Characterization of a Novel β‐thioglucosidase CpTGG1 in Carica papaya and its Substrate‐dependent and Ascorbic Acid‐independent O‐β‐glucosidase Activity

Abstract

Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases. These enzymes evolved more recently and are distributed mainly in Brassicales. Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits. In this study, we cloned a novel myrosinase gene, CpTGG1, from Carica papaya Linnaeus. and showed that it was expressed in the aboveground tissues in planta.

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

“…Velocities rapidly dropped at higher temperatures, reaching complete enzymatic inactivation between 75–85 °C. These optimal temperatures are supported by other accounts on thioglucosidase activity, which show variations based on species of origin: Brevicoryne brassicae , optimal temperature 35–45 °C [32], Carica papaya , optimal temperature 40–50 °C [30], mustard ( Sinapis ) variants have demonstrated relatively higher thermal stability [31,40]. …”

“…This pH represented a true maximum for non-natural glucosinolate 5 , which demonstrated a decline in normalized velocity with increasing pH; in contrast, 6 maintained a relatively constant normalized velocity over the same range. This optimal pH range, especially for 5 , is somewhat lower than what has been previously disclosed in the literature for thioglucohydrolases: Sinapis alba , optimal pH 6.5 [29], Carica papaya , optimal pH 6.5–8.5 [30]. Previous mechanistic studies suggest that pH-dependent changes in myrosinase activity do not result from conformational changes, but rather ionic changes to the enzyme active site [37].…”

“…Kinetic evaluation of 7 in unbuffered conditions demonstrated both time-dependence of increasing acidity and preferential formation of the nitrile product ( 3 ) at low pH [18]. Studies describing the pH-dependence of myrosinase catalytic activity on natural substrates have noted a maximum efficiency at pH 6.5 for enzyme isolated from Sinapis alba [29] and pH 6.5–8.5 for Carica papaya [30]. Similarly, thioglucohydrolases demonstrate an Arrhenius-type increase in the rate of catalysis of natural glucosinolates between 0–40 °C, an optimum temperat ure range between 40–55 °C, and complete thermal inactivation by 80 °C [30,31,32].…”

“…Studies describing the pH-dependence of myrosinase catalytic activity on natural substrates have noted a maximum efficiency at pH 6.5 for enzyme isolated from Sinapis alba [29] and pH 6.5–8.5 for Carica papaya [30]. Similarly, thioglucohydrolases demonstrate an Arrhenius-type increase in the rate of catalysis of natural glucosinolates between 0–40 °C, an optimum temperat ure range between 40–55 °C, and complete thermal inactivation by 80 °C [30,31,32]. If the ca talytic mechanism of myrosinase on non-natural glucosinolate substrates parallels the mechanism for natural substrates, it would be expected that similar dependence patterns on pH, temperature, and their effects on the ratio of organic product distribution (e.g.…”

High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase

“…While 4 has been used as a substrate for myrosinase in several studies, variances in the organismal source of myrosinase, isozyme, level of purity and the resultant effects on intrinsic activity, pH, and temperature limit the ability for direct comparison to known standard values [42]. In this study, the K m of 4 ranged from 122–233 µM and was supportive of prior findings: 117 µM ( Sinapis alba ) [26], 359 µM ( Brevicoryne brassicae ) [37], and 410 µM ( Brevicoryne brassicae ) [36].…”