樣品分析實(shí)驗(yàn)方法extraxcellular enzymes in soil

1、E + S ES E + P Fungi in BioremediationEDITED BY G.M. GADDEnzyme (E) - a protein produced by cells that acts as a catalystSubstrate (S) - a compound converted by the enzymeVolna energy(G) - Energy capable of performing work; G0 the free energy for the stand. conditionsA + B C + D G0 = Gf0C + D Gf0A +

2、 BExergonic, endergonick ReplyG0 versus G G = G0 + RT ln KAlthough G0 negative, no measurable water formation does not occurThe activation energy is needed- Enzymes contain a small non-protein moleculescoenzymesWeakly bound to the enzyme, a coenzyme molecule can move freely among the other enzymesDe

3、rivatives of vitamins, NAD + / NADH is a good example, is a derivative of vitamin niacinprosthetic groupsTied tightly to the enzyme, usually covalent and permanently, the enzymes can be transmitted over coenzyme- Enzymes contain a small non-protein moleculesSources of enzymes in soilPlants, soil fau

4、na, microorganismsThe enzymes accumulated in the soil are divided into : Free enzymes (exoenzyme) Endoenzyme (cytoplasmic, bacteria periplasm)Kiss et al. 1975Types of EnzymesConstitutive enzymes cell used often - always presentInductive enzymes are expressed only in the presence inducer / substrate

5、are produced only when the cell needsEnzymes can be destroyed by the spec. enzymes (proteases)Enzymes may be protected by clay and humus particles Enzyme-clay and organic polymer-enzyme complexes are very resistant to denaturation and protease attack1.1.Enzymes associated with living, metabolically

6、active cells in the Enzymes associated with living, metabolically active cells in the soil, usually in the cytoplasm, bound to the cell. Walls or as soil, usually in the cytoplasm, bound to the cell. Walls or as extracellular enzymes, the cells were being eliminatedextracellular enzymes, the cells w

7、ere being eliminated2.2.Enzymes associated with living cells but the nereprodukujcmi Enzymes associated with living cells but the nereprodukujcmi (disputes, plant seeds, etc.)(disputes, plant seeds, etc.)3.3.Enzymes, which are associated with dead cells or cells. Parts or Enzymes, which are associat

8、ed with dead cells or cells. Parts or diffuse from dying cells, which are produced.diffuse from dying cells, which are produced.4.4.Enzymes, which are permanently immobilized on clay particles Enzymes, which are permanently immobilized on clay particles and humic colloidand humic colloid5.5. - this

9、may be active for a very long - this may be active for a very long6.6. - may be originating from both prokaryotic and eukaryotic - may be originating from both prokaryotic and eukaryotic cellscellsThe basic categories of soil enzymesLocalization of enzymes in soil(i)Intracellular enzymes(ii) Peripla

10、smic enzymes(iii) Enzymes adhering to the surface membranes of B.(iv) The enzymes secreted during growth and cell division(v) Enzymes within the cell resting forms (disputes, cysts, plant seeds, endospores)(vi) Enzymes adhering to dead cells or their remnants(vii) Enzymes escaping from Hungary or de

11、ad intact cells zlyzovan(viii) Enzymes associated with the temporary substrate, which converts(ix) Enzymes adsorbed on the surface of clay minerals(x) The complex enzyme-humic colloidsPrepared by Dr. Klose according to Burns 1982, Nannipieri 1994Classification of enzymes Oxidoreductase carry out red

12、ox reactions transferase transfer groups (molecules, atoms) from one substrate / molecule to another hydrolase hydrolytic reaction carried out (Step biomolecules using H2O) Isomerase regroup molecules / atoms in one molecule / substrate Lyase removed or added to the group as a double bond ligase gro

13、up attached to the molecule using ATP hydrolysis as energy sourceThe cleavage of polymerThe cleavage of polymer1. Degradation Cellulose- a process that releases glucose subunits as a source of carbon and energy(i) endoglucanases / endocellulase - 1.4 randomly cleaves bonds within the polymer cellulo

14、se polymers to shorter(ii) exoglucanases/exocellulase demerged unit of two molecules celobiozy the end of the string, which was split off endo glucanase(iii) -glucosidases/celobiosidase - celobiozu hydrolyze the subunits of glucoseThe cleavage of polymer1. Degradation CelluloseThe cleavage of polyme

15、r2.Degradation lignin- Lignin is not primary metabolized as a carbon and energy source- expose the more digestible polysaccharides of wood so that these can be cleaved by fungal cellulases and hemicellulasesrandom, freeradicalcatalyzed synthesis of ligninrenders it resistant to hydrolyticcleavageapp

16、roach to degrade it: randomfree-radical oxidation (combustion)reactions involving molecular oxygen(Lignin) peroxidase use (ferric heme)enzyme systems utilize verystrong oxidantsManganese (Mn) peroxidase use (Mn3+)Laccase (phenoloxidase) use (Cu2+)The cleavage of polymer2.Degradation ligninBreakdown

17、of polymerslignin degradationattack CC bonds in the lignin, removing e-destabilized intermediates undergo ring-cleavage release low molecular weight lignin“structural monomers”Biodegradation of PAHsToxicity of PAHsMechanism of carcinogenity of PAHsIn microsomMicrobial degradation of PAHsMeasuring so

18、il enzyme activity Can be used as assay of microbial community enzyme activities Have been used to assay impact of environmental stresses on soil microbial activity Assays of soil enzyme activities sometimes designed to measure enzyme activities of enzymes both inside & outside of cells; or only enz

19、ymes that are outside of cells (= soil-bound enzymes) Measuring soil enzyme activity Direct methods- substrate is added to the soil solutionIndirect methods- extraction of enzyme, then substrate is added- extraction buffer, enzyme activity is then potential- activity is measured in defined/optimal c

20、onditions (pH, temperature)Measuring soil enzyme activity Direct methods- suitable for exo-cleaving hydrolytic enzymes- quantifitcation of the product of the reaction not suitable for endo-cleaving hydrolytic enzymes and oxidative enzymes substrate is covalently bound to the testing moleculeP-nitrop

21、henolSpectrophotometry, low sensitivity, not suitable for dense and dar suspensionsmetylumbellyferon (MUF)Detection of fluorescence, high sensitivity, depends on pH, decreases in presence of phenolicsamidomethylkumarin (AMC)Same as MUFMeasuring soil enzyme activity Indirect methods-Suitable for all

22、enzymes-Only about 10% of total enzyme amount is extractableSoil enzyme assaysspectrophotometrical assaysmeasuring of laccase, peroxidase and Mn-peroxidase, cellulase, phospatase, etc.principlesoil/litter sample is cultivated for the short period of time with substrate which the enzyme trasformes to

23、 color productsubstrate is homolog to the natural substrateSoil enzyme assaysspectrophotometrical assaysBeerLambert lawAtEnzymeSubstrate *ElementMacromolecule degradadArylsulphatase, E.C. 3.1.6.14-MUF-sulphateSulphurMineralization of organic sulphur-Glucosidase, E.C. 3.2.1.204-MUF-D-glucopyranosideC

24、arbonStarch and glycogenCellobiosidase, E.C 3.2.1.914-MUF-cellobiopyranosideCarbonCellulose-Xylosidase, E.C. 3.2.1.374-MUF-D-xylopyranosideCarbonXylane, xylobiose-Glucosidase, E.C. 3.2.1.214-MUF-D-glucopyranosideCarbonCellulosePhosphodiesterase (PDE), E.C. 3.1.4.1bis-(4-MUF)-phosphatePhosphorusHydro

25、lysis of phosphate diestersChitinase, E.C. 3.2.1.304-MUF-N-acetyl-D-glucosaminideCarbonBreaking -1-4-glycosidic bonds in N-acetyl-glucosaminide (chitin) and chitobiosePhosphomonoesterase (PME), E.C. 3.1.3.24-MUF-phosphatePhosphorusHydrolysis of phosphate estersLeucine-aminopeptidase, E.C. 3.4.11.1L-

26、leucine-AMCNitrogenOligopeptides aminoacidsAlanine-aminopeptidase, E.C 3.4.11.12L-alanine-AMCNitrogenOligopeptides aminoacidsSoil enzyme assaysfluorogenic substrates Soil enzyme assaysfluorogenic substrates 4-methylumbelliferone (MUF) 7-amido-4-methylcoumarin (AMC) excitation and emmision filtersmea

27、suring of fluorescence of released MUFcalibration with different MUF concentrations before is crucial !exitation at 355 nm emission at 460 nm 1. Chemicals (such as toluene) which inhibit microbial proliferation can be added to soil2. Duration period of enzyme assay is kept short, to reduce chance of

28、 an increase in microbial numbers3. Attempts are made to keep microbial cells in sample intact. Is not desirable to have cells in sample e leaky, so that cytoplasmic enzymes leak out into surrounding soil To help achieve such separation/differentiation:Immobilized soil enzymeProteins/enzymes that bi

29、nd to clay minerals - Are not as catalytically active as enzymes that are free in aqueous solution - But are more stable to some environmental stresses such as temperature extremes; digestion by proteases; etc. than enzymes that are free in aqueous solutionOxygen microenvironmentsBinding of enzymes

30、to soil surfaces (esp. clay & humic materials) may take place via:- Ionic interactions- Covalent bonds- Hydrogen bonding- Entrapment of enzyme by soil colloids- Other mechanismsImmobilized soil enzyme Immobilized soil enzymes can bring about very rapid (immediate”) changes in substrate molecules whi

31、ch are added to soil In such cases, little or no lag period may be noted before enzyme activity es apparent Compare this to cell-associated enzymes, which often need to be induced, so that minutes to hours often pass before cells produce a certain enzyme, causing a measurable effect on a specific su

32、bstrateImmobilized soil enzymeFigure 4: PhOx (a) and Perox (b) activity during the position of spruce needles, reedgrass and blueberry litter incubated at 0 and 10Cmultivar. anal.multivar. anal.Zdroj: Brta J, Applov M, Vank D, Kritfkov M, antrkov H (2010) Effect of available P and phenolics on miner

33、al N release in acidified spruce forest: connection with lignin-degrading enzymes and bacterial and fungal communities. Biogeochemistry, in press-46-34phenolics/Ntotphenolics/PtotPoxPhoxSTART_spruce_needlesSTART_blueberrySTART_reedgrassEND_spruce_needlesEND_blueberryEND_reedgrass30.7 %26.1 %b-33-23p

34、henolicsDOC/DNPoxPeroxSTART_spruce_needlesSTART_blueberrySTART_reedgrassEND_spruce_needlesEND_blueberryEND_reedgrass26.3 %23.8 %a1. DOC/DN2. POX3. Perox4. phenolics1. phenolics/NTOT2. phenolics/PTOT3. POX4. PhOxthe change in fungal community composition of low lignified litter (i.e. reedgrass) correlates mostly with bioavailable P (POX), the change in higher lignified litter (i.e. spruce needles) correlates mostly with phenolics/NTOT, phenolics/PTOT ratios and PhOx activity (i.e. fungal community of spruce needles at 10C) Zdroj: Brta