Lipases of germinating jojoba seeds efficiently hydrolyze triacylglycerols and wax esters and display wax ester-synthesizing activity

Abstract

Background: Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds.

Results: During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds.

Conclusions: In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Keywords: Jojoba; Lipase; Simmondsia chinensis; Triacylglycerols; Wax ester hydrolase; Wax ester synthesis; Wax esters.

Fig. 1

Wax ester mobilization during jojoba seed germination. Changes in wax ester content in germinating jojoba seeds of accession 144 (a), accession 145 (b), accession 146 (c), accession 147 (d). Data represent the mean of four biological replicates and error bars show standard deviation

Fig. 2

Immunoblot analysis of oleosin content in total protein extracts of jojoba seeds during germination. M1: oleosins from maize, M2: protein marker. Numbers on the right indicate the positions of molecular mass markers. The arrows indicate two isoforms of the maize oleosins. Similar changes were observed for accessions 145 and 146. The full-length blot is presented in Additional file 1: Fig. S6

Fig. 3

Protein mobilization during jojoba seed germination. Changes in protein content (relative to the seed initial weight) in germinating jojoba seeds of accession 144 (a), accession 145 (b), accession 146 (c), accession 147 (d). Data represent the mean of four biological replicates and error bars show standard deviation

Fig. 4

Fatty acid and fatty alcohol composition of germinating jojoba seeds. The relative content of main fatty acids (FA; a) and fatty alcohols (FA-OH; b) in lipids of jojoba seeds during germination (% of total FA and FA-OH). Data represent the mean of four biological replicates (accession 145) and error bars show standard deviation. The different letters denote statistical significance between FA or FA-OH content at different stages of germination (one-way ANOVA followed by Tukey’s post-hoc test, p < 0.05, n = 4)

Fig. 5

Biochemical characterization of the jojoba seed lipase activity . a. The impact of temperature on the jojoba seed lipase activity towards 18:1-TAG (left panel) and 18:1–18:1 WE (right panel). Data represent mean values and error bars show the range of duplicates. Assay conditions: aliquots (2.5 nmol of endogenous PC) of microsomal fraction mixture isolated from two individual jojoba seeds from each accession (35 dpg); 20 nmol of [¹⁴C]18:1-TAG or [¹⁴C]18:1–18:1 WE added to freeze-dried microsomes in 19 μl benzene; benzene evaporation, and addition of 100 μl 0.1 HEPES buffer (pH 7.0); incubation for 2.5 min (for TAG) or 1.5 min (for WE). b. The impact of the pH on the jojoba seed lipase activity towards 18:1-TAG (left panel) and 18:1–18:1 WE (right panel). Data represent mean values and error bars show the range of duplicates. Assay condition: as above with 0.1 M citrate buffer (pH 4.0–5.0), phosphate buffer (pH 6.0–8.0), or Tris-HCl buffer (9.0), 5 min incubation at 35 °C. c. The impact of pre-incubation conditions on jojoba seed lipase activity towards 18:1-TAG. Data represent mean values and error bars show the range of duplicates; nd – not detected. Assay conditions: as above, 5 min incubation at 60 °C. d. Substrate specificity of jojoba seed lipases towards saturated and unsaturated WEs. Data represent mean values and error bars show the range of duplicates. Assay condition: as above with 2.5 nmol of [¹⁴C] WE, 10 min incubation at 60 °C.

Fig. 6

WE-synthesizing activity of the jojoba seed lipases. a. Time course of WE synthesis from 18:1-TAG and 18:1-FA combined with 18:1-OH. Data represent mean values and error bars show the range of duplicates. Assay condition: aliquots (2.5 nmol of endogenous PC) of microsomal fraction mixture (isolated from two individual jojoba seeds from each accession, 35 dpg); substrates (20 nmol of [¹⁴C]18:1-FA or [¹⁴C]18:1-TAG and 20 nmol of fatty alcohol) added to freeze-dried microsomes in 19 μl benzene; benzene evaporation, and addition of 100 μl 0.1 M HEPES buffer (pH 7.0); incubation at 60 °C. b. WE synthesis from 18:1-FA and different fatty alcohols. Data represent mean values and error bars show the range of duplicates. Assays conditions: as above, 20 min incubation at 60 °C