Activity recovery, enzymes

S. viridosporus LiP has been concentrated by ultrafiltration (UF) for further studies on the enzyme purification and the determination of its chemical and biochemical properties [10, 11, 13, 14]. However, there is no report on the UF operational conditions and on the enzyme activity recovery. 

Figure 5.9 Recovery of enzyme activity after rapid dilution as described in Figure 5.8. Curve a represents the expected behavior for a control sample that was pre-incubated and diluted in the absence of inhibitor. Curve b represents the expected behavior for a rapidly reversible inhibitor. Curve c represents the expected behavior for a slowly reversible inhibitor, and curve d represents the expected behavior for an irreversible or very slowly reversible inhibitor. See color insert.

The initial hydroxylation of tryptophan, rather than the decarboxylation of 5-HTP, appears to be the rate-limiting step in serotonin synthesis. Therefore, the inhibition of this reaction results in a marked depletion of the content of 5-HT in brain. The enzyme inhibitor most widely used in experiments is parachlorophenylalanine (PCPA). In vivo, PCPA irreversibly inhibits tryptophan hydroxylase, presumably by incorporating itself into the enzyme to produce an inactive protein. This results in a long-lasting reduction of 5-HT levels. Recovery of enzyme activity, and 5-HT biosynthesis, requires the synthesis of new enzyme. Marked increases in mRNA for tryptophan hydroxylase are found in the raphe nuclei 1-3 days after administration of PCPA [6]. 

A (rapidly) reversible inhibitor will permit rapid and complete recovery of enzyme activity by dialysis. However, irreversible inhibitors are not removed by this procedure. Recovery from tight-binding inhibition is usually slow it is not uncommon for several dialysis bags containing enzyme to be prepared and for activity in each to be determined at various time points following the commencement of dialysis. The off-rate of these inhibitors is generally more rapid at higher temperatures. 

If an inhibitor has a slow ofif-rate , this may be observed in a continuous assay as a slow recovery in enzyme activity, with an initial rate equivalent to that expected with a reversible inhibitor increasing slowly until the rate is equivalent to that expected with a reversible inhibitor. In the earher example, the rate measured in group 4 would thus be expected to increase slowly almost ninefold, as inhibitor dissociates from the enzyme. 

The schematic below shows that in this dynamic system PAN could affect the synthetic process (site 1), the enzyme itself (site 2), or the degradation process (site 3). If the site of attack were site 2, the synthetic process might compensate for degradation of the enzyme by producing more. If the enzyme activity were measured as a function of time after exposure, there would be first a decrease and then recovery of activity. (Such a response has been observed for the effect of ozone on respiration.) Effects at site 3 would show first an increase in activity and en, if the system were regulated, a decline to normal, as the synthetic process slowed down. Effects at site 1 would cause a decrease in activity commensurate with the rate of enzyme degradation. 

Assays with Crude Extracts. Assays of the activities present in crude culture extracts were useful to indicate the enzymes available for recovery. Extracts from L. edodes typically exhibited a wide range of enzyme activities present in quantities apparently sufficient for isolation and characterization (Table I). 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Cabral and coworkers [253] have investigated the batch mode synthesis of a dipeptide acetyl phenylalanine leucinamide (AcPhe-Leu-NH2) catalyzed by a-chymotrypsin in a ceramic ultrafiltration membrane reactor using a TTAB/oc-tanol/heptane reverse micellar system. Separation of the dipeptide was achieved by selective precipitation. Later on the same group successfully synthesized the same dipeptide in the same reactor system in a continuous mode [254] with high yields (70-80%) and recovery (75-90%). The volumetric production was as high as 4.3 mmol peptide/l/day with a purity of 92%. The reactor was operated for seven days continuously without any loss of enzyme activity. Hakoda et al. [255] proposed an electro-ultrafiltration bioreactor for separation of RMs containing enzyme from the product stream. A ceramic membrane module was used to separate AOT-RMs containing lipase from isooctane. Application of an electric field enhanced the ultrafiltration efficiency (flux) and it further improved when the anode and cathode were placed in the permeate and the reten-tate side respectively. 

Endothelial cells recovered from both the enzyme (trypsin) recovery system (ERS) and the poly(IPAAm) temperature recovery system (TRS) were subcultured and examined comparatively for cell adhesivity, cell morphology and cell growth activity. The most important finding in the subcultured system was that the TRS exhibited much a higher activity of prostacyclin generation than ERS. It is known that prostacyclin generation is an important function of endothelial cells. 

The Sheldon group [87] prepared aquagels of different HNLs and compared them in the synthesis reaction of different cyanohydrins with the CLEAs and the free enzymes. The activity recovery for the aquagels and CLEAs measured by a photometric assay were quite low. Using the same loadings, the aquagels turned out to be much faster than the free enzyme. This confirms the underestimation of the recovery of activity by fast assays due to diffusion problems, as reported earlier [74, 75]. The stability and the catalytic performance of the immobilized HNLs are strongly influenced by the solvent, the immobilization method, and the enzyme source. 

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. Reversible inhibitors bind to enzymes through noncovalent bonds. Dilution of the enzyme-inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. Irreversible inhibition occurs when an inhibited enzyme does not regain activity on dilution of the enzyme-inhibitor complex. The two most commonly encountered types of inhibition are competitive and noncompetitive. 

As shown in the preceding section, in the presence of 5 M guanidine hydrochloride the six subunits of the enzyme were separated from one another and opened up to form randomly coiled, single polypeptide chains. These were catalytically inactive (13). When the guanidine hydrochloride was removed by dialysis, enzymic activity was restored, provided the protein was in a reducing environment during the dialysis (Table II). Under optimal conditions (No. 5 of Table II) there was 80-90% recovery of activity. If there was opportunity for persistent disulfide... 

Reoxidation of reduced S-protein results in potential activity recovery but only about 20% of that seen with RNase-A (206). In the presence of S-peptide the reoxidation of S-protein leads to much higher levels of activity. The addition of the rearranging enzyme to a solution of S-protein results in the immediate drop in potential activity to about 20% of the starting value (207). If S-peptide is added to this mixture the activity rapidly increases and approaches 90-100%. If the SS bonds were totally random in reoxidized S-protein the activity should have been much less than 20%. There is a bias in favor of an approximately correct structure therefore, the actual distribution of S-S bonds in reoxidized S-protein would be extremely interesting and might shed... 

The main data associated with the purification are summarized in table 6.4. This table indicates the total protein obtained in each step, the number of enzyme units3 for each enzyme, and the ratio of enzyme units to total protein, called the specific activity. In the absence of enzyme inactivation, the specific activity should be directly proportional to the enrichment. The percent recovery refers to the amount of enzyme activity in the indicated fraction, as compared with the amount present in fraction 1. This number is usually less than 100%. The apparent losses may reflect actual losses of enzyme during purification, or they may reflect inactivation (usually due to unknown causes) of the enzyme during purification. 

Basic Biological Processes rate of recovery of mice from anesthesia electrical resistance of plants rate of fungus growth rate of enzyme activity movement of protozoans healing rates of injured mice growth of plants. 

Hydrates have further applications in bioengineering through the research of John and coworkers (Rao et al., 1990 Nguyen, 1991 Nguyen et al., 1991, 1993 Phillips et al., 1991). These workers have used hydrates in reversed micelles (water-in-oil emulsions) to dehydrate protein solutions for recovery and for optimization of enzyme activity, at nondestructive and low-energy conditions. 

Ververidis, P. John, P. (1991). Complete recovery in vitro of ethylene-forming enzyme activity. Phytochemistry 30, 725-727. 

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