Mutant enzyme thermostability

When the cysteine443 was replaced with Ala, Gly, Ser, or Thr, the mutant enzymes folded the current conformations. Cys443T was the most thermostable mutant enzyme (Table 21). In this case the van der Waals volume of threonine is 93 A3, slightly larger than that of cysteine (86 A3). On the other hand, the hydrophobicities of cysteine and threonine are +2.5 and -0.7 respectively [134], The decrease in the Tm value of the C443T mutant is thought to be due to the low hydrophobicity of threonine. 

The increased thermostability of the disulfide mutant is also shown by denaturation temperature estimated by differential scanning calorimetry (Fig. 12.4). Tm (midpoint temperature in the thermally induced transition from the folded to the unfolded state) of the mutant enzyme is 63.0°C, which is 4.5°C higher than that of the wild-type enzyme (58.5 °C). Under reducing conditions, the Tm of the mutant enzyme is decreased to 50.5 ° C, which is lower than that of the wild-type enzyme, indicating that the disulfide bond formation is required for the thermostability and that the mutant enzyme with free cysteine residues does not have a stable structure like the wild-type enzyme. 

K15M <21> (<21> inactive mutant enzyme, increased thermostability and affinity for ATP when compared to the wild-type enzyme, the organization of the P-loop and flanking regions is heavily disturbed [21]) [21]... 

S. (2003) The structure of a mutant enzyme of Coprinus cinereus peroxidase provides an understanding of its increased thermostability. Acta Crys-tallogr.. Sect. D Biol. Crystallogr., 59, 997-1003. 

Various mutant enzymes have also been produced at high yields. For example, SNase-H124L, which is threefold less active but more thermostable than the wild-type enzyme (53), has been cloned and overexpressed under the control of phage T7 promoter in a T7 RNA polymerase expression vector pET3A (54). 

While wild-type PAMO was unable to convert 2-phenylcyclohexanone efficiently, all deletion mutants readily accepted this ketone as substrate. All mutants also displayed a similar thermostability when compared with the parent enzyme. The most active mutant (deletion of S441 and A442) was used for examining its enantioselective properties. It was found that the mutant preferably formed the (/ )-enantiomer of the corresponding lactone E = 100). While CHMO also shows a similar enantioselective behavior, this PAMO deletion mutant is a better candidate for future applications due to its superior stability. This clearly demonstrates that PAMO can be used as parent enzyme to design thermostable BVMO variants. It also illustrates that the available crystal structure of PAMO will be of great help for BVMO redesign efforts. ... 

The wild-type (WT) and cysteine mutants bound about one mole of Ca2+ per mole of protein after Ca2+-binding treatment (Table 20). The Ca2+-bound forms were also examined for thermostability, as shown in Figure 32. The half lives of the Ca2+-bound enzymes increased slightly, as compared to those of the Ca2+-free enzyme. 

As for subtilisin BPN, the first attempt at molecular modeling of disulfide mutants was performed with computer graphics using coordinates from the crystal structure of the enzyme,10-121 but increasing enzyme stability was unsuccessful. We have studied a thermostable subtilisin-type protease, aqualysin I, and the introduction site of a disulfide bond was chosen on structural homology between aqualysin I and subtilisin E. Here we describe a successful study to increase the stability of subtilisin E and others done for subtilisin enzymes. 

Thermostability of subtilisin E is increased by the introduction of the disulfide bond (Table 12.3). The half-life of the disulfide mutant is 2-3 times longer than that of the wild-type enzyme at 45°-60°C. On the other hand, those of the single-cysteine mutants are... 

Disulfide mutants of the alkaline protease were constructed on the basis of the positions of two disulfide bonds of aqualysin I. Cysl69-Cys200 disulfide bond, which corresponds to Cysl63-Cvsl94 of aqualysin I, increases the thermostability of the enzyme, but the other... 

If the enzyme of interest is essential for growth or survival, then it may be thermostabilized by selection in a thermophilic host (Oshima, 1994). The host growth temperature must be higher than the denaturation temperature of the enzyme to select for thermostable mutants. However, if the lower limit for growth is too far above the enzyme s... 

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

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