Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives

Abstract

The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at -20 or at -80 °C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5 M), and NaSO (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with NaSO (0.25 M) and NaCl (1 M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and NaSO are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.

Biocatalytic Synthesis of Natural Green Leaf Volatiles Using the Lipoxygenase Metabolic Pathway

“…The addition of additives such as NaCl, Na 2 SO 4 , and glycine to the reaction medium has increased the catalytic efficiency of the enzyme. During C6 aldehydes biosynthesis in the presence of these compounds, amounts of aldehydes equivalent to those obtained in their absence, and high molar conversion rates were achieved, while the amount of enzyme used was decreased from 1.5 to 2.5 fold [230].…”

“…The stabilization of the enzyme using selected chemical additives was also investigated. Jacopini et al [230] showed that about 100% of the HPL activity was maintained during five weeks of storage at −20 or at −80 • C in the presence of glycerol (10%, v/v). The addition of additives such as NaCl, Na 2 SO 4 , and glycine to the reaction medium has increased the catalytic efficiency of the enzyme.…”

Optimizing the Production of Recombinant Hydroperoxide Lyase in Escherichia coli Using Statistical Design

“…Moreover, scaling-up the culture conditions in a 1 L Erlenmeyer flask allowed to produce of higher soluble enzyme activities (up to 21,920 U L −1 of culture ) that were never reported before for any HPL, which are membrane-bound enzymes and having a tendency to form inclusion bodies in E. coli. This result represents a 79-fold increase compared to the production levels reported with the previous protocol [42,43]. To this end, the RSM approach is quite efficient and allows to show that high yields of recombinant protein can be reached when key parameters are tightly controlled.…”

“…Olive recombinant 13-HPL was expressed in Escherichia coli (E. coli) M15, purified by affinity chromatography and biochemically characterized [42]. In another work, we improved the stability and increased activity of the enzyme using selected chemical additives [43]. The use of olive recombinant 13-HPL for C 6 -aldehydes synthesis from 13-HPOD and 13-HPOT showed that it is an efficient and promising biocatalyst for the bioconversion process (conversion rates up to 94%) [42,43].…”

“…In another work, we improved the stability and increased activity of the enzyme using selected chemical additives [43]. The use of olive recombinant 13-HPL for C 6 -aldehydes synthesis from 13-HPOD and 13-HPOT showed that it is an efficient and promising biocatalyst for the bioconversion process (conversion rates up to 94%) [42,43]. Its use for C6-aldehyde production in a high-scale bioreactor requires an optimization of the heterologous expression of recombinant HPL in order to recover larger amounts of the soluble and active enzymes.…”