Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y from Yeast. The Significance of Thr60 and Met398 in Hydrolysis and Aminolysis Reactions

1995 ◽  
Vol 117 (22) ◽  
pp. 5944-5950 ◽  
Author(s):  
Steen Bech Sorensen ◽  
Mette Raaschou-Nielsen ◽  
Uffe H. Mortensen ◽  
S. James Remington ◽  
Klaus Breddam
Keyword(s):  
Site Directed Mutagenesis ◽  
Carboxypeptidase Y ◽  
Directed Mutagenesis ◽  
Serine Carboxypeptidase

scholarly journals Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis

1995 ◽  
Vol 308 (2) ◽  
pp. 405-409 ◽  
Author(s):  
Y Chiba ◽  
T Midorikawa ◽  
E Ichishima
Keyword(s):  
Amino Acids ◽  
Cell Extract ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Yeast Cells ◽  
Directed Mutagenesis ◽  
Serine Carboxypeptidase ◽  
Reading Frame ◽  
Sds Page

Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

2013 ◽  
Vol 37 (3) ◽  
pp. 330
Author(s):  
Yanan WANG ◽  
Xudong LIU ◽  
Linlin MU ◽  
Zhipeng LIU ◽  
Chunmei LI ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Cynoglossus Semilaevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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