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Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

Yuqi Qin|Yinbo Qu
생화학분자생물학회 (Korean Society Of Biochemistry And Molecular Biology)|2014.05|BMB Reports|제47권 제5호|pp. 256-261

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Saccharomyces cerevisiaeCel5ACircular dichroism spectroscopyDeglycosylationHypocrea jecorina
주제분류화학

초록

To investigate the function of N-glycosylation of Cel5A(endoglucanase II) from Hypocrea jecorina, two N-glycosylationsite deletion Cel5A mutants (rN124D and rN124H) wereexpressed in Saccharomyces cerevisiae. The weights of theserecombinant mutants were 54 kDa, which were lower than thatof rCel5A. This result was expected to be attributed todeglycosylation. The enzyme activity of rN124H was greatlyreduced to 60.6% compared with rCel5A, whereas rN124Dshowed slightly lower activity (10%) than that of rCel5A. rN124Dand rN124H showed different thermal stabilities compared withthe glycosylated rCel5A, especially at lower pH value. Thermalstabilities were reduced and improved for rN124D and rN124H,respectively. Circular dichroism spectroscopy showed that themodification of secondary structure by mutation may be thereason for the change in enzymatic activity and thermal stability.

Abstract

To investigate the function of N-glycosylation of Cel5A(endoglucanase II) from Hypocrea jecorina, two N-glycosylationsite deletion Cel5A mutants (rN124D and rN124H) wereexpressed in Saccharomyces cerevisiae. The weights of theserecombinant mutants were 54 kDa, which were lower than thatof rCel5A. This result was expected to be attributed todeglycosylation. The enzyme activity of rN124H was greatlyreduced to 60.6% compared with rCel5A, whereas rN124Dshowed slightly lower activity (10%) than that of rCel5A. rN124Dand rN124H showed different thermal stabilities compared withthe glycosylated rCel5A, especially at lower pH value. Thermalstabilities were reduced and improved for rN124D and rN124H,respectively. Circular dichroism spectroscopy showed that themodification of secondary structure by mutation may be thereason for the change in enzymatic activity and thermal stability.

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