Cloning, molecular characterization and heterologous expression of AMY1, an α-amylase gene from Cryptococcus flavus |
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| Authors: | Alexsandro S Galdino Cirano J Ulhoa Lídia Maria P Moraes Maura V Prates Carlos Bloch Jr & Fernando AG Torres |
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| Institution: | Laboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil;;Laboratório de Enzimologia, Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Goiás, Goiânia, GO, Brazil;and;Laboratório de Espectrometria de Massa, EMBRAPA-Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil |
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| Abstract: | A Cryptococcus flavus gene ( AMY1 ) encoding an extracellular α-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) 144DVVVNH149, (II) 235GLRIDSLQQ243, (III) 263GEVFN267, (IV) 327FLENQD332, placed the enzyme in the GH13 α-amylase family. Furthermore, sequence comparison suggests that the C. flavus α-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae . The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL−1) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme ( c . 67 kDa), part of which was due to N-glycosylation. |
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| Keywords: | Cryptococcus flavus α-amylase heterologous expression Saccharomyces cerevisiae |
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