Enzyme activity measurements

According to the Beer Law (Eq.(3)) the absorbance, or fluo-rescence or whatever optical parameter is being used, is directly

296 C.G. Whiteley, D.-J. Lee / Enzyme and Microbial Technology 38 (2006) 291–316 Table 1

Reagents, procedures and sensitivities of different protein assays Assay; Ref.; Sensitivity;

Accuracy

Interference; Principle Reagents Procedure

Biuret[26]1–10 mg Good Ammonium salts; Colour between Cu⁺and peptide bond

Sodium potassium tartrate [2.25 g] CuSO4[0.75 g] KI [1.25 g] in 100 ml 0.2 M NaOH and diluted to 250 ml

Biuret reagent salts; Colour between Cu⁺ and aromatic amino acids and phosphomolybdate allowed to stand (30 min), Folin reagent (100␮l, 1 M) added, incubated (30 min), absorbance read at 595 nm Bradford[26,161]

1–100␮g Good

Absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 to 595 nm when binding to protein occurs

Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95%

ethanol, add 100 ml 85%

(w/v) phosphoric acid. Dilute copper to the mono-valent ion under alkali conditions. A molybdenum/tungsten blue product is produced

Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm Na2CO3, 0.16 gm sodium tartrate, 0.4 gm NaOH, and 0.95 gm NaHCO3, in 100 ml distilled water. Adjust the pH to 11.25 with 10 M NaOH

Reagent B: 0.4 gm cupric sulfate (5× hydrated) in 10 ml water.

Cool the samples and read at 562 nm

Standard working solution (SWR): Mix 100 volumes reagent A with 2 volumes reagent B

proportional to the concentration of the reagent.

A = εlc (3)

where A is the absorbance,ε the extinction coefficient, l the path length and c is the concentration usually in␮mol ml⁻¹. In the case of enzymes, this concentration is per unit time and is the same as activity, i.e. ␮mol ml⁻¹min⁻¹. Consequently, if one knows the extinction coefficient of a substance at a particular wavelength and one is measuring the change in optical param-eter over time it is possible to dparam-etermine the activity (v1) of the enzyme (Eq.(4)):

v1= AV

εtv (4)

whereA is the change in absorbance, V the total volume in the assay mixture,ε the extinction coefficient in ml ␮mol⁻¹, t the time in min andv is the volume of the sample.

The extinction coefficient for the substance under investiga-tion can be found from the slope of a linear plot, usually by linear regression, between the absorbance and several concentrations of pure substance. For substances that do not absorb strongly they are usually reacted with a dye to produce a colour that is measured at some specific wavelength in the visible spectrum.

The units of enzyme activity per mass of protein is referred to

as the specific activity and the amount of protein in the biomass may be determined in several ways depending on the sensitivity range of the protein. (Table 1)[26].

The kinetics of simple enzyme catalysed reactions was first characterised in 1912 by two biochemists Michaelis and Menten as they derived the hyperbolic equation:

v1= Vmax[S]

Km+ [S] (5)

wherev1is the rate of the enzyme catalysed reaction, [S] the concentration of substrate, K_mthe Michaelis–Menten constant and V_maxis the maximum reaction rate. It can quickly be estab-lished that K_mis also equal to the substrate concentration that would give 50% V_max. At low substrate concentrations, the rate of an enzymatic reaction is of first-order and is directly pro-portional to substrate concentration (v = k[S]) (Fig. 4, point a) while at high substrate concentration the rate becomes inde-pendent of substrate concentration, is represented by zero order kinetics (v = Vmax) (Fig. 4, point b) and almost all of the enzyme molecules are bound to substrate. To avoid using this curvilinear plot Lineweaver and Burk[27]introduced an analysis of enzyme kinetics by a straight line double reciprocal plot of 1/v1against 1/[S] (Fig. 4, inset) with a slope of Km/Vmaxand an intercept on

C.G. Whiteley, D.-J. Lee / Enzyme and Microbial Technology 38 (2006) 291–316 297

Fig. 4. A typical Michaelis–Menten curve representing change in velocity of an enzyme catalysed reaction with respect to substrate concentration (Inset:

Lineweaver–Burke linear relationship).

the ordinate at 1/V_max, from the following relationship.

1 v1

=

 Km

Vmax

 1 S



+ 1 Vmax

(6)

Another term that is often used is Kcat. This is the catalytic formation of product by an enzyme and is the time required for an enzyme to ‘turnover’ a substrate molecule.

3.2. Enzyme classification

Enzymes are grouped into six functional classes by the Enzyme Commission of the International Union of Biochemists [28,29]assigning each enzyme a unique four-digit number. The first describes the enzyme class, the second to the class bond of the substrate, the third to a sub-class or functional group of the substrate and the fourth to the actual molecule. It is the inten-tion of this review not to present a detailed account of all of these enzymes but to report on those that are associated with biological remediation (Table 2).

3.3. Energy of reaction

Enzymes are biological catalysts responsible for supporting almost every type of chemical reaction. They are physiologically

Fig. 5. Energy of reactants and products and activation energy with, and without, an enzyme.

important because they speed up, by at least 1000-fold, the rates of reactions by decreasing the amount of energy required to form a complex of reactants, known as the transition state com-plex, that is competent to produce reaction products. The free energy required to form an activated complex is much lower in the catalyzed reaction and consequently at any instant a greater proportion of the molecules in the population can achieve the transition state. The result is that the reaction rate is increased (Fig. 5).

3.4. Enzyme–substrate interactions

Enzymes interact with their specific substrate to form an enzyme–substrate complex [ES] by either a ‘Lock-and-Key’ or

‘Induced Fit’ model (Fig. 6) which then passes to a transition state [ES^*] and eventually to an EP complex which dissociates into product and free enzyme.

E+ S ↔ ES ↔ ES^∗↔ EP ↔ E + P (7) In the ‘Lock-and-Key’ model, the active site of the enzyme is complementary in shape to that of the substrate. With the, more favoured, ‘Induced Fit’ model, however, an initial weak interaction between enzyme and substrate rapidly induces con-formational changes in the enzyme thereby strengthening the binding and bringing catalytic sites and scissile substrate bonds close together. Such catalysis takes place at the active site, within

Fig. 6. Enzyme–substrate complex with (a) Lock-and-Key and (b) Induced Fit model.

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Reagents, reactions and classification of enzymes associated with biological remediation

No. [Ref] Classification; Properties Reaction Reagents; Assay

1 Oxidoreductases Adds or removes H2

1.1.1 Dehydrogenases Assay vol: 6.25 ml; triethanolamine buffer, 0.1 M, pH 7.6; Mg Cl2,

0.1 M; substrate, 10 mg ml⁻¹, 12␮l; NADP Na salt, 10 mg ml⁻¹, 16

␮l; enzyme 20 ␮l

Donors: Oxo groups Assay monitored by change in A340

Acceptors: NAD(P)H

1.2.99[73] Dehydrogenase Assay vol: 1.0 ml; Tris buffer, pH 8.0, 50 mM; methyl viologen,

5 mM; HCOONa, 20 mM; enzyme 100␮l; sodium dithionite, 50 mM

Donors: Oxo groups Activity monitored by reduction of methyl viologen under H2at

604 nm Acceptors: Other

1.5.99[76] Reductase Assay vol: 1 ml; phosphate buffer, pH 6.8, 300 mM; Na2SO4,

2.2 M; DTT, 1 mM; coenzyme F42014␮M; Na2S2O40.7 mM;

incubate 4 min; HCHO, 15 mM added; incubate 1 min; H4-MPT 16␮M added to form methylene- H4-MPT; enzyme 50␮l

Donors: CH–NH groups Activity monitored by increase at 420 nm due to oxidation of

F420H2

Acceptors: Other 1 U of activity = oxidation of 1␮mol F420H2per minute

1.7.1.6 [114–116,168]

Azo reductase Assay vol: 2.0 ml; Tris–HCl, pH 7.4, 25 mM; NADH 0.21 mM;

FMN 20␮M; Mordant yellow, 25 ␮M; enzyme 50 ␮l. [AQDSH2

25␮M]

Donors: Azo compounds 1 U enzyme = reduction of 1␮mol of dye per minute

Acceptors: NAD(P)H. Monitor decrease in A430

1.7.2.2 Nitrite reductase Assay vol: 1.0 ml; phosphate buffer, pH 7.0, 50 mM; NADPH

0.2 mM; TNT 0.1 mM,; enzyme 50␮l. Nitrite monitored at 540 nm.

Sample 600␮l; PMS 10 mM, 1.5 ␮l; sulphanilamide, 1% in 0.68 M HCl, 200␮l; N-(naphthyl) ethylenediamine, 1%, 40 ␮l

1.7.99.3 [148–152]

Donors: Nitro compounds 1 U activity = consumption of 1␮mol NADPH per minute

Acceptors: cytochrome or copper

C.G.Whiteley,D.-J.Lee/EnzymeandMicrobialTechnology38(2006)291–316299

1.8.99[51] Sulphur reductases Assay vol: 1 ml; Tris–HCl, pH 8.0, 50 mM; AMP 2 mM;

K3Fe(CN)65 mM; Na2SO330 mM; enzyme 100␮l

Donors: sulphur groups Activity measured in the direction of APS formation and monitored

by decrease at 420 nm due to ferri-cyanide reduction Acceptors: NAD(P)H

1.10.3.2 [102]

Oxidase Assay vol: 2.0 ml; 2,2^-azinobis-3-ethyl

benz-thiazoline-6-sulphonic acid [ABTS], 0.5 mM; CH3COONa, pH 4, 50 mM; diphenol 0.5 mM; enzyme 100␮l

Donors: Diphenols 1 U enzyme = 1␮mol product per minute

Acceptors: O2 Monitoring oxidation of ABTS at 420 nm

1.11.1 Peroxidase Assay vol: 3 ml; malonate buffer, pH 4.5. 50 mM;

2,6-dimethoxyphenol, 0.1 mM, H2O2,0.2 mM; MnSO41.0 mM;

enzyme 100␮l

Donors: Phenols Activity monitored by oxidation of 2,6-dimethoxy-phenol at

470 nm Acceptors: Peroxide

1.12[169] Hydrogenases Assay vol: 3.15 ml; methyl viologen, 1 mM in Tris–HCl, 20 mM,

pH 7.8; sodium dithionite, 100 mM; enzyme 100␮l

Donor: H2 1 U enzyme = reduction of 1␮mol of methyl viologen per minute

under H2at 604 nm Acceptor: Fe-S protein

1.13.11.1 Dioxygenase Assay vol: 1.0 ml; Tris–HCl buffer, pH 8.0, 50 mM; catechol 1 mM;

enzyme 100␮l

Activity monitored at 260 nm

1 U enzyme =␮mole of catechol converted per minute

1.13.11.2 Dioxygenase Assay vol: 1.0 ml; Tris–HCl buffer, pH 8.0, 50 mM; catechol 1 mM;

enzyme 100␮l

Activity monitored at 375 nm

1 U enzyme =␮mole of catechol converted per minute

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No. [Ref] Classification; Properties Reaction Reagents; Assay

1.13.11.18.

[154–156]

Sulphur dioxygenase Assay vol: 1.0 ml; Tris–acetate buffer, pH 7.4, 70 mM; sulphur 2%;

enzyme 200␮l; sulphite + thio-sulphate or sulphide were determined colorimetrically

1.14.12[82] Dioxygenases Assay vol: 1.0 ml; Tris–HCl, pH 7.5, 50 mM, indole, 0.1 mM,

NADH, 0.3 mM, ferrous ammonium sulphate, 0.1 mM, enzyme 100␮l

Incorporation of both atoms of O2into the product.

Assayed by decrease in A340due to decrease of NADH

1.14 [83–85]

Monooxygenases Assay vol: 1.0 ml; Tris–HCl, pH 7.5, 50 mM, salicylate, 0.1 mM,

NADH, 0.3 mM, ferrous ammonium sulphate, 0.1 mM, enzyme 100␮l

Incorporation of one atom of O2into the product and the other reduced to H2O

Assayed by decrease in A340due to decrease of NADH

1.16.1.1 Reductase Assay vol. 1.2 ml; enzyme 100␮l; phosphate buffer, pH 7.5,

50 mM; NADPH, 100␮M; Mg (OAc)2, 200␮M; EDTA, 500 ␮M;

mercaptoethanol, 0.1%; HgCl2, 200␮M; 60 min in dark; phosphate buffer, 50 mM, pH 8.0 with 10 mg nitroblue tetrazolium (NBT) and 1.5 mg phenazine methosulphate (PMS)

Mercuric reductase [124,125]

Donors: Metal ions Acceptors: NAD(P)H

Enzyme activity determined at 590 nm from formazan standard curve

Cupric reductase [119–121]

Assay vol: 400␮l; sodium citrate pH 7.5, 200 mM; CuCl220␮M;

bathocuproine disulphonate 100␮M; FAD 40 ␮M; NADH 100 ␮M;

enzyme 100␮l

Assayed by increase in A480due to Cu⁺-batho-cuproine

1.97.1 [127–129]

Chromate reductase Assay volume, 1.0 ml; H2SO4, 0.1 M; 1,5-diphenyl-carbazide,

0.01%, Tris–HCl buffer, 50 mM, pH 7, K2CrO4, 0.05 mM;NADH, 0.1 mM; enzyme 100␮l

Electrons supplied by NAD(P)H or cytochrome c3

Assayed by decrease in A540due to decrease in chromate

C.G.Whiteley,D.-J.Lee/EnzymeandMicrobialTechnology38(2006)291–316301

2 Transferases Transfers groups from a donor to acceptor.

2.1.1.86 [79]

Transfer C1methyl group Assay vol: 100␮l; coenzyme M 6 mM; ATP 0.5 mM; CH3-H4MPT

3 mM; MgSO41 mM; Ti(III)citrate 0.7 mM; DTT 1 mM; Phosphate buffer pH 7, 40 mM

Analysed by release of thiol with dithiobisnitro-benzene

1 U enzyme =␮mole of coenzyme M methylated per minute by CH3-H4MPT

2.1.3.1. Transfer C1carboxy group Assay vol: 600␮l; Tris–HCl buffer, pH 7.0, 15 ␮M; glutathione

3␮M; NADH 0.15 ␮M; pyruvate 6 ␮M; methylmalonyl CoA 0.5␮M; malic dehydrogenase 0.2 U

Assay monitored by change in A340by coupling with the reduction of oxaloacetate to malate and NAD

2.2.1.6[63] Transketolase or transaldolase Assay vol: 1.0 ml; phosphate buffer, pH 7.8, 50 mM; pyruvate

100 mM; thiamine diphosphate 1 mM; MgCl210 mM; FAD 10␮M Activity monitored at 333 nm due to disappearance of pyruvate

1 U activity = 1␮mol acetolactate formed per minute

2.3.1.16 Transfers acyl group Assay vol: 1.0 ml; Tris–HCl, pH 8.8, 50 mM; NADH 0.1 mM;

3-hydroxyacyl-CoA dehydrogenase 0.5 U; CH3COCoA 1.5 mM;

Formation of CoA measured with 5,5^-dithiobis(2-nitrobenzoic acid) at 412 nm

Activity measured in the direction of ketoacylCoA formation by coupling with its reduction to 3-hydroxyacyl CoA and NAD

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No. [Ref] Classification; Properties Reaction Reagents; Assay

2.3.1.54 [61,62]

Transfers other than amino-acyl

Assay vol: 1.0 ml; phosphate buffer, pH 7.6, 100 mM; pyruvate 20 mM; CoA 80␮M; NAD 1 mM; malate 6 mM; DTT 2 mM;

citrate synthase 1 U; malate dehydrogenase 22 U; enzyme 100␮l Assay monitored by change in A340

2.6.1.

[70,71]

Transaminases Assay vol: 3.0 ml; phosphate buffer, pH 7.5, 100 mM; aspartate

120 mM; NADH 3 mM; malate dehydrogenase 200 U;

␣-ketoglutarate 100 mM; enzyme 100 ␮l Transfers ketoacids to amino

acids

Assay monitored by change in A340by coupling with the reduction of oxaloacetate to malate and NAD

2.7.1 Transfers phosphate to OH Assay vol: 1.0 ml; Tris–HCl, pH 8.0, 100 mM; glucose 20 mM;

ATP 1 mM; NADP⁺0.3 mM; glucose-phosphate dehydrogenase 2 U; enzyme 100␮l

Donor: ATP Activity monitored at 340 nm by coupling with oxidation of

glucose-6-phosphate to 6-phospho-gluconate and NADPH Acceptor: OH

2.7.7.

[49,50]

Transfers nucleotidyl phosphoryl groups.

Assay vol: 1.0 ml; Tris–HCl, pH 8.0, 100 mM; APS 1 mM; MgCl2

1 mM; PPi 1 mM; enzyme 20␮l; reaction terminated with NaOH, 6M, 100␮l. Centrifuged and aliquot used to measure ATP formation with standard hexokinase glucose-6-phosphate dehydrogenase coupled system

Activity measured in the direction of ATP formation

2.8.4[80] Transfers alkylthio groups Assay vol: 8.0 ml; MOPS NaOH buffer, pH 7.2, 500 mM; methyl

coenzyme M 10 mM; coenzyme B 1 mM; aquacobalamin 0.3 mM;

titanium citrate 30 mM; enzyme 100␮l; atmosphere 92% N2/8%

H2. CH4conc. determined by gas chromatography

1 U activity = produces 1␮mol methane per minute

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3 Hydrolases Adds or removes H2O

3.1.1[163] Lipases Assay vol: 5.0 ml; phosphate buffer, 0.1 M, pH 7.5; triacetin.

1% w/v; enzyme 1.0 ml; H2SO4, 5 M; NaIO4, 0.1 M; NaHSO3

50 mM; chromotropic acid, 2.5 ml Enzymatic cleavage of

glycerol from triacetin and reaction monitored at 570 nm. Glycerol then determined by standard curve

1 U activity = forms 1␮mol glycerol per minute

3.1.3 Phosphatases Assay vol: 5.0 ml; enzyme 1.0 ml;ρ-NPP, 60 ␮M; acetate

buffer, 10 mM, pH 8, 25^◦C, 20 min; NaOH, 0.5 M added Enzymatic cleavage of

ρ-nitrophenolphosphate (ρ-NPP) to

ρ-nitrophenol (ρ-NP) to generate ion, measured at 405 nm, in alkaline solution

1 U of activity = conversion 1␮mol ρ-NPP in 1 min

3.1.6 Sulphatases Assay vol: 5.0 ml; enzyme 1.0 ml;␳-NPS, 60 ␮M; acetate

buffer, 10 mM, pH 8, 25^◦C, 20 min; NaOH, 0.5 M added

Enzymatic cleavage of ρ-nitrophenolsulphate (ρ-NPS) to

ρ-nitrophenol (ρ-NP) to generate ion, measured at 405 nm, in alkaline solution

1 U of activity = conversion 1␮mol ρ-NPS in 1 min

3.2.1[164] Glucosidases Assay vol: 5.5 ml; glycine buffer, 0.4 M, pH 10.8; MUG,

1.5 mM; enzyme 1.0 ml; 30^◦C, 5 min; EtOH, 95%, 0^◦C

Methylumbelliferylgluco-pyranoside [MUG] is hydrolysed to methyl umbelliferone [MUF]

Fluorescence measured at 365 nm [excite] and 455 nm [emission]

3.4[165] Proteases Assay vol: 5.5 ml; enzyme 1.0 ml; phosphate buffer, 0.1 M pH

7.5; azocasein, 2% w/v, 37^◦C, 60 min; trichloroacetic acid (TCA), 10% w/v, 0^◦C added; centrifuged, 4000× g, 10 min A440of TCA soluble

peptides from enzymatic cleavage of azocasein

1 U activity =␮mol product that would increase A440by 1 in 60 min

304C.G.Whiteley,D.-J.Lee/EnzymeandMicrobialTechnology38(2006)291–316 Table 2 (Continued )

No. [Ref] Classification; Properties Reaction Reagents; Assay

3.5.4[78] Acting on carbon nitrogen bonds of cyclic amidines

Assay vol: 700␮l; Tricine-KOH, pH 8.0, 50 mM; K2HPO4pH 8.0, 1.5 M; methenyl-H4-MPT⁺, 30␮M; enzyme 50 ␮l.

Activity measured in the direction of formyl H4MPT and monitored by decrease at 335 nm due to disappearance of methenyl H4-MPT⁺

1 U of activity = 1␮mol methenyl H4-MPT⁺hydrolysed to formyl H4MPT per minute

3.8.1 Acting on C-halide bonds Assay vol: 1.0 ml; chlorobenzene, 0.3 pmol; phosphate buffer, pH

7.4, 300 pmol; enzyme 200␮l; reduced glutathione, 13 pmol, 0^◦C, in phosphate buffer, 0.1 ml

Dehalogenase Enzyme activity monitored asA260per unit time

4 Lyases Adds or removes a group other than H2O.

4.1.1.5 Carboxy lyase Assay vol: 1.0 ml; phosphate buffer, pH 6.0, 50 mM; acetolactate

10 mM; enzyme 100␮l. After 5 min reaction quenched with NaOH, 0.5 mM

Acetoin measured at 522 nm with naphthol, 1% + creatine 0.5% in 1 M NaOH. Aliquot, 400␮l + reagent 4.6 ml

1 U of activity = 1␮mol acetoin produced per minute

4.1.2 Aldehyde lyase Assay vol: 3.1 ml; fructose-1,6-biphosphate 12 mM; hydrazine

sulphate 3.5 mM; EDTA, pH 7.5, 10␮M; enzyme 100 ␮l Activity monitored at 240 nm due to formation of hydrazone with 3-phosphoglyceraldehyde

4.2.1 Carbon oxygen lyase Assay vol: 1.0 ml; Tris–HCl, pH 8.0, 100 mM; MgSO42 mM;

2-phosphoglycerate 5 mM; enzyme 100␮l

Activity monitored from formation of phosphoenol-pyruvate at 240 nm

C.G.Whiteley,D.-J.Lee/EnzymeandMicrobialTechnology38(2006)291–316305 4.99.1.2

[124,125, 162,166]

p-Chloromercuribenzoate (pCMB) absorbs at 250 nm which shifts as it is cleaved by organo-mercurial lyase into Hg (II) and benzoate

Assay vol: 1.0 ml; Tris–HCl, pH 7.5, 50 mM; p-CMB, 100␮M;

L-cysteine, 1.0 mM; enzyme 100␮l

Enzyme activity is measured asA250with respect to time

5 Isomerases/mutases Catalyse geometric/structural changes within a molecule

5.3.1 Intramolecular

oxidoreductases

Assay vol: 1.0 ml; HEPES buffer, pH 7.3, 100 mM; NADH 0.5 mM;

glyceraldehydes-3-phosphate 4 mM; glycerol-phosphate dehydrogenase 4 U, enzyme 50␮l

Interconverts aldoses and ketoses

Assay monitored by change in A340

5.4.2 Intramolecular transferases Assay vol: 1.0 ml; HEPES buffer, pH 7.4, 25 mM; NADH 0.25 mM;

3-phosphoglycerate 5 mM; MgSO41 mM; ADP 1 mM; enolase 1 U;

pyruvate kinase 1 U; lactate dehydrogenase 1 U; enzyme 100␮l 1 U activity = converts 1␮mol 3-phosphoglycerate to

2-phosphoglycerate per minute Assay monitored at 340 nm

6 Ligases Joins groups together

6.2.1 [88,167]

Forming acid–thiol bonds Formation of AMP is coupled enzymatically to myokinase (1) pyruvate kinase (2) and lactate dehydrogenase (3)

Assay vol: 1.0 ml; MgCl2, 2.5 mM; ATP 0.5 mM; CoASH, 0.25 mM;

KCl, 10 mM; PEP, 10 mM; NADH, 0.35 mM; 2 U of myokinase, pyruvate kinase, lactate dehydrogenase

Activity monitored by decrease A340

AMP + ATP→ 2ADP (1)

2ADP + 2PEP→ 2 pyruvate + ATP (2) 2 pyruvate + 2NADH→ 2 lactate + NAD (3)

306 C.G. Whiteley, D.-J. Lee / Enzyme and Microbial Technology 38 (2006) 291–316

Fig. 7. Acyl covalent bond formation between an enzyme-serine and typical substrate.

the enzyme, which provides specific, high affinity binding of substrates and provides a favourable environment for catalysis.

After enzyme–substrate binding one, or more mechanisms of catalysis generates transition-state complexes and reaction products. (a) Destabilisation of ES complex may take place either by strain, distortion or desolvation. The zone of solvation around the substrate, while it is in solution, is lost as the substrate binds to the enzyme. The induced structural rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds, which more easily attain the transition state. (b) The substrate and enzyme when separate in solution are free to undergo translational motion leading to a disorientated high entropy situation while in the form of the ES complex they are both restricted and possess low entropy. (c) The substrate is orientated within the active site on the enzyme forming a covalent enzyme–substrate intermediate (Fig. 7)[30].

(d) General acid/base catalysis may occur where there is a trans-fer of a proton in the transition state either specifically by an acid or generally by some acidic/basic amino acid. (e) Some enzymes require metal ions to act as electrophilic catalysts that stabilise the increased electron density. (f) Enzyme substrate interactions induce strain in the substrate and orientate reactive groups into proximity with one another.

3.5. Cofactors/coenzymes

In many cases, a second component (cofactor, coenzyme or metal-ion activator) needs to be present on the active enzyme

Fig. 8. Binding of substrate and cofactor to an enzyme.

before catalysis can occur. The apoenzyme is a term given to the protein portion of the inactive enzyme that initially binds to a prosthetic group, coenzyme or metal ion to produce the active holoenzyme. Even if the substrate is present at the active region of the enzyme catalysis does not occur until the second component is present (Fig. 8)[31].

4. Enzymology of biological remediation 4.1. Aerobic digestion

In the process of activated sludge floc-forming microbes degrade wastewater sludge, pollutants or any organic material, under aerobic conditions, to effect a biosolid–liquid separation.

While a fraction of the organic material is used for the synthe-sis of new microorganisms, resulting in an increase in biomass, the remaining material is channeled into metabolic energy and oxidized to carbon dioxide, water, nitrates, sulphates and phos-phates to provide energy for both synthesis and cellular functions (Fig. 9). The settled biosolids are subsequently recycled to aer-ation tanks in order to maintain the biomass concentraer-ation and the supernatant is discharged. Once the organic waste material becomes exhausted then the organisms will begin endogenous respiration to oxidize cellular material. Unfortunately, a dis-advantage of an aerobic treatment is the production of large amounts of biosolids (sludge) which contains volatiles, organic solids, nutrients, pathogens, heavy metals, inorganic ions, toxic organic chemicals and the original problem of dissolved organic waste is now transformed into a problem of particulate waste.

Aerobic respiration is defined as the aerobic catabolism of nutrients to carbon dioxide and water involving glycolysis, the tricarboxylic cycle, an electron transport system and molecu-lar oxygen as the final electron acceptor: this type of aerobic digestion is notable in organisms that require molecular oxygen,

Fig. 9. Aerobic digestion of waste involving enzymes and microorganisms.

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