Selective heat inactivation

Another method which may become a useful technique for selective inactivation of cellulases in enzyme mixtures is the use of selective heat inactivation. While establishing the thermostability properties of crude xylanases from a fungal strain Y-94, Mitsuishi et al. (80) observed differential heat labilities of the cellulase and xylanase activities in the culture filtrate. After an incubation period of 20 minutes at 65°C, the xylanase activity was reduced by 5-10% whereas the Avicelase and /3-glucosidase activities were reduced by 100% and 60%, respectively. We have observed a similar temperature dependency of xylanase and cellulase activities in T. auranti-acus. As indicated in Figure 2, treatment of the culture filtrate at 70°C for 20 minutes resulted in less than a 5% loss in xylanase activity whereas cellulase activities were reduced by 40-50%. A similar effect has also been observed for the xylanases and cellulase enzymes produced in culture filtrates from T. harzianum (93). Further work in the area of heat treatments may improve the effectiveness of cellulase inactivation. Since the cellulase activities of some enzyme preparations can be more rapidly inactivated on... 

In addition to having the required spedfidty, lipases employed as catalysts for modification of triglycerides must be stable and active under the reaction conditions used. Lipases are usually attached to supports (ie they are immobilised). Catalyst activity and stability depend, therefore, not only on the lipase, but also the support used for its immobilisation. Interesterification reactions are generally run at temperatures up to 70°C with low water availability. Fortunately many immobilised lipases are active and resistant to heat inactivation under conditions of low water availability, but they can be susceptible to inactivation by minor components in oils and fats. If possible, lipases resistant to this type of poisoning should be selected for commercial operations. 

Primary CLL cells purified from peripheral blood of volunteer patients (see Note 3) CLL cells are purified by Ficoll gradient centrifugation, by negative selection, or by a combination of both (see Note 4). CLL cells can be used immediately or frozen in 90% heat/inactivated FBS supplemented with 10% DMSO and stored at -80°C or in liquid nitrogen for short-and long-term storage, respectively. CLL cells are cultured in complete RPMI medium and maintained in a humidified atmosphere of 5% CO at 37°C (see Note 5). 

In the range of temperatures selected for evaluation, the soluble invertase presented detectable instability only at 55°C (Table 3 and Fig. 4). Hence, the thermodynamic parameters for the heat inactivation of invertase (AG, AH, A S and Ea ) were not determined, because the correlation log k x T 1 might necessarily be established, as proposed by Owusu and... 

Several examples have shown that the degree of activity resulting from synthesis is reproducible, as is the amino acid composition. In other cases, e.g., with p-nitrophenyl acetate, activity was quite variable. Nearly total inactivation by heat in aqueous solution has been demonstrated for some pyropolyamino acids other such systems are heat-stable in aqueous solution. In the p-nitrophenyl acetate system, the nature of the heat inactivation, if not the mechanistic reason for enhanced activity, is understood to involve both imide and imidazole residues. Differing interactions of these residues to produce loci of varying degrees of efficiency could help to explain the quantitative nonreproducibility of activity in separate syntheses. With OAA, selectivity of action was strict, in that several a-keto acids were not measurably acted upon under controlled conditions. The identification of the active locus for hydrolysis of the substrate p-nitrophenyl acetate supports the general inference of specificities, inasmuch as similarly prepared polymers have been shown not to be operative for other reactions, e.g., decarboxylation of OAA (17). 

PMN had been isolated by centrifugation through PolymorphPrep and positive selection by anti-CD15 beads (MAGS) and were suspended in AIM-V containing 2.5% autologous heat-inactivated serum. 

The other approach is the selective inactivation of specific isoenzymes. Placental alkaline phosphatase, for instance, is remarkably stable to heat inactivation. Incubation of the enzyme at 65 °C has no effect on its activity, unlike the other isoenzymes which are inactivated. Other isoenzymes can be differentiated by their stability in other conditions. For instance phenylalanine inhibits placental and intestinal isoenzymes but has little effect on the bone and liver isoenzymes. 

The dehydration reaction of aldoxime to form nitriles using the resting cells of Rhodococcus sp. YH3-3 was optimized. We found that the enzyme was induced by aldoxime and catalyzed the stoichiometric synthesis of nitriles from aldoximes at pH 7.0 and 30°C. Phenylacetonitrile once synthesized from phenylacetaldoxime was hydrolyzed to phenylacetic acid, since the strain has nitrile degradation enzymes such as nitrile hydratase and amidase. We have been successful in synthesizing phenylacetonitrile and other nitriles stoichiometrically by a selective inactivation of nitrile hydratase by heating the cells at 40°C for 1 h. Various nitriles were synthesized under optimized conditions from aldoximes in good yields. 

Methods. Samples of from 0.1-10 mg of the oligosaccharide were dissolved in 0.05-0.2 ml of water, mixed with an equal volume of enzyme solution, and incubated at room temperature. In the experiments the concentrations of the oligosaccharide and enzymes were selected in a ratio such that saturation levels of the substrates were present during the incubations (8). Samples of 5 / liters were removed from the various digests at specified time intervals, placed on paper chromatograms, and heated in an oven at 100°C for 5 minutes to inactivate the enzyme. Upon completion of the experiment, the chromatograms were developed... 

Sample Preparation. Broiled beefsteaks were selected as the basic experimental material. Steaks 5/8 inch thick and 3 inches in diameter were prepared from major round muscles of choice grade beef and were oven-broiled 3 inches from the heating elements for 12 minutes on the first side and 10 minutes on the second. These conditions produced an internal temperature of about 75°C., which was sufficient for complete enzyme inactivation (8). This cooking schedule produced a medium well done steak. Either all steaks to be used in a given test series were prepared from the same carcass or steaks prepared from different carcasses were randomized with respect to treatment variables. All samples were prepared this way unless otherwise indicated. 

Screening and selection of the source plasma will only avoid contamination by known pathogens. The protein purification steps and specific virus reduction methods used in production processes, however, will inactivate and/or remove both known and unknown viruses. Terminal virus inactivation treatments are applied to product in final container and must balance virus inactivation with any modifications to protein immunogenicity, activity, and yield. While many upstream virus inactivation steps rely on chemical methods that involve the addition and subsequent removal of toxic agents (e.g., solvent/detergent), physical methods for virus inactivation, such as pH and heat, are used for terminal steps. 

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