Enzyme microenvironment

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

Depending on the immobilization procedure the enzyme microenvironment can also be modified significantly and the biocatalyst properties such as selectivity, pH and temperature dependence may be altered for the better or the worse. Mass-transfer limitations should also be accounted for particularly when the increase in the local concentration of the reaction product can be harmful to the enzyme activity. For instance H2O2, the reaction product of the enzyme glucose oxidase, is able to deactivate it. Operationally, this problem can be overcome sometimes by co-immobilizing a second enzyme able to decompose such product (e.g. catalase to destroy H202). 

It has been suggested that the inhibition of ALDH by AGP 17 starts with an interaction between the amino group of 17 and the cysteinyl thiolate side chain of the enzyme to form a modified holoenzyme, or that the enzymic reaction may either proceed through the cyclopropanone 20 yielding 24 a or through the imi-nium ion 18, yielding the modified enzyme 24b [17]. Thus, the covalent he-mithioacetal 24a or hemithioaminal enzyme derivatives 24b rapidly accumulate in the enzyme microenvironment and lead to the observed activity loss, Eq. (9) [21]. 

Several factors may limit the overall rate of enzymatic reductive reactions. First, the electron transfer to the reactive metal (e.g., Co, Fe, or Ni) may be limiting. It is also possible that access of the organic substrates to the reduced metals contained within enzyme microenvironments may be limited. Mass transfer limitation is even more important in intact bacterial cells. For example, Castro et al. (1985) found that rates of heme-catalyzed reductive dehalogenations were independent of the heme content of the cells. 

The limited work with LM encapsulated enzyme systems shows that the method is indeed viable for enzyme immobilization. The ability to encapsulate multiple enzyme / cofactor systems (32) while maintaining a selective permeation barrier is a distinct advantage of LMs. Since enzymes in emulsions can associate with a hydrocarbon layer, there is some degree of hydrophobicity present in the enzymes microenvironment. A hydrophobic environment is known to be beneficial for the stability of many potentially useful enzymes which deactivate in aqueous solution. Another advantage of LM encapsulation is that the enzyme can be protected from conditions and/or reagents present in the bulk external aqueous phase which might be deleterious to its activity. 

As evident from Fig. 8.4, an increase in the selectivity has been observed in IL/ scCOj biphasic systems media (>99.5%) with respect to scCO assayed alone (95%). These results could be explained by the use of water-immiscible ILs which have a specific ability to reduce water activity in the enzyme microenvironment. The synthetic activity of the immobilized lipase in IL/scCO biphasic systems is lower than that in scCO assayed alone. Similar results were found by Mori et al. [40] in IL/ hexane biphasic systems. These authors reported that the enzymatic membranes prepared by simple adsorption of CaLB onto the surface were more reactive than membranes prepared with ILs. As can be observed in Fig. 8.4, the initial reaction rate in the assayed IL/scCO biphasic systems increased in the following sequence [bdimim ][PF ]<[bmim ][PFg ]<[bmim ][NTfj ]<[omim ] [PF ], which was practically in agreement with flie activity sequence reported by these authors using free Candida antarctica lipase B in homogeneous ionic liquid systems ([bmim ] [PF ]<[bdmim+][PFg ]<[bmim+][NTfj ]<[omim ][PF ]), with the exception of [bmim [PF ] and [bdimim+][PFg ]. These results were explained taking into account that biotransformation occurs within the ionic liquid phase, so substrates have to be transported from scCOj to the ionic liquid phase. The mechanism of substrate transport between the ionic liquid and the supercritical carbon dioxide could be by three consecutive steps diffusion of the substrates through the diffusion... 

Often, one of the products can be partitioned from the enzyme microenvironment, leading to a favorable shift in equilibrium and completion of the reaction (see Chapter 18). 

Barreiro 1999). However, the reaction becomes readily reversible when the concentrations of sucrose within the enzyme microenvironment drop to values lower than 4 mM (Figure 5-1). 

ILs with NTf or PF - anions, besides their mild hydrogen-bond accepting properties, are also hydrophobic in nature. Hydrophobic ILs are superior to hydrophilic ones, in terms of stability, since they tend to preserve the essential water layer around the protein, thus maintaining the catalytically active conformation of enzymes [26, 27, 31, 45, 49, 53, 55, 89, 90,102] or reducing direct protein-ion interactions [53,55, 105]. It has been specifically pointed out, in the case of water/[bmim]PF microemulsions, that a small amount of the IL could be soluble in the aqueous microphase, thus exerting a protective effect on the enzyme microenvironment [106]. On the other hand, MD studies reveal a significant reduction of the size of water clusters at the protein surface and their replacement by the IL in media (such as [bmimJCl or [bmim]N03) may result a destabilization effects [104,105]. 

Acid hydrolase activity is poised for optimal activity and control by the nature of the enzyme microenvironment within the lysosome-vacuolar apparatus. A significant feature of this control has already been discussed in Section VI A.l. It was demonstrated that an interposed membrane precluded normal access of substrate to active site. In this section we will consider those factors which allow for optimal hydrolase activity within the compartmentalized vacuolar system. It must be realized that the lysosome-vacuolar system is maintained in a dynamic state and that trans-membrane diffusion of intermediates and metabolites may be oc-... 

The kinetics of enzymatic reactions in microemulsions obey, as a rule, the classic Michaelis-Menten equation [6,26,35], but difhculties arise in interpreting the results because of the distribution of reactants, products, and enzyme molecules among the microphases of the microemulsion [8,36-38], In addition, there are some enzymes in reverse micelles that exhibit enhanced activity as compared to that expressed in water this has given rise to the concept of superactivity [6,26,39], The superactivity has been explained in terms of the state of water in reverse micelles, the increased rigidity of the enzymes caused by the surfactant layer, and the enhanced substrate concentration at the enzyme microenvironment [36,40],... 

Coprecipitation of enzymes and a polymer rich in amino groups such as poly-ethyleneimine or pentaethylenehexamine were proposed as one solution to the problems outlined above [56,60,61]. Lipases from Alcaligenes sp. and Candida antarctica fraction B were coprecipitated with polyethyleneimine and polyeth-yleneimine sulfate-dextran, which changed the enzyme microenvironment and also physically stabilized the biocatalyst. The greater stability of copredpitated lipase CLEA was apparent from the absence of free lipase after samples were boiled in detergent (sodium dodecylsulfate) and analyzed by gel electrophoresis. By conUast, conventional CLEAs rdeased free enzyme into solution under... 

---