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Directed evolution of Aspergillus niger glucoamylase to increase thermostability.

McDaniel A, Fuchs E, Liu Y, Ford C - Microb Biotechnol (2008)

Bottom Line: Irreversible inactivation tests indicated that CR2-1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol(-1) compared with that of wild-type GA.Thus, CR2-1 is more thermostable (by 5 kJ mol(-1) at 80°C) than the most thermostable A. niger GA variant previously described, THS8.In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol(-1), respectively, at 80°C.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA.

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Mentions: The thermostability of Glu408Lys was further tested by adding it to the thermostable genetic background of RE5. RE5+E408K had an increased ΔG of 1 and 2 kJ mol−1 at 72.5°C and 80°C, respectively, compared with RE5 alone (Table 3). These results confirm that Glu408Lys is a thermostabilizing mutation. Lys408 is located near residues Asp403 and Asp406 on the enzyme surface in contact with the surrounding solvent. A molecular dynamics simulation modelling the potentially stabilizing interaction of Lys408 with nearby residues showed that the NH3+ group on Lys408 is close enough (1.81 Å) to form a stabilizing salt bridge with the COO‐ group on Asp406, with potential to contribute to enzyme thermostability (Fig. 6). Salt bridges can be very stabilizing at higher temperatures, and are more prevalent in thermophilic than mesophilic proteins (Kumar et al., 2000).


Directed evolution of Aspergillus niger glucoamylase to increase thermostability.

McDaniel A, Fuchs E, Liu Y, Ford C - Microb Biotechnol (2008)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815294&req=5

Mentions: The thermostability of Glu408Lys was further tested by adding it to the thermostable genetic background of RE5. RE5+E408K had an increased ΔG of 1 and 2 kJ mol−1 at 72.5°C and 80°C, respectively, compared with RE5 alone (Table 3). These results confirm that Glu408Lys is a thermostabilizing mutation. Lys408 is located near residues Asp403 and Asp406 on the enzyme surface in contact with the surrounding solvent. A molecular dynamics simulation modelling the potentially stabilizing interaction of Lys408 with nearby residues showed that the NH3+ group on Lys408 is close enough (1.81 Å) to form a stabilizing salt bridge with the COO‐ group on Asp406, with potential to contribute to enzyme thermostability (Fig. 6). Salt bridges can be very stabilizing at higher temperatures, and are more prevalent in thermophilic than mesophilic proteins (Kumar et al., 2000).

Bottom Line: Irreversible inactivation tests indicated that CR2-1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol(-1) compared with that of wild-type GA.Thus, CR2-1 is more thermostable (by 5 kJ mol(-1) at 80°C) than the most thermostable A. niger GA variant previously described, THS8.In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol(-1), respectively, at 80°C.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA.

Show MeSH