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Interestingly, the four mutation sites are clustered at a certain region of the enzyme, suggesting a potential "hot zone" for regulating the pH optima of xylanases.
Mutation sites are underlined.
These mutation sites are presented in the exon four regions instead of exon one, two and three that have been previously identified [7].
The mutation sites are basically located in the centre of the probe so as to obtain a Tm shift around 2 6°C as estimated by the software.
In this study we have generated several mutants of αB-crystallin in which the mutation sites are located in the C-terminal region of the protein, in particular the flexible C-terminal extension.
Although our hANT4 peptides contain mutations required for proper assembly and stability of the proteins in yeast mitochondrial membrane, these mutation sites are distant from the ADP/ATP binding pocket and unlikely to substantially change the substrate binding affinity per se.
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The Ser47Ala, Thr530Ala and stop mutation sites were mutated by using the QuikChange II XL Site-Directed Mutagenesis Kit.
Mutation sites were chosen to be long distances (>8.5 Å) away from the catalytic sites.
Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima.
Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element.
With the 16 pairs of primer combinations, 54 space-induced mutation sites were observed in 971-5, 86 in 972-4, and 5 in R955 compared to their controls, and the mutation rates were 4.3%, 6.9% and 0.4%, respectively.
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