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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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Three‐dimensional structure of the catalytic domain of GA showing the location of the mutations in CR2‐1.
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f1: Three‐dimensional structure of the catalytic domain of GA showing the location of the mutations in CR2‐1.

Mentions: Glucoamylase from A. niger has been characterized both biochemically and genetically (Aleshin et al., 1992; 1994; 1996). The enzyme has two domains and a linker: an N‐terminal catalytic domain of 440 amino acids with an (α,α)6‐barrel structure that has been structurally characterized by X‐ray crystallography (Fig. 1); a C‐terminal starch‐binding domain of 108 amino acids, necessary for hydrolysis of insoluble starch granules (but not necessary for hydrolysis of soluble substrates); and a heavily O‐glycosylated linking region of 68 amino acids. The GA gene from A. niger has been cloned and can be expressed conveniently in the yeast Saccharomyces cerevisiae (Cole et al., 1988). Numerous approaches have successfully been taken to improve the thermostability of A. niger GA by site‐directed mutagenesis (Ford, 1999; Reilly, 1999; Liu and Wang, 2003). Recently, Wang and colleagues (2006) have used directed evolution in conjunction with site‐directed mutagenesis to construct the most thermostable A. niger GA previously described (THS8). THS8 contains eight mutations (Asp20Cys, Ala27Cys, Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala and His391Tyr) with a cumulative increase in thermostability (ΔG) over wild‐type GA of 5 kJ mol−1 at 80°C without a reduction in catalytic activity.


Directed evolution of Aspergillus niger glucoamylase to increase thermostability.

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

Three‐dimensional structure of the catalytic domain of GA showing the location of the mutations in CR2‐1.
© Copyright Policy
Related In: Results  -  Collection

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

f1: Three‐dimensional structure of the catalytic domain of GA showing the location of the mutations in CR2‐1.
Mentions: Glucoamylase from A. niger has been characterized both biochemically and genetically (Aleshin et al., 1992; 1994; 1996). The enzyme has two domains and a linker: an N‐terminal catalytic domain of 440 amino acids with an (α,α)6‐barrel structure that has been structurally characterized by X‐ray crystallography (Fig. 1); a C‐terminal starch‐binding domain of 108 amino acids, necessary for hydrolysis of insoluble starch granules (but not necessary for hydrolysis of soluble substrates); and a heavily O‐glycosylated linking region of 68 amino acids. The GA gene from A. niger has been cloned and can be expressed conveniently in the yeast Saccharomyces cerevisiae (Cole et al., 1988). Numerous approaches have successfully been taken to improve the thermostability of A. niger GA by site‐directed mutagenesis (Ford, 1999; Reilly, 1999; Liu and Wang, 2003). Recently, Wang and colleagues (2006) have used directed evolution in conjunction with site‐directed mutagenesis to construct the most thermostable A. niger GA previously described (THS8). THS8 contains eight mutations (Asp20Cys, Ala27Cys, Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala and His391Tyr) with a cumulative increase in thermostability (ΔG) over wild‐type GA of 5 kJ mol−1 at 80°C without a reduction in catalytic activity.

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