A Two-Step Bioconversion Process for Canolol Production from Rapeseed Meal Combining an Aspergillus niger Feruloyl Esterase and the Fungus Neolentinus lepideus

Abstract

Rapeseed meal is a cheap and abundant raw material, particularly rich in phenolic compounds of biotechnological interest. In this study, we developed a two-step bioconversion process of naturally occurring sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid) from rapeseed meal into canolol by combining the complementary potentialities of two filamentous fungi, the micromycete Aspergillus niger and the basidiomycete Neolentinus lepideus. Canolol could display numerous industrial applications because of its high antioxidant, antimutagenic and anticarcinogenic properties. In the first step of the process, the use of the enzyme feruloyl esterase type-A (named AnFaeA) produced with the recombinant strain A. niger BRFM451 made it possible to release free sinapic acid from the raw meal by hydrolysing the conjugated forms of sinapic acid in the meal (mainly sinapine and glucopyranosyl sinapate). An amount of 39 nkat AnFaeA per gram of raw meal, at 55 °C and pH 5, led to the recovery of 6.6 to 7.4 mg of free sinapic acid per gram raw meal, which corresponded to a global hydrolysis yield of 68 to 76% and a 100% hydrolysis of sinapine. Then, the XAD2 adsorbent (a styrene and divinylbenzene copolymer resin), used at pH 4, enabled the efficient recovery of the released sinapic acid, and its concentration after elution with ethanol. In the second step, 3-day-old submerged cultures of the strain N. lepideus BRFM15 were supplied with the recovered sinapic acid as the substrate of bioconversion into canolol by a non-oxidative decarboxylation pathway. Canolol production reached 1.3 g/L with a molar yield of bioconversion of 80% and a productivity of 100 mg/L day. The same XAD2 resin, when used at pH 7, allowed the recovery and purification of canolol from the culture broth of N. lepideus. The two-step process used mild conditions compatible with green chemistry.

Release of phenolic acids from sunflower and rapeseed meals using different carboxylic esters hydrolases from Aspergillus niger

“…Esterase activity was assayed spectrophotometrically at either 37°C and 55°C for AnFaeA or 37°C and 50°C for AnFaeB and ChlE, as previously described (Odinot et al, 2017), by monitoring the A335 with respect to the rate of hydrolysis of 0.032 mM of the enzyme substrate in 88 mM of a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 5.5 for AnFaeA and pH6 for AnFaeB and ChlE). The synthetic substrates used were methyl sinapate for AnFaeA, methyl caffeate and methyl p-coumarate for AnFaeB, and chlorogenic acid for ChlE.…”

“…In this context, SFM and RSM, which are cheap, abundant, and rich in phenolics, make perfectly suitable candidates. It is worth noting that only few studies have reported the use of these two raw materials for the production of phenolic acids and derivatives like vinyl phenols (Kreps et al, 2014;Odinot et al, 2017).…”

“…Historically, pure AnFaes were shown to be essentially inactive when used directly on natural raw substrates. However, we recently studied the enzymatic hydrolysis of an industrial rapeseed meal (I-RSM) using a recombinant AnFaeA to produce sinapic acid (Odinot et al, 2017) and demonstrated that this enzyme was highly effective on the raw RSM. In the best conditions, AnFaeA hydrolyzed most of the SAEsters present in the RSM, with an overall yield of 68-76 % (100% hydrolysis of sinapine).…”

“…Then, the same approach was applied to a non-industrial rapeseed meal (NI-RSM) and the corresponding phenolic extract, by using AnFaeA to release free SA. The results (hydrolysis yield and highest released SA amount) were compared with our previous data obtained on an I-RSM (Odinot et al, 2017). All cases were compared and discussed based on the following parameters: hydrolysis yields, qualitative and quantitative composition of hydrolyzed hydroxycinnamoyl esters, and free CA or SA amount released per gram of substrate.…”

In situ oxidation of canola meal sinapic acid by horseradish peroxidase (type II) and tyrosinase

“…This is based on a previous report that sinapine esterase catalyzed the hydrolysis of sinapine to sinapic acid (Nurmann & Strack, ). In addition, a recent study compared the impacts of three different recombinant strains from Aspergillus niger on enzymatic hydrolysis of sinapine, and found that the feruloyl esterases from A. niger (BRFM451) showed highest hydrolysis sinapic acid yields of 68% to 76% (Odinot et al, ). Therefore, for continuous generation of sinapic acid, sinapine esterase or feruloyl esterase could be a more effective alternative than alkaline hydrolysis of canola meal.…”