Extracellular Enzyme Hydrolysis

Extracellular enzymes are generally defined as enzymes that have crossed the cytoplasmic membranes of the microbial cell. These enzymes are divided into two groups. 

A majority of the extracellular enzymes released by the microbes are bound to the solid surface (such as particulate detrital matter or soil organic matter in clays), but a small fraction remains in the soil pore water (Eigure 5.17). Those free in soil pore water are most susceptible to microbial degradation and chemical alteration. Surface-bound enzymes may not be as effective as free enzymes because of a slow rate of substrate diffusion to the sites where enzymes are present. 

Because extracellular enzyme activity is the rate-limiting step in microbially mediated decomposition of plant detritus in wetlands and aquatic systems, many researchers have studied enzyme 

FIGURE 5.17 Fate of extracellular enzymes after they are released form microorganisms. 

FIGURE 5.18 Substrate bonded to p-nitrophenyl glucoside is hydrolyzed hy enzymes and released as p-nitrophenol, which determined by colorimetric methods. 

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X particulate substrate (hydrolyzable substrate) available as substrate for the microorganisms after hydrolysis by extracellular enzymes and diffusion of the products into the cell... 

On the other hand, Haines and Alexander reported that PEG 20,000 might be hydrolyzed by an extracellular enzyme of Pseudomonas aeruginosa ( ). According to our experiments, PEG 6000 was not hydrolyzed by an extracellular enzyme (the supernatant fluid of a culture), but it was metabolized by washed cells and the culture could not utilize ethylene glycol or diethylene glycol which might be the hydrolysis products of PEG. 

Second, the polymer needs to be broken down into small fragments. Microorganisms excrete extracellular enzymes that cleave the polymeric chains [4]. This enzymatic cleavage reaction, on the one hand, needs functional sites within the polymer backbone where the enzymes can catalyze the cleavage of chemical bonds. On the other hand the polymeric chain needs to be flexible enough that the chain can enter the catalytic site of the enzyme. In most cases, the chemical reaction catalyzed by the exo-enzymes is a hydrolysis process that converts the polymer chain into smaller oligomers and monomers [4]. 

Chylomicrons transport dietary triacylglycerol and cholesteryl ester from the intestine to other tissues in the body. Very-low-density lipoprotein functions in a manner similar to the transport of endogenously made lipid from the liver to other tissues. These two types of triacylglycerol-rich particles are initially degraded by the action of lipoprotein lipase, an extracellular enzyme that is most active within the capillaries of adipose tissue, cardiac and skeletal muscle, and the lactating mammary gland. Lipoprotein lipase catalyzes the hydrolysis of triacylglycerols (see fig. 18.3). The enzyme is specifically activated by apoprotein C-II, which... 

The hydrolysis of tomatine by an inducible extracellular enzyme from Fusarium oxysporum f. sp. lycopersici has been described.56... 

Several general approaches have been used to measure the activities of extracellular enzymes in aquatic systems. These methods typically measure a potential activity, inasmuch as a substrate added to a sample to measure enzyme activity is in competition with naturally occurring substrates (whose concentration is usually unknown) for enzyme active sites. The most commonly applied method involves a small substrate proxy, typically consisting of a monosaccharide or an amino acid covalently linked to a small fluorophore substrates frequently used include methyumbellifery- (MUF-) monosaccharides and 4-methyl-coumainylamide (MCA)- amino acids. Upon hydrolysis of the bond between the monomer and the fluorophore, the fluorophore becomes fluorescent, and hydrolysis is measured as an increase in fluorescence signal with time (Hoppe, 1983 Somville and Billen, 1983). 

FIGURE 3 Several hypothetical scenarios which might explain resistance of organic macromolecules to extracellular enzymatic hydrolysis (a) natural substrates are not a good fit for enzyme active sites, perhaps because of biological or chemical modifications (b) specific substrates are too dilute to induce enzymes under most circumstances (c) substrates are physically protected from hydrolysis (e.g., Mayer, 1994 Keil et al., 1994). Enzymes may also be complexed, hindering their activities (Wetzel, 1993). 

Keith, S. C., and C. Arnosti. 2001. Extracellular enzyme activity in a river-bay-shelf transect Variations in polysaccharide hydrolysis rates with substrate and size class. Aquatic Microbial Ecology 24 243—253... 

The present study indicates that the extracellular enzyme, pepsin, exhibits striking differences from its mammalian homologue with respect to optimum pH, Ea for catalysis, thermal stability, and substrate affinity. These data are interesting from the viewpoint of biological adaption at low temperatures, but they also provide some substance to our contention that enzymes from fish plant wastes can have sufficiently unique properties to justify their use over conventional sources of enzymes used as food-processing aids. The relatively low Eas for protein hydrolysis by fish pepsins indicate they may be especially useful for protein modifications at low temperatures. Alternatively, the poor thermal stability of the fish pepsins studied indicate that the enzymes can be inactivated by relatively mild blanching temperatures. The reality of this concept will have to await studies where the pepsins are used as food-processing aids. Such studies are currently underway in our laboratory. 

Lipoprotein lipase, an extracellular enzyme, causes the hydrolysis of VLDL and chylomicron triglycerides. Hormone-sensitive lipase catalyzes the hydrolysis of intracellular (storage) triglyceride. 

Pullulanase is an extracellular enzyme of Aerobacter aerogenes that causes essentially quantitative hydrolysis of pullulan to maltotriose. The enzyme is readily prepared in a crude form that is free from other carbohydrases, and is important in structural studies because it debranches amylopectin and glycogen. When the A. aerogenes is grown in continuous culture, the enzyme is bound to the cells, but it can be released by detergents, and purified by ion-exchange chromatography. This purified enzyme has been crystallized. ... 

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