Enzymic reaction, controlled modification

Thus, rather than trial-and-error development of functionality, it should be possible to design functionality based on the principles of protein structure and function and the specificities of the enzymes used for modification. Use of immobilized exo- and endopeptidases in such technology could be especially attractive for the reasons listed in Table I, particularly since problems associated with autolysis would be eliminated and the extent of proteolytic reactions could be controlled with some precision. 

The catalytic activity of enzymes is controlled in several ways. Reversible allosteric control is especially important. For example, the first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate product of the pathway. The inhibition of aspartate trans carbamoyl as e by cytidine triphosphate (Section 10.1) is a well-understood example offeedback inhibition. This type of control can be almost instantaneous. Another recurring mechanism is reversible covalent modification. For example, glycogen phosphorylase, the enzyme catalyzing the breakdovm of glycogen, a storage form of sugar, is activated by phosphorylation of a particular serine residue when glucose is scarce (Section 21.2.1). 

Two methods were used one is the iodine method that was used to determine dextrinization or the ratio of hydrolysis of the starch, and the other is the phenolphifaalein method lhat was used to determine CD formation. Starch-dextrinizing activity was determined in accordance with Fuwa (19) and Pongasawasdi and Yagisawa (20) with slight modifications. The reaction mixture containing 100 (iL of diluted enzyme aliquot and 300 pL of 0.5% soluble starch prepared in 0.1 M acetate buffer, pH 5.5, was incubated at 55 °C for 10 min. The enzyme reaction was stopped by the addition of 4.0 mL of 0.2 M HCl solution. Then, 0.5 mL of iodine solution (0.3 g/L I2 and 3.0 g/L KI) was added to form an amylose-iodine complex with residual amylose. The final volume was adjusted to 10 mL with distilled water. The absorbance of the blue color of the amylose-iodine complex was measured by spectrophotometer at 700 nm, and a decrease in absorbance was verified, when compared to a control tube with heat-inactivated enzyme. One unit of enzyme activity was defined as the quantity of enzyme that reduces the blue color of the starch-iodine complex by 10% per minute. 

CGTase activity was measured as 3-CD-forming activity based on the phenolphthalein method (21) with slight modifications as described in Alves-Prado et al. (18). One hundred microliters of diluted enzyme aliquot was added to 800 pL of 1% soluble starch prepared in 100 mM acetate buffer, pH 5.5, and incubated at 55°C for 10 min. The enzyme reaction was stopped by the addition of 4.0 mL of 0.25 M Na2C03 solution, and 0.1 mL of 1 mM phenolphthalein solution was added to the reaction mixture. The absorbance was measured at 550 nm, and a decrease in absorbance was compared to a control reaction mixture with inactive enzyme (100°C for 30 min). One unit of enzyme aetivity was defined as the amount of enzyme that produced 1 pmol of (3-CD per minute using a standard curve with 3-CD. 

The complex structure of natural fibres, especially of wool, complicates enzymatic fibre modification. Enzymes like proteases and lipases catalyse the degradation of different fibre components of a wool fibre, thus making reaction control difficult. 

Fig. 10). Traditionally, solid enzymes in which the enzyme is fixed onto a solid substrate have been used in enzyme reactions. However, due to fixation of multiple locations, enzyme activity can be sacrificed. In fact, if the polymer was bonded to an enzyme at multiple locations, enzyme activity was reduced after repeated use [140]. On the other hand, if the polymer was fixed to the enzyme at only one terminal, the denaturation of the enzyme is almost eliminated. A new bioreactor can be made by performing enzyme reactions at a dissolution temperature of PNIPAAm, thereby increasing the temperature after completion of the reaction, recovering the enzyme, and separating it fi om the product [139, 140, 143-145]. The advantage of modifying the peptide at one end of the polymer is to bring the modification site nearer the active site of the peptide. Hoffman et al. demonstrated that it is possible to control the bonding of the substrate to the active site by changes of soluble/insoluble behavior in response to temperature changes [142, 144].

Ubiquitin ligases largely control the substrate specificity of ubiquitin-conjugation reaction. The temporal specificity of ubiquitin conjugation to substrates by these enzymes is provided by regulation of the ligase activity. Activity of ubiquitin ligases can be modulated by posttranslational modification such as phosphorylation and by allosteric modification of the enzyme, or by attachment to UbL proteins. 

The control via activation or inhibition of the rate(s) of an enzyme-catalyzed reaction(s). This control includes the increase or decrease in the stability or half-life of the enzyme(s). There are many different means by which control can be achieved. These include 1. Substrate availability and reaction conditions (e.g., pH, temperature, ionic strength, lipid interface activation) 2. Magnitude of Vraax sud valucs) 3. Activation (particularly, feedforward activation) 4. Isozyme formation 5. Com-partmentalization and channeling 6. Oligomerization/ polymerization 7. Feedback inhibition and cooperativity (particularly, allosterism and/or hysteresis) 8. Covalent modification and 9. Gene regulation (induction repression)... 

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