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

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their “fresh green” aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box–Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L−1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L−1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported.

Download Full-text

Tea green leafhopper, Empoasca vitis, chooses suitable host plants by detecting the emission level of (3Z)-hexenyl acetate

Bulletin of Entomological Research ◽

10.1017/s000748531600064x ◽

2016 ◽

Vol 107 (1) ◽

pp. 77-84 ◽

Cited By ~ 10

Author(s):

Z.-J. Xin ◽

X.-W. Li ◽

L. Bian ◽

X.-L. Sun

Keyword(s):

Host Plants ◽

Oviposition Preference ◽

Green Leaf Volatiles ◽

Hydroperoxide Lyase ◽

Future Research ◽

Suitable Host ◽

Leaf Volatiles ◽

Green Leafhopper ◽

Hexenyl Acetate ◽

Empoasca Vitis

AbstractGreen leaf volatiles (GLVs) have been reported to play an important role in the host-locating behavior of several folivores that feed on angiosperms. However, next to nothing is known about how the green leafhopper, Empoasca vitis, chooses suitable host plants and whether it detects differing emission levels of GLV components among genetically different tea varieties. Here we found that the constitutive transcript level of the tea hydroperoxide lyase (HPL) gene CsiHPL1, and the amounts of (Z)-3-hexenyl acetate and of total GLV components are significantly higher in tea varieties that are susceptible to E. vitis (Enbiao (EB) and Banzhuyuan (BZY)) than in varieties that are resistant to E. vitis (Changxingzisun (CX) and Juyan (JY)). Moreover, the results of a Y-tube olfactometer bioassay and an oviposition preference assay suggest that (Z)-3-hexenyl acetate and (Z)-3-hexenol offer host and oviposition cues for E. vitis female adults. Taken together, the two GLV components, (Z)-3-hexenol and especially (Z)-3-hexenyl acetate, provide a plausible mechanism by which tea green leafhoppers distinguish among resistant and susceptible varieties. Future research should be carried out to obtain the threshold of the above indices and then assess their reasonableness. The development of practical detection indices would greatly improve our ability to screen and develop tea varieties that are resistant to E. vitis.

Download Full-text

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

Catalysts ◽

10.3390/catal9100873 ◽

2019 ◽

Vol 9 (10) ◽

pp. 873 ◽

Cited By ~ 3

Author(s):

Sophie Vincenti ◽

Magali Mariani ◽

Jean-Christophe Alberti ◽

Sabrina Jacopini ◽

Virginie Brunini-Bronzini de Caraffa ◽

...

Keyword(s):

Higher Plants ◽

Green Leaf Volatiles ◽

Added Value ◽

Hydroperoxide Lyase ◽

Green Leaf ◽

Enzymatic Reactions ◽

Leaf Volatiles ◽

Structural And Functional Properties ◽

Natural Flavors ◽

Volatile Aldehydes

In higher plants, the lipoxygenase enzymatic pathway combined actions of several enzymes to convert lipid substrates into signaling and defense molecules called phytooxylipins including short chain volatile aldehydes, alcohols, and esters, known as green leaf volatiles (GLVs). GLVs are synthesized from C18:2 and C18:3 fatty acids that are oxygenated by lipoxygenase (LOX) to form corresponding hydroperoxides, then the action of hydroperoxide lyase (HPL) produces C6 or C9 aldehydes that can undergo isomerization, dehydrogenation, and esterification. GLVs are commonly used as flavors to confer a fresh green odor of vegetable to perfumes, cosmetics, and food products. Given the increasing demand in these natural flavors, biocatalytic processes using the LOX pathway reactions constitute an interesting application. Vegetable oils, chosen for their lipid profile are converted in natural GLVs with high added value. This review describes the enzymatic reactions of GLVs biosynthesis in the plant, as well as the structural and functional properties of the enzymes involved. The various stages of the biocatalytic production processes are approached from the lipid substrate to the corresponding aldehyde or alcoholic aromas, as well as the biotechnological improvements to enhance the production potential of the enzymatic catalysts.

Download Full-text