Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.
A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-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) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-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.
Galdino
Alexsandro S
AS
Laboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil.
Ulhoa
Cirano J
CJ
Moraes
Lídia Maria P
LM
Prates
Maura V
MV
Bloch
Carlos
C
Torres
Fernando A G
FA
D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000516:alpha-Amylases