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Effects of Structural Changes in Bile Salt Hydrolase Enzyme on Biocatalytic Efficiency and Activation Energy at Working pH and Temperature Conditions
Copyright policy )Effects of Structural Changes in Bile Salt Hydrolase Enzyme on Biocatalytic Efficiency and Activation Energy at Working pH and Temperature Conditions
Microbial bile salt hydrolases (BSHs) catalyse the hydrolysis of glycine and taurine-linked bile salts in the small intestine of humans. Achieving the effects of structural changes in BSH molecules on biocatalytic efficiency (kcat/Km) and activation energy (Ea) is necessary to determine biocatalytic performances of the enzymes. Amino acids responsible for biocatalytic activity or substrate specificity in BSH molecules were modified to determine the effects of structural changes on kcat/Km values and Ea values of the bioconversion reactions. Purified wild type positive control enzyme (pCON2) and mutant recombinant target enzymes (F18L and Y24L) reacted with six conjugated pure bile salt substrates at working temperature and pH conditions. The results of the hydrolysis conversion analysis conducted at various pH conditions were used to estimate kcat/Km, and the assays conducted at various temperature conditions were used to approximate Ea of the biocatalytic reactions. The quantified kcat/Km value was found remarkably highest with mutant recombinant enzymes (Y24L), while the efficiency value with wild type (pCON2) was determined as lowest, indicating that the structural modifications in BSH molecules showed higher values. The alterations with the mutant-type enzymes F18L and Y24L resulted in decreasing kcat/Km and increasing Ea estimations of the hydrolysis conversion reactions.
Mikrobne hidrolaze žučnih soli (BSH) kataliziraju hidrolizu soli glicina i taurina u tankom crijevu ljudi. Aminokiseline odgovorne za biokatalitičku aktivnost ili specifičnost supstrata u BSH molekulama modificirane su da bi se odredio utjecaj strukturnih promjena BSH molekula na biokatalitičku učinkovitost (kcat/Km) i energiju aktivacije (Ea) reakcija biokonverzije. Pročišćen prirodni enzim pCON2, upotrijebljen kao kontrola, a mutantni enzimi (F18L i Y24L) reagirali su sa šest čistih supstrata konjugiranih žučnih soli pri radnim uvjetima pH i temperature. Eksperimenti provedeni pri različitim pH vrijednostima primijenjeni su u procjeni kcat/Km vrijednosti biokatalitičkih reakcija, dok su eksperimenti provedeni pri različitim temperaturama primijenjeni u aproksimaciji njihovih Ea vrijednosti. Iznos kcat/Km bio je najveći kod mutantnih enzima (Y24L), a najniži kod kontrole (pCON2), što ukazuje da su strukturne modifikacije u BSH molekulama povezane s većom učinkovitosti. Preinake kod mutantnih enzima F18L i Y24L rezultirale su smanjenjem vrijednosti kcat/Km te povećanjem procijenjene Ea vrijednosti reakcija hidrolize.
bile salt; wild hydrolase; recombinant hydrolase; biocatalytic efficiency; activation energy, žučne soli; prirodna hidrolaza; preinačena hidrolaza; biokatalitička učinkovitost; energija aktivacije, General Chemical Engineering, General Chemistry
bile salt; wild hydrolase; recombinant hydrolase; biocatalytic efficiency; activation energy, žučne soli; prirodna hidrolaza; preinačena hidrolaza; biokatalitička učinkovitost; energija aktivacije, General Chemical Engineering, General Chemistry
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Microbial bile salt hydrolases (BSHs) catalyse the hydrolysis of glycine and taurine-linked bile salts in the small intestine of humans. Achieving the effects of structural changes in BSH molecules on biocatalytic efficiency (kcat/Km) and activation energy (Ea) is necessary to determine biocatalytic performances of the enzymes. Amino acids responsible for biocatalytic activity or substrate specificity in BSH molecules were modified to determine the effects of structural changes on kcat/Km values and Ea values of the bioconversion reactions. Purified wild type positive control enzyme (pCON2) and mutant recombinant target enzymes (F18L and Y24L) reacted with six conjugated pure bile salt substrates at working temperature and pH conditions. The results of the hydrolysis conversion analysis conducted at various pH conditions were used to estimate kcat/Km, and the assays conducted at various temperature conditions were used to approximate Ea of the biocatalytic reactions. The quantified kcat/Km value was found remarkably highest with mutant recombinant enzymes (Y24L), while the efficiency value with wild type (pCON2) was determined as lowest, indicating that the structural modifications in BSH molecules showed higher values. The alterations with the mutant-type enzymes F18L and Y24L resulted in decreasing kcat/Km and increasing Ea estimations of the hydrolysis conversion reactions.
Mikrobne hidrolaze žučnih soli (BSH) kataliziraju hidrolizu soli glicina i taurina u tankom crijevu ljudi. Aminokiseline odgovorne za biokatalitičku aktivnost ili specifičnost supstrata u BSH molekulama modificirane su da bi se odredio utjecaj strukturnih promjena BSH molekula na biokatalitičku učinkovitost (kcat/Km) i energiju aktivacije (Ea) reakcija biokonverzije. Pročišćen prirodni enzim pCON2, upotrijebljen kao kontrola, a mutantni enzimi (F18L i Y24L) reagirali su sa šest čistih supstrata konjugiranih žučnih soli pri radnim uvjetima pH i temperature. Eksperimenti provedeni pri različitim pH vrijednostima primijenjeni su u procjeni kcat/Km vrijednosti biokatalitičkih reakcija, dok su eksperimenti provedeni pri različitim temperaturama primijenjeni u aproksimaciji njihovih Ea vrijednosti. Iznos kcat/Km bio je najveći kod mutantnih enzima (Y24L), a najniži kod kontrole (pCON2), što ukazuje da su strukturne modifikacije u BSH molekulama povezane s većom učinkovitosti. Preinake kod mutantnih enzima F18L i Y24L rezultirale su smanjenjem vrijednosti kcat/Km te povećanjem procijenjene Ea vrijednosti reakcija hidrolize.