Exoenzyme activity

Sinsabaugh, R. L., and A. E. Linkins. 1988. Exoenzyme activity associated with lotic epilithon. Freshwater Biology 20 249-261. 

Hoppe H. G. (1983) Significance of exoenzyme activities in the ecology of brackish water measurments by means of methylumbelliferyl-substrates. Mar. Ecol. Prog. Ser. 11, 299-308. 

Christian, J.R. and Karl, D.M. (1995) Measuring bacterial exoenzyme activities in marine waters using mercuric chloride as a preservative and a control. Marine Ecology Progress Series 123, 217-224. 

The bacterial culture converts a portion of the supplied nutrient into vegetative cells, spores, crystalline protein toxin, soluble toxins, exoenzymes, and metabolic excretion products by the time of complete sporulation of the population. Although synchronous growth is not necessary, nearly simultaneous sporulation of the entire population is desired in order to obtain a uniform product. Depending on the manner of recovery of active material for the product, it will contain the insolubles including bacterial spores, crystals, cellular debris, and residual medium ingredients plus any soluble materials which may be carried with the fluid constituents. Diluents, vehicles, stickers, and chemical protectants, as the individual formulation procedure may dictate, are then added to the harvested fermentation products. The materials are used experimentally and commercially as dusts, wettable powders, and sprayable liquid formulations. Thus, a... 

In this chapter we describe the use of pea seeds to express the bacterial enzyme a-amylase. Bacterial exoenzymes like the heat stable a-amylase from Bacillus licheni-formis are important for starch hydrolysis in the food industry. The enzymatic properties of a-amylase are well understood [13,14], it is one of the most thermostable enzymes in nature and it is the most commonly used enzyme in biotechnological processes. Although fermentation in bacteria allows highly efficient enzyme production, plant-based synthesis allows in situ enzymatic activity to degrade endogenous reserve starch, as shown in experiments with non-crop plants performed under greenhouse conditions [12,15]. Finally, the quantitative and sensitive detection of a-amylase activ-... 

In Azotobacter vinelandii, urease appears to be synthesized only when urea or thiourea is present (75). A study of the urease constitutive in Corynebacterium renale (76) did not reveal features remarkably different from the plant enzyme. A similar conclusion was reached in the characterization of a highly purified enzyme from B. pasteurii (77). Stewart (78) has devised a medium for the detection of urease activity in pseudomonads and has resolved uncertainties that have developed in the literature. It has been reported that Sarcina ureae produces urease as an exoenzyme (79). 

Rates of supply may be controlled, in part, by the activity of hydrolytic enzymes that degrade combined molecules in dissolved or particulate organic matter to monomeric constituents prior to enzymatic transport into bacterial cells (Skoog et al., 1999 see also Chapter 13). At present, in situ rates of such exoenzymes have not been accurately measured, but enzyme potential has been estimated with the addition of artificial substrates in saturating... 

The only known lipase that is nonspecific is the enzyme of a mold. Geotrichum candidum. It has a decided preference for fatty acids with a cis-A9 double bond such as oleic acid 14). There is no explanation for this specificity. The lipases of most other microorganisms are very similar to pancreatic lipase. For example, the lipase of the mold Rhizopus arrhizus is an exoenzyme which contains carbohydrates that are not essential for activity. It is not inhibited by DFP, it is specific for primary ester groups, and it does not distinguish between diflEerent fatty acids (15). Other microbial lipases are similar in these respects but may diflFer in their recognition of steric hindrance. The lipase of Stapyhlococcus aureus... 

ADP-ribosylation by C3 and its isoenzymes is a reversible reaction, i.e. in the presence of high concentrations of nicotinamide (lOm/Vl) and at low pH (pH <7) it can be reversed (Habermann et ai, 1991). The de-ADP-ribosylation reaction has been exploited to identify the acceptor amino acid of ADP-ribosylation by C3-like isoforms. Like other ADP-ribosyltransferases, C3-like exoenzymes exhibit NAD gly-cohydrolase activity (Aktories et ai, 1988b). However, this enzyme activity is at least 100-fold lower than the transferase activity and most likely has no physiological significance. 

Akfories K, Jung M, Bohmer J et al. (1995) Studies on the active site structure of C3-like exoenzymes Involvement of glutamic acid in catalysis of ADP-ribosylation. In Biochimie 77 326-32... 

Laudanna C, Campbell JJ, Butcher EC (1996) Role of Rho in chemoattractant-activated leukocyte adhesion through integrins. In Science 271 981 -3 Machesky LM, Hall A (1996) Rho a connection between membrane receptor signalling and the cytoskeleton. In Trends Cell Biol. 6 304—10 Maehama T, Ohoka Y, Ohtsuka T et al. (1990) Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids. In FEBS Lett 263 376-80 Malcolm KC, Ross AH, Qiu R-G etal. (1994) Activation of rat liver phospholipase D by the small GTP-binding protein RhoA. In J. Biol. Chem. 269 25951 -4 Moriishi K, Syuto B, Yokosawa N efal. (1991) Purification and characterization of ADP-ribosyltransferases (Exoenzyme C3) of Clostridium Botulinum type C and D strains. In J. Bacferiol. 173 6025-9... 

Saito Y, Nemoto Y, IshizakiTetal. (1995) Identification of Glu as the critical amino acid residue for the ADP-ribosyltransferase activity of Clostridium botulinum C3 exoenzyme. In FEBS Lett. 371 105—9... 

Figure 1 shows a time course for ADP-ribosylation using 1 g of recombinant Rho fused with glutathione S transferase and 3 ng of C3 exoenzyme. The reaction proceeds in a time-dependent manner and reaches a plateau at 30 min. In contrast, no ADP-ribosylation is observed in the reaction using the E173Q mutant (Fig. 1). The recombinant wild-type enzyme has a catalytic activity of 1.3 pmol of ADP-ribose transferred/ng/h and the Km value for NAD is 0.125 [iM (Fig. 2).

In the past decade, toxins produced by various species of Clostridium were reported to disrupt the ACTSK by ADP-ribosylation of either actin, as does the C2-toxin from C. botulinum (Aktories et al., 1986 Reuner et al., 1987), or the small GTPase Rho, as does exoenzyme C3 from the same bacterium (Aktories et al., 1987 Chardin et al., 1989). However, neither of the C. difficile toxins was found to have any ADP-ribosyltransferase activity (Florin and Thelestam, 1991 Just et al., 1994b Popoff eta/., 1988). 

Microbial fermentation of CSM, which was proposed to detoxify FG in the CSM (Zhang et ah, 2006a,b), seems promising because some exoenzymes such as cellulolytic enzymes, amylase, protease, and lipolytic enzymes that are secreted by certain microorganisms, and some vitamins, as well as some unknown active substances are produced in the fermented CSM (Brock et ah, 1994), which adds nutritional value of the fermented CSM. Recently, Qian et ah (2008) reported that in situ alkaline-catalyzed transesterification could produce a CSM with FG and TG contents below the FAO standard. However, the requirement for a high amount of methanol usage in the in situ transesterification and the potential energy consumption to remove the methanol in the meal may be an obstacle for its practical application. 

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