Optimum incubation period

Lipases are sensitive to extremes of pH, and even in the vicinity of the pH optimum, where enzymes are supposedly more stable, marked inhibition may occur (Frankel and Tarassuk 1956B). Thus, it must also be borne in mind that the length of the incubation period and the prior history of the preparation can influence the range and perhaps the shape of the pH activity curve. 

It is essential (Kendall et al., 1983 Yolken et al., 1983) to biotinylate correctly the antibody or enzyme, since there is a pronounced optimum (Section 3.2.2). However, current avidin/biotin methods may have to be changed with respect to biotinylation procedures to attain their full potential. The optimal dilutions of the biotinylated antibody, biotinylated enzyme and avidin solutions have to be determined and the test performed as detailed in Table 14.4. It is essential to use incubation periods of about 20 min (pronounced optimum) for the reaction between the biotinylated proteins and avidin (Kendall et al., 1983). 

A 60 min incubation period was chosen as optimum since it gave the highest level of light output in the shortest period of time. 

The formation of a SAM on gold electrode surface may take longer than 12 h due to the surface roughness and the concentration of the thiolated oligonucleotides. In certain cases, incubation periods of 5 days for the immobilization of 1 pM 23-mer oligonucleotides on 10 pm gold microelectrodes (i.d.) have been found as the optimum period. 

Soil/Plant. In plants and soils, the nitro groups reduced to amino groups (Hartley and Kidd, 1987). When 4,6-dinitro-o-cresol was statically incubated in the dark at 25°C with yeast extract and settled domestic wastewater inoculum, no significant biodegradation and necessary acclimation for optimum biooxidation within the 4-week incubation period was observed (Tabak et al., 1981). 

The major drawback of this process is the long incubation periods needed for optimum oil recovery. The additional cost of enzymes on top of solvent cost could also be of concern although it is possible that solvent usage could be drastically reduced for enzyme treated materials. The quality characteristics of the extracted oil and the meal may, on the other hand, be important factors in considering this process for industrial use. 

The E. coli systems of both Nisman and of Spiegelman, as well as the A. faecalis system of Beljanski, have been purified to the point where only 100-300 Mgm of protein per milliliter are required for activity. [As a matter of fact, in all of these systems there seems to be an optimum concentration of protein, beyond which considerable inhibition is observed for normal periods of incubation (60 min) however, Nisman and Fukuhara 297) found that higher concentrations of enzyme produce a considerable enhancement of activity if longer incubation periods (120-180 min) are used. This, again, is quite similar to the behavior of the pea seedlings system 147), and illustrates well the complexity of both.]... 

It is necessary to estabUsh a criterion for microbial death when considering a sterilization process. With respect to the individual cell, the irreversible cessation of all vital functions such as growth, reproduction, and in the case of vimses, inabiUty to attach and infect, is a most suitable criterion. On a practical level, it is necessary to estabUsh test criteria that permit a conclusion without having to observe individual microbial cells. The failure to reproduce in a suitable medium after incubation at optimum conditions for some acceptable time period is traditionally accepted as satisfactory proof of microbial death and, consequentiy, stetihty. The appHcation of such a testing method is, for practical purposes, however, not considered possible. The cultured article caimot be retrieved for subsequent use and the size of many items totally precludes practical culturing techniques. In order to design acceptable test procedures, the kinetics and thermodynamics of the sterilization process must be understood. 

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

Most enzymes show a bell-shaped pH-velocity profile and a characteristic pH at which their activity is maximal. Figure 5.13 shows V0 vs pH curves for three enzymes. Note that both the pH optimum and the form of the velocity profile vary with the enzyme. Such curves must be interpreted with caution, as they give no indication why the velocity declines above and below the pH optimum. The decline in rate may be due to the formation of improper forms of the enzyme or substrate (or both) or inactivation of the enzyme, or it may be due to a combination of these factors. The possibility of enzyme inactivation is frequently overlooked, although a pH stability curve is necessary for enzyme characterization. A pH stability curve is readily obtained by preincubating the enzyme at a specified pH for a period of time equal to the assay incubation time and then assaying activity at the optimum pH. 

After 5 min at 30 C, add 1/5 volume 0.03% H2O2 and continue incubation for desired period (10-100 min Table 10.2). It might be useful to determine the optimum peroxide concentration for a particular system. 

It was necessary to find a better procedure for obtaining and maintaining the cultures free of dissolved oxygen during the entire period of incubation and then to repeat the experiments to find the most eflFective bacteria and the optimum conditions. 

Spawn jar discolored with yellowish droplets of fluid. Spawn jar incubated for an overly long period of time, at higher than optimum temperatures, or both, causing the exudation of metabolites ("sweat") and the build-up of fluids in which bacteria thrive. Incubate at temperature and for period of time recommended for species being cultivated. 

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