Enzymes in the Environment: Activity, Ecology and Applications - Chapter 2 pptx

ORIGIN AND ASSOCIATION OF ENZYMES WITH AQUATIC MICROORGANISMS Three common terms are used for the enzymes involved in the transformation and degrada-tion of polymeric substrates outside

2 Ecology of Microbial Enzymes in Lake Ecosystems

Ryszard Jan Chro ´ st and Waldemar Siuda

University of Warsaw, Warsaw, Poland

I INTRODUCTION

During the past decade, an increasing number of ecological studies have considered thecomplexity of freshwater ecosystems One major outcome of these studies has been anaccelerated interest in the role of heterotrophic microorganisms (particularly bacteria) inthe functioning of aquatic environments and the processes by which organic matter ismade available to them (1–4) These heterotrophic microorganisms are the key trophiclevel at which the metabolism of the whole ecosystem is affected, i.e., organic matterdecomposition, nutrient cycling, and structure of aquatic food webs The demonstration

of the importance of heterotrophic bacteria as a particulate carbon source for higher trophiclevels and a major respiratory sink has created a renewed interest in the production andutilization of organic substrates by these microorganisms

Most organic compounds produced in natural waters have a polymeric structure(5,6) and they are too large to be readily assimilated The transport of organic moleculesacross microbial cell membranes is an active process mediated by specific enzymes called

permeases Only the low-molecular-weight organic molecules (monomers or oligomers)

can therefore be taken up (7) In order to be available for microbial metabolism, polymericcompounds must be transformed into smaller molecules through enzymatic depolymeriza-tion

Besides the physicochemical conditions of aquatic environments, the compositionand availability of organic matter are the major factors that influence the development andactivity of heterotrophic bacteria (8,9) The heterotrophic bacteria are the only biologicalpopulations capable of significantly altering both dissolved (DOM) and particulate (POM)organic matter Microbial enzymes associated with these processes are the principal cata-lysts for a large number of biochemical transformations of organic constituents in aquaticenvironments Many of these transformations can be mediated only by heterotrophic bac-teria because the enzyme systems required for these reactions are not found in other or-ganisms

Microorganisms have adopted essentially two strategies that enable them to utilizemacromolecular compounds The macromolecule can be engulfed by the cytoplasmic

membrane to form a vacuole within the cytoplasm Enzymes are secreted into this vacuoleand the polymeric compounds are hydrolyzed and subsequently taken up Uptake of sub-

strate solutions by this method is referred to as pinocytosis; uptake of particulate substrates

is termed phagocytosis However, many microbial cells are unable to carry out these

pro-cesses, and therefore pinocytosis and phagocytosis are restricted to only those eukaryoticmicroorganisms that lack a cell wall, e.g., many protozoa Those eukaryotic and prokary-otic microorganisms that possess a cell wall have developed an alternative strategy forthe assimilation of polymeric substrates Hydrolytic enzymes are secreted outside the cyto-plasmic membrane, where they hydrolyze macromolecules in close vicinity to the cell.The resulting low-molecular-weight products are then transported across the cell mem-brane and utilized inside the cytoplasm

The hydrolysis of polymers is an acknowledged rate-limiting step in the utilization

of organic matter by microorganisms in aquatic and soil environments Prior to tion into microbial cells, polymeric materials undergo stepwise degradation by a variety

incorpora-of cell surface–associated enzymes and/or enzymes secreted by intact living cells or ated into the environment through the lysis of microorganisms The importance of micro-bial enzymatic activities to the mobilization, transformation, and turnover of organic andinorganic compounds in freshwater and marine environments has been shown in manystudies (10–18) Results of these studies have shown that studying enzymatic processesprovides powerful information that helps in understanding basic processes of decomposi-tion and microbial activity in both freshwater and marine ecosystems

liber-II ORIGIN AND ASSOCIATION OF ENZYMES WITH AQUATIC MICROORGANISMS

Three common terms are used for the enzymes involved in the transformation and

degrada-tion of polymeric substrates outside the cell membrane: ectoenzymes (19), extracellular

enzymes (20), and exoenzymes (21) In this chapter, the term ectoenzyme is used to refer

to any enzyme that is secreted and actively crosses the cytoplasmic membrane and remainsassociated with its producer Ectoenzymes are cell-surface-bound or periplasmic enzymesthat react outside the cytoplasmic membrane with polymeric substrates that do not pene-trate the cytoplasm Extracellular enzymes occur in free form dissolved in the water and/

or are adsorbed to surfaces (e.g., detrital particles, organic colloids, humic complexes,minerals in suspension) Extracellular enzymes in water may be secreted actively by intactviable cells, they can be released into the environment after cell damage or viral lysis,and/or they may result from zooplankton grazing on algal cells and from protozoan grazing

on bacteria

Ectoenzymes and extracellular enzymes (in contrast to intracellular enzymes) reactoutside the cell, and most of them are hydrolases The ectoenzymes that cleave polymers

by splitting the key linkages on the interior of the substrate molecule and form intermediate

sized fragments are called endoectoenzymes (e.g., aminoendopeptidases act on the

cen-trally located peptide bonds and liberate peptides) (22) Those ectoenzymes that hydrolyzethe substrate by consecutive splitting of monomeric products from the end of the molecule

are termed exoectoenzymes (e.g., aminoexopeptidases hydrolyze peptide bonds adjacent

to terminalα-amino or α-carboxyl groups and liberate free amino acids) (23)

There are three pools of microbial enzymes in water samples: intracellular enzymesare located and react with substrates inside the cytoplasmic region and are mostly responsi-

Figure 1 Percentage distribution of cell-bound and extracellular activity of microbial chitinase(CHTase), deoxyribonuclease (DNase), 5′-nucleotidase (5′-nase), alkaline phosphatase (APase), β-glucosidase (GLCase), and aminopeptidase (AMPase) in water samples from eutrophic Lake Mi-kołajskie (Chro´st, unpublished.)

ble for internal cell metabolism; extracellular enzymes are in the surrounding environmentand catalyze reactions without control from their producers; and ectoenzymes are cell-surface-bound enzymes, mostly hydrolases, that degrade polymeric substrates, yieldingreadily utilizable monomers All pools are composed of both endo- and exoenzymes.Distribution between ecto- and extracellular activity for selected enzymes (amino-peptidase,β-glucosidase, alkaline phosphatase [APase], 5′-nucleotidase [5′-nase], deoxyri-bonuclease [DNase], and chitinase [CHTase]) has shown that ectoenzymes contributed

on average from 75% (APase) to 98% (chitinase) of the total activity in lake water (Fig.1) Activities of the intracellular and extracellular pool enzymes are low Intracellularenzymes contributed from 0.5% (chitinase) to 10.7% (aminopeptidase) to the total activity

of water samples Activity of the extracellular enzymes, dissolved in the water, constitutedfrom 1% (5′-nase) to 16.5% (APase) of the total activity An interesting observation isthat extracellular enzyme activity as a percentage of total activity is higher in lake sedi-ments than in the water column (Table 1) This was particularly evident in the case ofchitinase and lipase activities

Enzyme activities bound to the 0.2- to 1.0-µm-size fraction of microplankton(mainly composed of bacteria) make up a greater fraction of activity by microorganisms

in lake water High ectoenzyme activity found in this size fraction has correlated with

Table 1 Percentage Contribution of Extracellular Enzyme Activities to the Total Activity ofLake Water and Lake Sediment Samples

Percentage

Leucine-aminopeptidase Plußsee/eutrophic 9.5⫾ 3.7 12.6⫾ 6.4

Scho¨hsee/mesotrophic 8.4⫾ 2.1 11.2⫾ 5.1α-Glucosidase Plußsee/eutrophic 10.2⫾ 4.9 15.1⫾ 7.3

Mikołajskie/eutrophic 8.7⫾ 2.6 13.3⫾ 3.8Szymon/hypereutrophic 10.8⫾ 3.3 14.6⫾ 4.9

Bełdany/eutrophic 21.4⫾ 6.8 33.1⫾ 8.3Kisajno/mesotrophic 22.4⫾ 6.2 35.8⫾ 9.1Alkaline phosphatase Plußsee/eutrophic 24.5⫾ 6.3 32.6⫾ 7.3

Scho¨hsee/mesotrphic 1.7⫾ 0.5 23.2⫾ 8.4

⫾ Standard deviation of an average value.

Source: Data from Chro´st, unpublished.

bacterial abundance and/or bacterial production of lake water A variety of isms produce ectoenzymes in waters and sediments in freshwater ecosystems However,many studies have reported that bacteria are the major producers of ectoenzymes amongaquatic microorganisms (24–33)

microorgan-III CONTROL OF ECTOENZYME SYNTHESIS AND ACTIVITY

The conditions in the aquatic environment, as in the soil aqueous phase, are unfavorablefor enzymes First, the substrate concentration is usually very low and highly variable.Many substrates may be insoluble, exist in intimate association with other compounds,and/or be bound to humic substances, colloidal organic matter, and detritus Therefore,these conditions are suboptimal for the coupling of an enzyme to its substrate Second,

an enzyme may be lost from the parent cell and may be bound to suspended particles andhumic materials, or it may be exposed to a variety of inhibitors present in the water.Finally, an enzyme may be denaturated by physical and chemical factors in the aquaticenvironment or hydrolyzed by proteases

Obviously, for an enzyme to be of benefit to its producer microorganism, it mustavoid degradation long enough to associate with its substrate Moreover, even if an enzymeovercomes these obstacles and binds with its substrate, the physical and chemical condi-tions of the reaction medium may be unsuitable for catalysis (e.g., nonoptimal pH or tem-perature, presence of inhibitors, absence of activators, suboptimal ionic strength) Never-theless, there is strong evidence that various aquatic microorganisms produce ectoenzymes

in freshwaters that encounter a number of polymeric substrates (31) and that microbialgrowth is dependent on the products of ectoenzymatic reactions (13,14,16,24, 34–36)

A microbial cell living in an aquatic ecosystem is influenced by a variety of mental factors The signal for appropriate gene expression and consequent ectoenzymeproduction within a cell is in response to the surrounding environment Depending on the

environ-regulatory control of gene expression, two types of microbial enzymes are synthesized inwaters and sediments: constitutive enzymes, whose synthesis is constant regardless of thepresence or absence of the substrate in the environment, and inducible enzymes, whoserates of synthesis are strongly dependent on the presence of their substrates (or substratederivatives) Many inducible enzymes are synthesized at a low basal rate (i.e., are constitu-tive) in the absence of a substrate When the substrate is available in the environment,there is a dramatic increase in the production rate of the particular enzyme Synthesiscontinues at this amplified rate until the inducer is removed and/or the product of enzy-matic catalysis accumulates (24) and it then returns to the basal rate.

Most of the ectoenzymes synthesized by aquatic microorganisms are catabolic zymes involved in degradation of polymeric substrates that are not continuously available

en-in the water or sediments Therefore, the constant synthesis of ectoenzymes en-in the absence

of substrates is unnecessary, because it requires the expenditure of energy that otherwisemay be channeled into other useful activities Since microorganisms have been competingwith each other for millions of years, the evolutionary advantages of induction are readilyapparent Most of the ectoenzymes found in freshwaters are inducible, and only a fewhave a constitutive nature (e.g., some amylases or proteases in bacteria)

A Induction and Repression/Derepression of Ectoenzyme Synthesis

The efficient induction of ectoenzymes is more complicated than that of intracellular zymes First, many of the ectoenzyme substrates present in fresh water are polymericcompounds, and they are too large to enter the cell and serve as inducers of synthesis.Second, for an ectoenzyme to be secreted at appropriate rates, the microorganism must

en-be able to monitor the activity of the ectoenzyme outside the cell We suggest that theseproblems are overcome by a low constitutive rate of ectoenzyme secretion If the substrate

is present, then low-molecular-weight products accumulate to a certain level, enter thecell, and serve as the inducer (20) When environmental conditions inhibit an ectoenzymeactivity (e.g., unsuitable pH, absence of activating cations Mg2 ⫹, Zn2 ⫹), the induction ofits synthesis does not occur because the product of catalysis is not generated However,since the microorganisms in freshwater ecosystems are in a complex relationship with avariety of readily utilizable compounds of autochthonous and allochthonous origin, theinduction of a particular ectoenzyme by an end product resulting only from degradation

of a single polymeric substrate seems to be questionable Until now, it has appeared thatone ectoenzyme may have several inducing compounds (24,37)

It is well documented that synthesis of many ectoenzymes produced by aquaticmicroorganisms is repressed by the end product that accumulates in the cell or in sur-rounding environment The repression of alkaline phosphatase synthesis by inorganicphosphate (the end product of phosphomonoester hydrolysis) in microalgae and bacteria

is probably one of the best-known examples (11,13,38,39) In Lake Plußsee, the specificactivity of APase significantly decreased when the ambient orthophosphate concentrationswere higher than 0.5 µM (13) APase activity was inversely related to the amount of

intracellular phosphorus stored (Pst) in algal cells When Pstconstituted less than 10% ofthe total cellular phosphorus, the algae produced alkaline phosphatase with a high specificactivity, and when Pstwas higher than 15% and the ambient orthophosphate concentrationsexceeded 0.6µM, this activity rapidly decreased.

The synthesis of virtually all ectoenzymes in most aquatic microorganisms is

re-Figure 2 Effect of water supplementation with dissolved organic matter extracted from plankton on growth of bacteria (A) and specific activity of bacterialβ-glucosidase (B) and aminopep-tidase (C) in Lake Mikołajskiet (Chro´st, unpublished.)

phyto-pressed when they are grown on sources of readily utilizable dissolved organic matter

(UDOM) This mode of regulation is called catabolic repression When water samples

from eutrophic Lake Mikołajskie were supplemented with dissolved organic matter tracted from phytoplankton (both UDOM and polymeric compounds) the bacterial cellnumbers increased markedly during 96 hours of incubation (Fig 2A) Contrary to that incontrol samples, supplementation of lake water with phytoplankton organic matter resulted

ex-in a significant decrease ex-in the rates of specific activities of bacterial β-glucosidase andaminopeptidase (calculated per bacterial cell) during the first period of bacterial growth(6–48 hours) However, in both of these enzymes, specific ectoactivity began to increaseafter 48 hours of bacterial growth In control samples, where bacteria grew solely onnaturally present DOM in lake water, the specific activity of these ectoenzymes increasedwithin the incubation period (Figs 2B, 2C)

The repression of ectoenzymes is tightly coupled to the availability of UDOM in

lake water.Figures 2Band2Cshow that ectoenzyme synthesis in DOM-enriched sampleswas no longer repressed when the concentration of the readily utilizable low molecular-weight molecules fell below a critical level, and polymeric substrates had to be used tosupport the growth and metabolism of bacteria Similar in situ observations during phyto-plankton bloom development and breakdown were reported forβ-glucosidase activity ineutrophic Lake Plußsee (24), for β-glucosidase and aminopeptidase activities in meso-trophic Lake Scho¨hsee (25), and for lipase activity in eutrophic Lake Mikołajskie (40).Despite the widespread occurrence of catabolic repression, with the exception ofthose for enteric bacteria, the molecular details of the repression are poorly understood.Some studies have indicated that cyclic adenosine monophosphate (cAMP), together withits receptor protein, may play a central role in control of catabolic repression (41,42).Using the repression strategy for ectoenzyme synthesis, microorganisms can avoid thewasteful production of inducible enzymes, which are not useful when their growth is notlimited by UDOM (3,19,24,35).

B Inhibition of Activity

It is important to consider that the repression/derepression of an ectoenzyme not beequated to the reversible inhibition of activity Even if an ectoenzyme is synthesized, itsactivity may be inhibited by the accumulation of the end product or by high concentrations

of the substrate (19) Two general types of reversible inhibition are known: competitiveand noncompetitive inhibition

Competitive inhibition occurs when an inhibiting compound is structurally similar

to the natural substrate and, by mimicry, binds to the enzyme In doing so, it competeswith an enzyme’s natural substrate for the active substrate-binding site The hallmark ofcompetitive inhibition of many ectoenzymes (e.g., alkaline phosphatase,β-glucosidase,aminopeptidase) is that it decreases the affinity of an ectoenzyme (an increase of theapparent Michaelis constant is observed) for the substrate and, therefore, inhibits the initialvelocity of the reaction (Fig 3) (13,26,37) Competitive inhibition is reversible and can

be overcome by increased substrate concentration, and therefore the maximum velocity

(Vmax) of the reaction is unchanged (Fig 3A)

Noncompetitive inhibition generally is characterized as an inhibition of enzymaticactivity by compounds that bear no structural relationship to the substrate Therefore, theinhibition cannot be reversed by increasing the concentration of the substrate It may

be reversed only by removal of the inhibitor Unlike competitive inhibitors, reversiblenoncompetitive inhibitors cannot interact at the active site but bind to some other portion of

an enzyme-substrate complex This type of inhibition encompasses a variety of inhibitorymechanisms and is therefore not amenable to a simple description Noncompetitive inhibi-tion of the activity of exoproteases by Cu2⫹ions (43) and inhibition ofα-glucosidase, β-

glucosidase, N-acetyl-glucosaminidase, and alkaline phosphatase by H2S in natural watershave been described (12)

C Environmental Control of the Synthesis and Activity

synthe-Figure 3 Competitive inhibition of β-glucosidase activity by glucose (end product of enzymereaction) in water samples from eutrophic Lake Mikołajskie (A) Hyperbolic relationship betweenenzyme activity and increasing substrate concentrations, (B) Lineweaver-Burk’s linear transforma-

tion of the relationship between enzyme activities (1/v) and increasing concentrations of substrate

(1/S) (Chro´st, unpublished.)

sis and activity were under different control mechanisms, which were dependent on thephysical-chemical conditions of the habitat There is ample evidence for general catabolicrepression of ectoenzyme synthesis in bacteria due to readily utilizable carbon sources(24,37,44,45) as well as more specific repression by end products of enzyme catalysis(34,46) However, control of aminopeptidases appears to be distinct and more complexthan that of other ectoenzymes In some bacteria, amino acids, peptides, and/or proteinsseem to induce aminopeptidase synthesis (45,47) It is not known specifically how amino-peptidase induction operates, especially since amino acids are reported to act as inducers

in some bacteria, rather than acting in their more predictable role as end-product inhibitors.The ability of bacteria living in the euphotic zone of the lakes to produce ecto-enzymes seems to be strongly affected by the availability of the low-molecular-weight,readily utilizable substrates exudated by algae (eg., excreted organic carbon-EOC), whichare known to be excellent substrates for bacteria (48–50) Chro´st and Rai (25) found thatthe rates of leucine-amino-peptidase and α-glucosidase production by aquatic bacteriastrongly depend on bacterial organic carbon demand When the amount of EOC fulfilledthe bacterial organic carbon requirement, microorganisms did not synthesize enzymesneeded for hydrolysis of the polymeric substrates because their utilization was unneces-sary Moreover, the specific activity of aminopeptidase correlated negatively to the rates

of algal EOC

During the active growth of phytoplankton, algal populations excrete into the water

a variety of photosynthetic products, including easily assimilable low-molecular-weightsubstrates (51), which support bacterial growth and metabolism These substrates inhibitthe activity and repress the synthesis of ectoenzymes in bacteria On the other hand, whenlow levels of readily available substrates limit bacterial growth and metabolism, bacteriaproduce ectoenzymes with high specific activity to degrade polymers and other nonlabilesubstrates Such a situation occurs in lake water during the breakdown of phytoplanktonbloom Senescent algae liberate, through autolysis of cells, a high amount of polymericorganic compounds (polysaccharides, proteins, organophosphoric esters, nucleic acids,lipids, etc.), which induces synthesis of ectoenzymes Another mechanism that causesrepression cessation of enzyme synthesis is low level of directly utilizable organic com-pounds in the water during bloom breakdown (52)

Bacteria living in the profundal zone of the lakes are often substrate-limited (2,53)because the amount of substrate in deep waters depends primarily on the sedimentationrates of the organic matter that is produced in the euphotic zone There is no direct supply

of labile organic compounds exudated by algae In the profundal zone, sedimentationprovides labile monomeric organic compounds that are mostly polymers that are utilized

by bacteria Under such environmental circumstances, bacterial metabolism is stronglydependent on the presence and amount of polymeric substrates and the activity of synthe-sized ectoenzymes that catalyze the release of readily utilizable monomers

Microbial ectoenzymatic activity in natural waters is also strongly dependent onenvironmental factors, such as temperature, pH, inorganic and organic nutrients, ultraviolet

B (UV-B) radiation, and presence of activators and/or inhibitors (3,13,21,54–59) Severalstudies have shown that ectoenzymes display the highest activities in alkaline waters of

pH 7.5 to 8.5 (24,40) or acid waters of pH 4.0 to 5.5 (55) In contrast to the pH response,many ectoenzymes exhibit no obvious adaptation to ambient temperature, because theoptimal temperature is often considerably higher than in situ temperature of waters (13,33,40) The optimal temperatures for alkaline phosphatase andβ-glucosidase are unchangedwhen they are produced by planktonic microorganisms in lake water under different insitu temperatures (13,24)

In light of these aforementioned studies, the environmental regulation of ectoenzymesynthesis and activity is complex and usually no single factor is involved in this process It

is important to realize that environmental regulation of ectoenzymes, induction, synthesisrepression, and inhibition are related to concentration, period of exposure, and such factors

as temperature, pH, oxygen level, and chemical characteristics of regulatory molecules.The same molecule that is an inducer under one set of circumstances may be a repressorunder other environmental conditions, or at different concentrations

A Methods

There are significant difficulties in measuring ectoenzyme activities in heterogeneous ronments such as natural waters and soil, which include questions about methodologyand data interpretation For example, should assays be performed according to the well-established principles of enzymology (e.g., excess substrate, optimal pH and temperature,shaking of reaction mixtures) or in situ conditions encountered in an aquatic environment(e.g., limiting and unevenly distributed substrate, suboptimal and fluctuating physical con-ditions, stationary incubation)? How are the optimal assays related to those assays doneunder more ‘‘realistic’’ conditions?

envi-A variety of methods are available for monitoring the enzyme activities when ing with microbial cultures or isolated enzymes in biochemical laboratories However,most classical enzymatic methods cannot be applied directly in aquatic environments Theenzyme amount and activity in natural waters are usually much lower than those measured

work-in cultures or work-in enzyme extracts, and therefore the classical biochemical methods oftenare inadequate for measuring low ectoenzyme reaction velocity Furthermore, the environ-mental conditions of ectoenzyme assays in water samples often are suboptimal (e.g., un-suitable temperature, pH, presence of interfering compounds) and the choice of substrateused to study ectoenzymes of natural microbial assemblages in aquatic environments often

is problematic

Depending on the chemical nature of the ectoenzyme substrate, there are three gories of methods for measurement of ectoenzyme activity in aquatic environments: spec-trophotometric, fluorometric, and radioactive The most commonly used in the past werespectrophotometric methods (60–63) The major disadvantage of spectrophotometricmethods is long incubation time necessary for enzyme reactions, which is due to theirrelatively low sensitivity (micromolar [µM] to millimolar [mM] concentrations of thefinal product of enzyme reaction are required) However, spectrophotometric assays can

cate-be used when measuring high enzyme activity in samples, or when working with purifiedand/or concentrated enzymes

During the last two decades, fluorometric methods have been widely used for zyme activity determinations in aquatic environments (3,21,24,33,52,64,65) Fluorometricassays are very sensitive, and they measure the final products of enzymatic reactions in

en-nanomolar (nM ) to micromolar ( µM) concentrations When using a modern

spectroflu-orometer to measure enzyme activity in water samples, the incubation time for ing substrate-enzyme reaction can be shortened to a few minutes Several authors haveapplied radiometric methods for enzyme activity determination in aquatic environments

monitor-(66–69) Although these methods are extremely sensitive, they are seldom used because

of greater costs and precautions needed when handling radioactive materials

B Substrates

When studying the significance of ectoenzyme activities in relation to in situ substrateturnover in aquatic (and in soil) environments, one should be able to determine the realrates of the process In this case, the substrate should have an affinity for the ectoenzymesimilar to that of the natural substrates in situ Moreover, the enzyme should be assayed

by using low substrate concentrations comparable to the concentrations of the naturalsubstrate Application of low substrate concentrations results in low levels and difficultdetection of end products Generally, in enzyme reactions, the length of incubation re-quired for end product formation is related to product detection sensitivity

A large variety of commercially produced ectoenzyme substrates are now available.Depending on their chemical structure and the enzyme assay, two types of organic com-pounds can serve as substrates: natural and artificial substrates Natural substrates arenative compounds (nonlabeled) or their chemical structure is only slightly modified bylabeling with chromophores, fluorophores, or radiolabeling with14C,3H,32P,35S, or125I.Most natural substrates have an affinity for the enzyme that is complementary to that ofthe natural substrates in aquatic samples Monitoring of enzyme activity by means ofnatural nonlabeled substrates requires a sensitive analytical method to measure the endproduct or substrate remaining after incubation time (70) Modern analytical methods offerprecise and rapid determination of several natural compounds that can be used as enzymesubstrates or products (e.g., amino acids, proteins, deoxyribonucleic acid [DNA], carbohy-drates) The application of the labeled natural substrates requires very sensitive and accu-rate methods for quantitative determination of the label bound to the substrate molecule(e.g., spectrophotometry, fluorometry, radiometry) Most suitable natural substrates areradiolabeled compounds because their end products can be measured after a short incuba-tion period (minutes) (66–69) Until now, this approach has been limited by the reducedavailability of radiolabeled substrates and the high handling costs of radioactive materials.Except for some cases of analytical difficulties, natural substrates are promising for study-ing ectoenzymes in aquatic environments

Artificial substrates are synthesized in laboratories and their chemical structure (e.g.,chemical bonds) only mimics that of natural compounds Ectoenzymes react with artificialsubstrates by splitting specific chemical bonds between an organic moiety and its chromo-phore or fluorophore, yielding colored or fluorescent products, respectively Because theseare not natural substrates, enzyme activities obtained are not necessarily identical to thosemeasured by using natural substrates However, their application allows for low costs andsimpler and more rapid measurements of ectoenzymatic activity

In the past, chromogenic artificial substrates were used intensively in the studies ofectoenzyme activity in fresh waters (10,11,60–63,71) It is advantageous to use chromo-genic substrates because they can be measured easily by spectrophotometry However,low sensitivity is a major disadvantage of this technique, and long incubation times of 72

to 96 hours often are required (62,71) This may result in microbial proliferation andectoenzyme synthesis during the assay, changes, which must be prevented They usuallyare avoided by adding plasmolytic or antiseptic agents to assays, such as toluene or chloro-form (10,38,71) However, these agents change the membranes, thereby leading to release

of ecto- and intracellular enzymes In cases in which some enzymes are located intra- andextracellularly (e.g., phosphatase, arylsulfatase), ectoenzyme activity may be significantlyoverestimated (72).

Recently, fluorophore-labeled artificial substrates have been commonly used forsensitive assays of ectoenzyme activity in aquatic environments (13–16,21,25,33,36,37,54,55,64,65) and are advantageous when it is necessary to perform a large number ofassays Fluorogenic substrates yield highly fluorescent, water-soluble products with opticalproperties significantly different from those of the substrate Many substrates are degraded

to products that have longer wavelength excitation or emission spectra Therefore, thesefluorescent products typically can be quantified in the presence of an unreacted substrate

by using a fluorometer Three types of substrates derived from water-soluble fluorophoresare commercially available: blue, green, and red (73)

Hydroxy- and amino-substituted coumarins are the most widely used fluorogenicsubstrates Coumarin-based substrates produce highly soluble, intensely blue fluorescentproducts Phenolic dyes such as 7-hydroxycoumarin (umbelliferone) and the more com-mon 7-hydroxy-4-methylcoumarin (methylumbelliferone) are not fully deprotonated andtherefore not fully fluorescent unless the reaction mixture has pH⬎ 10 (64) Substratesderived from these fluorophores are not often used for continuous measurement of enzy-matic activity Products of substrates containing aromatic amines, including the commonlyused 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin, and 6-aminoquino-line, are partially protonated at pH⬍ 5 but fully deprotonated at neutral pH Thus, theirfluorescence is not subject to variability due to pH-dependent protonation/deprotonationwhen assayed near or above physiological pH

Substrates derived from water-soluble green fluorophores, fluorescein, and mine, provide significantly greater sensitivity in fluorescence-based enzyme assays Inaddition, most of these longer-wavelength fluorophores have excitation coefficients thatare 5 to 25 times that of coumarins, nitrophenols, or nitroanilines, making them potentiallyuseful as sensitive chromogenic substrates (73) Substrates based on the derivatives offluorescein and rhodamine usually incorporate two moieties, each of which serves as asubstrate for the enzyme Consequently, they are cleaved first to the monosubstituted ana-log and then to the free fluorophore Because the monosubstituted analog often absorbsand emits light at the same wavelength as that of the ultimate hydrolysis product, this initialhydrolysis complicates the interpretation of hydrolysis kinetics (often biphasic kinetics areobserved) However, when highly purified, the disubstituted fluorescein- and rhodamine-based substrates have virtually no visible-wavelength absorbance or background fluores-cence, making them extremely sensitive substrates

rhoda-Substrates derived from water-soluble red fluorophores (long-wavelength phores) often are preferred because background absorbance and autofluorescence generallyare lower when longer excitation wavelengths are used Substrates derived from the redfluorescent resorufin and dimethylacridinone contain only a single hydrolysis-sensitivemoiety, thereby avoiding the biphasic kinetics

fluoro-The majority of fluorophore-labeled substrates produce very low background rescence and can be used without any loss in sensitivity at the high concentrations (milli-molar) that are sometimes needed for enzyme saturation (65) It also is possible to workwith substrate concentrations in the nanomolar range, close to the presumed range ofnatural substrate concentrations in aquatic environments It has been shown that substrateslinked to fluorophores provide a very sensitive system for detecting and quantifying manyspecific and nonspecific hydrolases in aquatic environments (21) The potential ectoenzy-

fluo-matic activity of water or sediment samples can be measured over a short incubationtime without problems of microbial proliferation, low activity, and nonsaturation of theectoenzyme In spite of this advantage in using the fluorescent substrates in ectoenzymeassays, their use is controversial (as are chromogenic substrates), because of their unknownaffinity for the ectoenzymes in comparison to that of natural substrates.

C Potential Enzyme Activity—Kinetic Approach

If information about the potential activity of the ectoenzyme in the aquatic habitat isrequired, there are reasons for using high concentrations of the substrate in assays Theenzyme should be substrate-saturated Possible competition with co-occurring natural sub-strates should be prevented, as should competitive inhibition of the substrate with inhibi-tors in samples (Fig 3)

Many hydrolytic ectoenzymes follow Michaelis-Menten kinetics:

v ⫽ (Vmax ⫻ [S])/(K m ⫹ [S]) where a plot of the initial velocity of reaction (v) against increasing concentrations of substrate ([S]) gives a rectangular hyperbola For such assays, the kinetic approach is

recommended; it allows calculation from the experimental data of the kinetic parameters

characterizing an enzyme-substrate reaction They are Vmax, the maximal velocity of zyme catalysis that theoretically is attained when the enzyme has been saturated by an

en-infinite concentration of substrate, and K m, the Michaelis constant, which is numerically

equal to the concentration of substrate for the half-maximal velocity (Vmax), which indicatesthe enzyme affinity to the substrate (74) The kinetic approach requires substrate concen-trations ranging from low to high for first-order (the reaction velocity increases linearlywith the increase in substrate concentrations) and zero-order (reaction velocity remainsconstant, not affected by the concentration of substrate) enzyme reactions

A typical ectoenzyme kinetic experiment may be described as follows: Data arecollected as a function of at least five triplicate reactant concentrations of substrate, andthe experimental dependence on this function is determined and plotted graphically Theresults depend essentially on the shape of the hyperbolic curve described by the data, thus

making determination of Vmaxand K mdifficult (Fig 3A) To obtain these kinetic

parame-ters, the Michaelis-Menten equation often is rearranged to the linear form and Vmaxand

K mare obtained from the slope and intercept (Fig 3B) (74)

Such graphical methods produce correct values for the parameters only in the sence of error Unfortunately, all the measurements are subject to some degree of impreci-sion, and therefore use of linearized equations such as that of Lineweaver-Burk, Eadie-Hofstee, and Woolf may give inaccurate or biased experimental data (75,76) The bestsolution to this problem is to perform a nonlinear regression analysis on the original experi-mental data The kinetic parameters then can be calculated from the direct plot of reaction

ab-velocity (v) versus substrate (S) concentration by using a computer program to determine

the best fit of the rectangular hyperbola (77)

D In Situ Enzyme Activity—Direct Approach

True ecological information requires the detection of environmental processes under insitu conditions, which cannot be fully controlled and, therefore, cannot be simulated inthe laboratory The composition of naturally occurring substrates in water samples usually

is unknown, and concentrations may vary widely over short sampling times This conditioncomplicates the choice of the substrate concentration being monitored in ectoenzymeassays because of the potential interference or competition with natural substrates and/orinhibitors.

Ideally, to prevent these problems and to measure the real in situ rates of ectoenzymeactivity, one should follow the decrease in naturally occurring substrate concentration orthe increase in ectoenzyme product formation under in situ conditions Because of theanalytical difficulties, this approach is very seldom used in aquatic studies Moreover, theincrease in concentration of ectoenzyme product in samples simply cannot be measuredbecause liberated product is simultaneously utilized by microorganisms To overcome thisproblem and to be able to measure the amount of product released from its substrate, it

is necessary to inhibit product assimilation by intact living microorganisms Several tory agents that do not inhibit enzyme activity can be used to prevent the microbial assimi-lation of low-molecular-weight products of ectoenzymatic hydrolysis of polymeric sub-strates (e.g., antibiotics or chemotherapeutics blocking active transport systems, somefixing agents such as sodium azide)

inhibi-Figure 4 Direct estimation of enzymatic hydrolysis of natural DNA by means of decrease insubstrate concentration in water samples from eutrophic Lake Mikołajskie (A) Concentration ofremaining DNA in samples after incubation times, (B) amount of DNA hydrolyzed (see text fordescription of kinetic parameters) (Chro´st, unpublished.)

Figure 4 presents an example of direct estimation of natural DNA hydrolysis ineutrophic Lake Mikołajskie To prevent microbial growth and utilization of products ofDNA degradation in the course of its hydrolysis, water samples were fixed with 0.3%sodium azide The concentration of DNA decreased from 4.75⫾ 0.08 µg L⫺1 (at timezero) to a plateau of 3.79⫾ 0.17 µg L⫺1(after 55 hours) (Fig 4A) A plot of the amount

of DNA-hydrolyzed [s] versus time of hydrolysis [t] gave a rectangular hyperbola (Fig.

4B):

s ⫽ [S n]⫻ [t])/(khydrolysis⫹ [t])

By applying a nonlinear regression analysis to the experimental data it was possible to

estimate hydrolysis parameters: [S n], concentration of DNA naturally present in water

samples, which theoretically is attained after infinite time of hydrolysis, and khydrolysis, thehydrolysis constant, i.e., the hydrolysis time of the half-concentration of natural DNA

(S n) The preceding data provide an example for determining hydrolysis parameters of anaturally occurring enzyme substrate by analysis of substrate concentration evolution inwater samples during the course of its enzymatic degradation

Using a direct approach, it also is possible to estimate the hydrolysis parameterscharacterizing in situ enzymatic degradation of natural substrates when the concentrations

of the final product are determined during the course of hydrolysis In situ hydrolysis ofproteins by proteolytic enzymes yielded increasing concentrations of free, dissolved aminoacids in lake water samples when microbial uptake was inhibited by 0.3% sodium azide(Fig 5)

Figure 5 Direct estimation of enzymatic hydrolysis of natural protein by means of release ofreaction products (amino acids) in water samples from eutrophic Lake Mikołajskie (Siuda and Kier-sztyn, unpublished.)

ACTIVITY

Both spatial and seasonal ectoenzymatic activities fluctuate markedly in lake waters(13,24,28,29,36,38) The production of ectoenzymes by microorganisms is strongly corre-

Figure 6 Seasonal aminopeptidase activity and chlorophyllaconcentration in the surface watersamples (0- to 1-m depth) from Lake Plußsee (Chro´st, unpublished.)

lated to the influx of polymeric organic matter and/or the depletion of readily utilizableUDOM in the environment (3,24,52)

Ectoenzyme production and activity show marked seasonal variation in both surfaceand deep waters of lakes In surface waters, the maximal ectoenzymatic activities occurduring the late stage of phytoplankton bloom and after its breakdown, and minimal activi-ties occur during the clearwater phase in lakes (Fig 6) (13,24,38,78)

Ectoenzymatic activity is especially lower during summer thermal stratification inthe hypolimnion than in the epilimnion of a lake, and the activity is strongly dependentupon the sedimentation rates of detritus produced in the euphotic zone (Fig 7) Usually,

a lag period is observed between the maximal ectoenzymatic activity in surface and that

in deep waters of a lake (Fig 7B,D) (78)

There also are known high diurnal fluctuations in enzyme activity in lake water(78,79) because dynamic environmental factors affect enzyme production and/or activity,

as well as microbial growth, metabolism, and biomass of microbial enzyme producers(3,25,36) In the summer epilimnion of eutrophic Lake Mikołajskie, APase activity varied

by a factor 1.8 during 24 hours (Fig 8) Minimal rates of APase activity were measuredduring the night period, and the enzyme activity continuously increased from morning

to evening The rates of enzyme activity were positively correlated with fluctuations inchlorophyllaconcentrations, indicating that phytoplankton was a major producer of alka-line phosphatase in an epilimnion of the lake In contrast to alkaline phosphatase activity,the highest rates of aminopeptidase activity in the surface water layer of eutrophic LakeGłe˛bokie were recorded during the night when the maximal concentrations of the totaland dissolved proteins were determined (78)

Current research clearly shows that orthophosphate ions (Pi) are major factors for ling microbial primary and secondary production in many freshwater environments (80–

control-Figure 7 Aminopeptidase (A, B) andβ-glucosidase (C, D) activities in the thermal stratified watercolumn of Lake Mikołajskie during summer phytoplankton bloom (A, C) and after bloom breakdown(B, D) in the epilimnion (epi-), metalimnion (meta-), and hypolimnion (hypo-) (Chro´st, unpub-lished.)

85) As confirmed by several independent approaches, the ambient Pi concentration is fartoo low to meet plankton phosphorus (P) requirements in the euphotic zone of lakes, andtherefore most (80–90%) of the P used for production of microbial biomass originatesfrom dephosphorylation of P organic compounds during their degradation

A variety of aquatic organisms (bacteria, algae, cyanobacteria, protozoa, plankton, benthic animals, and aquatic angiosperms) release Pi from organic compounds.Although contributions of the last two groups of aquatic organisms to P cycling can beimportant in some fresh waters (86), these are not discussed here

macrozoo-In this review, enzymatic microbial cycling of P is defined as the process of phorylation of organic P compounds by hydrolytic enzymes produced by microorganismsthat leads to the release of Pi into the environment surrounding microbial cells This defi-

dephos-Figure 8 Summer diurnal fluctuations of alkaline phosphatase activity (A) and concentration ofchlorophyllain the surface water samples (0–0.5 m) from eutrophic Lake Głe˛bokie (Chro´st, unpub-lished.)

nition excludes the release of Pi and dissolved organic phosphorus (DOP) compounds byzooplankton (87,88) However, since these planktonic animals can affect significantly thewhole microbial community as well as dynamics of P compounds in aquatic environments,

it is necessary to discuss selected aspects of their influence on enzymatic Pi release.Only 30%, or less, of the total organic P pool in freshwaters is composed of easilyhydrolyzable dissolved or colloidal P constituents (86) The remainder constitutes particu-late organic P that is not directly available for microbial metabolism but can be utilizedafter ingestion of food particles by herbivorous zooplankton Transformation of particulate

P into DOP compounds (incomplete digestion of food particles, zooplankton grazing)effectively accelerates enzymatic Pi release by increasing the substrate pool for phospho-hydrolases in an environment (89) One interesting and extensively studied aspect of Precycling by zooplankton entails the production of specific phosphohydrolases by theseplanktonic animals and the liberation of the intracellular phosphohydrolytic enzymes fromgrazed phytoplankton (67)

Almost all (except phosphoamides) natural DOP compounds in aquatic ments are chemically stable phosphate esters Phosphorus in these compounds is notreadily available for microorganisms because the majority of phosphate ester moleculescannot be transported directly through microbial cell membranes Limited quantities of

environ-β-glycerophosphate and several phosphorylated monosaccharides can be taken up by someaquatic bacteria (90,91); orthophosphate ions, however, are the dominant forms of phos-phorus for microbial assimilation Therefore microbial utilization of Pi from almost allits organic compounds must be preceded by their enzymatic dephosphorylation (3,13,68).Release of Pi into aquatic ecosystems is affected by a great variety of abiotic andbiotic environmental factors and processes The most important are activity of phosphohy-drolytic enzymes (3,11), zooplankton grazing (92,93), viral and spontaneous lysis of mi-croplankton cells (94,95), and UV light (96) However, it is now well founded that enzyme-mediated hydrolysis of naturally occurring phosphate esters is the most significant mecha-nism for P release in aquatic environments.

There are three main groups of hydrolytic enzymes responsible for Pi release: specific and/or only partially specific phosphoesterases (mono- and diesterases), nucleo-tidases (mainly 5′-nucleotidase), and nucleases (exo- and endonucleases) Most of them aretypical ectoenzymes (3,67) However, some of phosphohydrolytic enzymes are activelysecreted by planktonic microorganisms into surrounding water (e.g., extracellular phos-phoesterases and some nucleases) (Fig 9)

non-Figure 9 Conceptual model of enzymatic decomposition of various organic phosphorus pounds in lake water Pathways that are crucial for Pi regeneration in scale of the whole ecosystemare illustrated by bold arrows ‡@, 5′-nucleotidase; ‡A, alkaline and acid phosphatases; ‡B, exo-nucleases; ‡C, endonucleases; ‡D, phytase; ‡E, cyclic 3′,5-nucleotide phosphodiesterases and 2′,3-nucleotide phosphodiesterases; ‡F, liberation and release of DOP compounds from disrupted andliving cells; ‡G, direct uptake of organic P source (Siuda, unpublished.)