Cloning, molecular characterization and heterologous expression of AMY1, an α-amylase gene from Cryptococcus flavus

Authors


  • Editor: Diethard Mattanovich

Correspondence: Fernando Torres, Laboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil 70910-900. Tel.: +55 61 3307 2423; fax: +55 61 3349 8411; e-mail: ftorres@unb.br

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. flavusα-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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