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.

scholarly journals Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing.

1986 ◽  
Vol 6 (4) ◽  
pp. 1343-1348 ◽  
Author(s):  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Gene Product ◽  
Signal Sequence ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Platelet Derived Growth Factor ◽  
Directed Mutagenesis

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.

scholarly journals Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis

2020 ◽  
Author(s):  
Xue Bai ◽  
Daixi Li ◽  
Fuqiang Ma ◽  
Xi Deng ◽  
Manjie Luo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Renal Function ◽  
Catalytic Activity ◽  
Homology Modeling ◽  
Alcaligenes Faecalis ◽  
Industrial Applications ◽  
Consensus Method ◽  
Structural Homology ◽  
Wide Range ◽  
Quadruple Mutant

Abstract Enzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 ºC and a 4.2 ºC higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability.

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