Multi Site-Directed Mutagenesis of Human Flavin-Containing Monooxygenase 5 (FMO5) Produces a Mutant with 20-fold Higher Affinity for Methimazole: Evidence that Ile61 is Crucial for Catalytic Activity

2011 ◽  
Vol 1 (1) ◽  
pp. 14-22
Author(s):  
Erin White ◽  
Emmanuel Atta-Asafo-Adjei
Keyword(s):  
Catalytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis

2019 ◽  
Vol 20 (4) ◽  
pp. 916 ◽  
Author(s):  
Diana Humer ◽  
Oliver Spadiut
Keyword(s):  
Catalytic Activity ◽  
Horseradish Peroxidase ◽  
Signal Sequence ◽  
Enzyme Engineering ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Promising Alternative ◽  
Wide Range ◽  
Quadruple Mutant ◽  
Commercial Applications

Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.

Enhancement of the catalytic activity of d-lactate dehydrogenase from Sporolactobacillus laevolacticus by site-directed mutagenesis

2018 ◽  
Vol 133 ◽  
pp. 214-218
Author(s):  
Kento Nakano ◽  
Shoichi Sawada ◽  
Ryosuke Yamada ◽  
Takashi Mimitsuka ◽  
Hiroyasu Ogino
Keyword(s):  
Catalytic Activity ◽  
Lactate Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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