich complexes

were developed as mediators for GOx since their sizes are similar to

that of ferrocene derivatives and they contain a -ligand for interaction

with the enzyme co-factor (Forrow and Walters, 2004). Screen-printed

electrodes modified with ruthenium dioxide were investigated as amperometric

sensors to immobilize GOx onto the electrode surface using Nafion films

(Kotzian et al., 2005).

Recently there has been considerable interest in the development of single-

use sensor strips especially in the fields of decentralized medical diagnostics

or on-site environmental monitoring (Wang et al., 1995). These strips can be

regarded as disposable electrochemical cells onto which a sample droplet is

placed. A miniaturized total analytical system (TAS) for on-line monitoring

of glucose in biological systems based on integrated microdialysis sampling

and photolithographically-prepared glucose electrodes has been developed

(Wang, 1999).

Interest in optical fibre biosensors is growing fast since they confer several

advantages compared to biochemical and clinical analyses due to their simple

and flexible construction. Malhotra et al. (2005) presented a glucose biosensor in

which GOx was immobilized onto poly-3-hexyl thiophene mixed with stearic acid

(P3HT/SA) LB films. The activity of GOx-containing LB films was measured by

a colorimetric method using o-dianisidine as a chromogenic dye with absorbance

at 540 nm. Capillary electrophoresis integrated with an optical biosensor was

built by modifying the detector part of the electrophoresis equipment with the

redox-sensitive polymer film polyaniline (PANI) containing immobilized GOx

(Bossi et al., 2003). Measuring molecular fluorescence is a very promising

technique in biosensing because of its high sensitivity. Fibre-optic glucose sensors

based on this phenomenon have been developed by immobilizing GOx onto an

oxygen optrode composed of either decacyclene in silicone (ex 385 nm, em 450–

600 nm) or the ruthenium complex tris(1,10-phenanthrolene)ruthenium chloride

(ex 447 nm, em 604 nm). The formation of hydrogen peroxide can be followed

by measuring chemiluminescence (luminol) or via the formation of a fluorescent

oxidation product from non-fluorescent p-hydroxyphenyl acetic acid (HPA) or

homovanillic acid (HVA) in the presence of HRP. A decrease in the pH in

the enzyme layer can be determined taking advantage of the light absorption

(e.g. bromocresol green) or fluorescence (e.g. hydroxypyrenetrisulphonate) of

acid/base indicators. In a different type of glucose biosensor GOx itself (the

prosthetic group, FAD), or GOx chemically modified with a fluorescein derivative

(GOx-FS), served as an indicator for recording changes in fluorescence (Pickup

et al., 2005).

450 ADÁNYI ET AL.

3.2. Multienzyme Biosensors Based on Glucose Oxidase

Multienzyme systems involving GOx have been developed for the rapid, repro-

ducible and cheap determination of different disaccharides and polysaccharides.

For example, the concentration of maltose was determined using a bienzyme cell

in which amyloglucosidase and GOx were co-immobilized on a protein membrane

using glutaraldehyde followed by amperometric detection. For lactose sensing,

-galactosidase, GAO and GOx enzymes were co-immobilized either on the surface

of a measuring electrode using a triacetate cellulose membrane, or on the surface

of different resins, or on the controlled-pore glass used in analytical reactors, or on

protein membranes by covalent coupling (Adányi et al., 1999). Sensors for total

D-glucose  + and sucrose have been developed using pectin as a novel matrix

to enhance enzyme entrapment and stabilisation on rhodinised carbon electrodes.

Total D-glucose can be measured by enzymatically transforming all -glucose

anomers to the  form using mutarotase. For sucrose detection, a multienzyme

system involving invertase, mutarotase and GOx were used (Jawaheer et al., 2003).

With the incorporation of lysozyme during the immobilization step, considerable

enhancement of the operational stability of a biosensor has been demonstrated in the

case of sucrose determination (Gouda et al., 2002). Marconi et al. (2004) developed

a method for the determination of gelatinised starch in processed cereal foods by

co-immobilization of amyloglucosidase and GOx on a Pt electrode surface while

the third enzyme, -amylase, was added to the solution under analysis. Wu et al.

(2005) proposed a biosensor for the determination of glucosinolates employing a

bienzyme system involving myrosinase and GOx co-immobilized onto an eggshell

membrane on the surface of a dissolved oxygen electrode.

3.3. Biosensors Based on Galactose Oxidase

GAO is used in biosensors developed for the determination of galactose or dihydrox-

yacetone in different biofermentation processes, or for measuring lactose primarily

in food samples. Schumacher et al. (1994) constructed an electrode for the deter-

mination of galactose and galactose-containing disaccharides in which GAO was

immobilized in gelatine between two dialysis membranes and stuck to an electrode.

Malhotra et al. (2005) prepared an amperometric biosensor to measure galactose

in milk and blood serum by immobilizing the enzymes in Langmuir–Blodgett

(LB) films of poly-3-hexyl thiophene (P3HT) mixed with stearic acid (SA), and

depositing the LB film onto indium tin-oxide (ITO) coated glass plates. Hasebe

and Uchiyama (2000) demonstrated that the substrate specificity of GAO from

F. dendroides changed dramatically in the presence of L-histidine resulting in

catalysis of the air oxidation of L-ascorbate. Using this observation a flow-

type system was developed for the indirect measurement of histidine. Since the

catalytic activity of GAO is inhibited by free radicals a biosensor based on enzyme

inhibition was developed to detect superoxide and nitrogen oxide radicals using

GAO entrapped in a gel-like -carrageenan membrane coupled to an amperometric

oxygen electrode (Campanella et al., 2000).

PRODUCING AND DECOMPOSING ENZYMES 451

3.4. Biosensors Based on Cholesterol Oxidase

To measure free and total cholesterol contents in biological samples, ChOx-

based biosensors and bienzyme cells containing immobilized cholesterol esterase

(ChEt) and ChOx were developed, respectively. Ram et al. (2001) reported a

biosensor in which ChOx and ChEt were bound to either a collagen membrane

or to conducting polymer matrices. The electrochemical redox processes taking

place in enzyme-layered films deposited either on platinum or ITO-coated glass

plates have been investigated. Gobi and Mizutani (2001) constructed a direct

amperometric biosensor by layer-by-layer nanothin film formation using ChOx

and poly(styrenesulfonate) on a monolayer of HRP covalently immobilized on

Au-alkanethiolate electrodes. ChOx and ChEt were entrapped within polypyrrole

(PPy) films on a platinum disc electrode during electrochemical polymerisation

(Singh et al., 2004). Situmorang et al. (1999) studied the conversion of cholesterol

esters by flow injection potentiometry using a tungsten electrode and monitoring

ferricyanide/ferrocyanide conversion. Hall and Turner (1991) described an amper-

ometric organic-phase enzyme electrode (OPEE) using ChOx to determine the

concentration of cholesterol in a chloroform/hexane mixture. Pena et al. (2001)

reported on a bienzyme amperometric composite biosensor for the determination of

free and total cholesterol in food samples. ChOx and HRP together with potassium

ferrocyanide as a mediator were incorporated into a graphite-Teflon (30%–70%)

matrix. The enzyme membrane was prepared from ChEt and ChOx using a photo-

sensitive polymer and an ultra-thin dialysis membrane, and this was applied in a flow

system with a Clark-type oxygen electrode (Endo et al., 2003). An amperometric

cholesterol biosensor using carbon nanotubes has been produced by layer-by-layer

deposition of a cationic polyelectrolyte (poly(diallyldimethylammonium) chloride;

PDDA) and ChOx on a multi-walled carbon nanotube-modified gold electrode (Guo

et al., 2004). The hybrid composite film was used to immobilize ChOx on the

surface of PB-modified glass carbon electrode (Tan et al., 2005).

The FIA method was investigated by covalent co-immobilization of enzymes

to the silica in packed-bed reactors (IMMER) and using an amperometric

HRP electrode or photometric determination of H

(Baticz and Tömösközi,

2002). Optical cholesterol biosensors have also been constructed by immobilizing

ChOx and octadecyl silica (ODS) particles in either hydrogel network matrices

of copolymers of poly(vinyl alcohol)/hydroxyethyl carboxymethyl cellulose

(PVA/HECMC) or sol-gel (Wu and Choi, 2003).

3.5. Biosensors Based on Catalase

CAT decomposes H

and remains active when kept in appropriate organic

solvent environments. Wang et al. (1995) demonstrated the applicability of CAT

for organic-phase biosensing by immobilising the enzyme onto a glassy carbon

amperometric transducer by casting a mixed enzyme/Eastman-AQ polymer solution.

Horozova et al. (2002) prepared an OPEE by immobilizing the enzyme within a