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- Título:
- Improvement of Aspergillus niger glucoamylase thermostability by directed evolution
- Iowa State Univ
- Univ Calif Berkeley
- Universidade Estadual Paulista (UNESP)
- 0038-9056
- Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch-plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply-mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild-type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site-directed mutations D20C/A27C (forming a disulficle bond), S30P, and G137A to create a multiply-mutated GA designated THS8. THS8 GA is substantially more thermostable than wild-type GA at 8OoC, with a 5.1 kJ/mol increase in the free energy of therrnoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly-mutated GAs have specific activities and catalytic efficiencies (k(cat)/K-m) similar to those of wild-type GA.
- 1-Out-2006
- Starch-starke. Weinheim: Wiley-v C H Verlag Gmbh, v. 58, n. 10, p. 501-508, 2006.
- 501-508
- Wiley-Blackwell
- thermostability
- directed evolution
- glucoamylase
- enzyme
- mutation
- http://dx.doi.org/10.1002/star.200600493
- Acesso restrito
- outro
- http://repositorio.unesp.br/handle/11449/36250
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