The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus
The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0....
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Published in | Carbohydrate research Vol. 344; no. 13; pp. 1682 - 1686 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
Netherlands
Elsevier Ltd
08.09.2009
|
Subjects | |
Online Access | Get full text |
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Summary: | The yeast
Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5
μg
mL
−1 affected
C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after
C. flavus growth in the presence of 0.5
μg
mL
−1 TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5
kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The
K
M values of native and non-glycosylated α-amylases were 0.052 and 0.098
mg
mL
−1, and
V
max values were 0.038 and 0.047
mg
min
−1, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg
2+ and Cu
2+ suggesting that N-glycosylation is involved in the folding of Amy1. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0008-6215 1873-426X |
DOI: | 10.1016/j.carres.2009.06.006 |