Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

doi:10.21203/rs.3.rs-1975355/v1

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Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

https://doi.org/10.21203/rs.3.rs-1975355/v1

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Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, pulse meal contains anti-nutritional and anti-palatability compounds, including (poly)phenolics (tannic and non-tannic), such that improvements in pulse meal processing are still needed to increase its uptake by the industry. Microbial fermentation is currently used as a strategy to decrease tannin content, but results in the undesirable accumulation of monophenolics. Here we investigate the viability of cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba) meal. A representative catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Its amenability to large scale recombinant production was established, and its ongoing stability in complex environments including resuspension in slurries of faba bean meal demonstrated. Reaction results suggest that BLC23O is effective for biocatalytic phenol reduction in faba bean meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (tannins, proteins, carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry.

pulse meal

Vicia faba

biocatalysis

catechol 2

3 dioxygenase

phenol reduction

Alternative feed ingredients and innovation of feed technology are progressively being investigated and incorporated into processing pipelines toward enhanced digestibility and efficiency, decreased environmental impact and maximized economic opportunity (Behnke, 1996; der Poel et al., 2020).

Pulses (Fabaceae family) and their by-products are examples of advantageous sources of animal feed that could be further optimized to increase their value and applicability (Sherasia, Garg, & Bhanderi, 2018; Singh, 2017). Currently, pulses play an important role in sustainable development based on their ability to fix nitrogen, reducing the need for costly nitrogen fertilizers when used in crop rotations, and sequestering greenhouse gas from the environment (Irisarri et al., 2021). However pulse nutrient and protein-rich profiles significantly furthers their potential. For example, pulse meal, a by-product of the industry, is approximately 40–45% protein on a dry mass basis and thus holds great potential to serve as animal feed, contributing to food security and reducing competition with human food (Sherasia et al., 2018). Such applications of by-products also reduces costs related to disposal and allows for the conversion of low value products into higher value feed/food (Ominski et al., 2021).

Tannins and other non-tannic (poly)phenols are intrinsic to pulses, and in the context of animal nutrition, are considered anti-nutritional factors (ANFs) based on observed negative effects on digestion and the bioavailability of nutrients (Kardum & Glibetic, 2018; Kumar et al., 2021). These effects are due to (poly)phenols forming complexes with storage proteins, interfering with peptide bond proteolysis, and serving as direct inhibitors of digestive enzyme, together decreasing protein and carbohydrate availability (Punia, Siroha, & Kumar, 2021). Chelation of metal ions by polyphenolics has also been shown to reduce absorption and decrease uptake of vitamins and minerals (Singh, 2017). Finally, the palatability of pulse meal is reduced by tannins in particular, due to undesirable astringency, which lowers food intake and animal performance (Singh, 2017). Thus, reducing phenol content (tannic and non-tannic) in pulse products remains an important goal for increased nutritional and economic value.

Currently, microbial fermentation is used as a strategy to lower ANFs, including tannins, in feeds (der Poel et al., 2020; Olukomaiya, Fernando, Mereddy, Li, & Sultanbawa, 2019). For example, Lactobacillus plantarum, a Gram-positive lactic acid bacterium commonly used in the food industry, specifically possesses genes encoding tannin acyl hydrolase (commonly known as tannase) and gallate decarboxylase, yielding the monophenol pyrogallol (Jimenez, Esteban-Torres, Mancheno, de Las Rivas, & Munoz, 2014). However, the observed accumulation of such monophenols in L. plantarum fermented feeds is also undesirable (Kardum & Glibetic, 2018; Kumar et al., 2021). The next step in degradation of monophenols involves breaking the phenolic ring. In nature, this activity relies on a family of microbial catechol dioxygenases that carry out the oxidative cleavage of hydroxylated aromatic rings, to yield linear compounds that feed into the citric acid cycle (Kamimura et al., 2017). Phenolic ring cleavage can be intradiol (between two consecutive hydroxyl groups on the ring (positions 1 and 2)), proximal extradiol (next to the hydroxyls; position 2 and 3) or distal extradiol (removed from the hydroxyls; position 4 and 5) (Hou, Patel, & Lillard, 1977). A brief review of the current literature and a search of the NCBI database suggests L. plantarum does not encode any members of this family of enzymes, consistent with the high accumulation of phenolics in such fermentations.

Thus, as an alternative or complement to microbial fermentation, cell-free biocatalysis has been proposed for reduction of phenolic content. A biocatalytic strategy would decouple tannin and phenolic degradation from cellular physiology, reducing any downregulation imposed by the carbohydrate rich environment and feedback inhibition, as well as allowing absolute control of enzyme types and concentrations applied (Claassens, Burgener, Vogeli, Erb, & Bar-Even, 2019).

One example of a potential candidate for cell-free phenolic degradation of pulse meal is a Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O; (Adewale et al., 2021)). B. ligniniphilus L1 is a halotolerant and alkaliphilic bacterium isolated from sediments from the South China Sea, known to use polyphenolic lignin as its sole carbon source. Catechol 2,3-dioxygenases elicit proximal extradiol cleavage. Three catechol 2,3-dioxygenases encoded by B. ligniniphilus L1 have been identified and the shortest one, a protein of 283 amino acids (BLC23O, NCBI accession WP_017726464.1) with a calculated molecular mass of approximately 32 kDa, was recently characterized (Adewale et al., 2021). This enzyme was found to have unusually broad substrate specificity and good thermostability, traits proposed to be associated with its atypical monomeric structure.

Here the application of BLC23O to cell-free reduction of phenolic content in pulse meal is assessed. Scaled-up recombinant production and purification of the enzyme is described, followed by evaluation of the enzyme’s potential to reduce phenolics in 3 different meal fractions from Vicia faba (faba bean).

Large-scale (4 L fermentative) production and Ni-NTA enrichment of BLC23O

Shake flask production of BLC23O from Echerichia coli has been previously reported (Adewale et al., 2021). Here production of BLC23O was scaled up to a 4 L fermentative format. Wet weight determination of the obtained cell pellet was 49.8 g, or 12.4 g cell pellet / L of culture. The pellet was re-suspended in lysis buffer and high-pressure homogenization was used to extract proteins. Following centrifugation, the supernatant was collected and BLC23O was further enriched using the fused His-tag. Outcomes from the Ni-NTA affinity chromatography were visualized by SDS-PAGE (Fig. 1A). Thick bands at the expected Mw of approximately 35 kDa are present in the three elution fractions and the original supernatant fraction. Small amounts of protein were also detected at 35 kDa in the washes and flow-through, suggesting minor losses during the enrichment. No BLC23O was detected in the lysis pellet, indicating that cell lysis was successful. Contaminating proteins were largely eliminated from the final sample. The elution fractions were pooled and protein quantified, yielding 116 mg of enzyme / L of cell culture, or 9.3 mg of BLC23O / g of wet weight cells.

Kinetic analysis of BLC23O.

A small portion (4%) of the obtained protein from Ni-NTA enrichment was subjected to further purification by size exclusion chromatography. The chromatographic separation curve for BLC23O (Supplemental Figure S1A) showed a clear and steep peak at 82 min. Calibration of indicated this is consistent with the expected molecular weight of 35 kDa (Supplemental Figure S1B), falling in between ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Collected fractions spanning this peak were visualized by SDS-PAGE, yielding a single significant band around the expected Mw of 35 kDa (Supplemental Figure S1C). A Michaelis-Menten analysis of the obtained purified BLC23O against varying concentrations of the known substrate, 3-methycatechol, yielded a K_M of 379 ± 86 µM, a turnover number (kcat) of 0.13 ± 0.01 s^− 1, and a catalytic efficiency (kcat/K_M) of 340 ± 80 M^− 1 s^− 1 (Fig. 1B). Comparison of these results with a previous kinetic analysis for BLC23O highlights non-significant differences (previously: K_M 418 ± 25 µM, kcat 0.20 ± 0.03 s^− 1and kcat/K_M 480 ± 80 M^− 1 s^− 1; (Adewale et al., 2021)).

Measurement of phenolic content in faba bean meal by BLC23O

Initially, the phenolic content in three distinct samples of faba bean was meal was assessed, including the original ‘total’ meal, as well as ‘fine’ and ‘course’ fractions of the meal obtained from air-classification. Following reaction of re-suspended meal fractions with the Folin-Ciocalteu (F-C) reagent, the absorbance at 725 nm wavelength was measured and quantified by comparison to a calibration curve made with pure tannic acid (Fig. 2 and Supplemental Figure S2A). Statistically significant differences in phenolic content were detected, where the fine fraction had the highest total phenolic content, followed by total meal, and then the coarse fraction. This is consistent with the fine fraction being protein enriched and the coarse fraction being carbohydrate enriched (Coda et al., 2015). Higher-phenolics in the ‘fine’ protein fraction is consistent with the protein amino acids introducing significant phenolic content, in addition to other (poly)phenolics being fractionated into the sample due to linkages with the protein. Some phenolic content was also observed in the ‘coarse’, carbohydrate rich fraction, likely due to some association between carbohydrates and (poly)phenols (Kardum & Glibetic, 2018; Smeriglio, Barreca, Bellocco, & Trombetta, 2017). That this assay was able to detect the differences between the meal fractions, demonstrates that the F-C reagent is appropriate for use in phenolic content determination assays in faba bean meal.

The kinetics of the F-C reagent were evaluated in more detail at the maximum amount of phenolics detected in meal reactions, using 14 µg/mL tannic acid (Supplemental Figure S2B). Under these conditions the reaction was complete within 60–70 min and phenolic content remained constant for an additional 120 min thereafter. These outcomes highlight the minimum amount of time solutions should be left to react before measuring the absorbance, ensuring an efficient and robust assay.

Biocatalytic reduction of phenolics in faba bean meal

The ability of BLC23O to degrade polyphenols in total faba bean meal and the air classified fractions was evaluated by monitoring phenolic content over time. Following addition of the enzyme to the meal suspensions, samples were taken at 15 min intervals and reacted with the F-C reagent and absorbance obtained at 725 nm. In all cases, BLC23O-treatment led to detectable decreases in phenol content over time, compared to untreated meal samples (Fig. 3A-C). Importantly, in both the meal-alone and enzyme-alone samples, phenolic content remained constant or even displayed a slight increase over time. That increases in phenol content were observed in the meal fractions (in the absence of any enzyme) is likely representative of the effect of chemical hydrolysis over time. (Poly)phenol content can be inaccessible due to being embedded in large complex structures (Smeriglio et al., 2017; Soares et al., 2020). Water hydrolysis is known to release phenols from the large complexes and break up interactions with proteins and sugars, increasing phenol available to react with the enzyme and assay reagents. Thus to quantify the effect of the enzymatic treatment on phenolic levels, the ratio between phenolics in the BLC23O-treated samples and phenolics in the meal-only samples were determined. Additionally, background phenolics detected in the enzyme-only sample were subtracted. This analysis revealed a 49.6% decrease in phenolic content in the course meal fraction over 3 h (Fig. 3F). Phenolic content in the total meal was reduced by 26.7%, while only a 9.0% reduction was observed in the fine fraction (Fig. 3E & 3D). The coefficient of determination (R²) values for the linear regressions of each fraction type are low, especially for the assay with the coarse flour, indicating a high amount of variability in the data.

To ascertain the stability of the reaction, overnight assays were performed for the total meal fraction in the presence and absence of BLC23O, as well as for the enzyme-alone (background control). Samples were taken at 0 and 24 h, reacted with the F-C reagent for 60 min, and absorbances obtained at 725 nm wavelength. Consistent with previous experiments, a small but significant increase in phenolics was detected in the meal-only sample, while decreases in both the enzyme-only and the BLC23O-treated meal samples were observed (Fig. 4). Quantification (as described above) indicates a 68% decrease in phenolics in this meal fraction upon treatment with BLC23O for 24 hrs. This represents a 41% further decrease in phenolic content compared to values obtained after only 3 h, and emphasizes the catalytic stability of BLC23O over time in this complex environment.

In conclusion, this study demonstrates the viability of cell-free biocatalytic reduction of phenolic content in faba bean meal. The representative catechol dioxygenase, BLC23O, enzyme used in the proof-of concept was selected based on its known stability and broad substrate specificity. Its amenability to high yield fermentative recombinant production has now been established, and its ongoing stability in complex environments including resuspension in a slurry of pulse meal demonstrated. While results suggest that BLC23O is effective for biocatalytic phenolic reduction, the upstream release of phenolics from higher molecular weight species (tannins, proteins, carbohydrates) is likely a limiting factor in the absence of other enzymes or microbial fermentation. Analyses of the effect of including other enzymes with BLC23O to improve substrate availability, and the effect of adding BLC23O to fermentative processes is ongoing, toward optimization of the nutritional and economical value of pulse meal in the animal feed industry.

All chemicals were obtained from Sigma Aldrich, except where indicated below. Faba beans were obtained from Faba Canada Ltd, de-hulled at the Canadian International Grains Institute (Winnipeg, Canada), and milled/air classified at the Richardson Center for Food Technology and Research (Winnipeg, Canada), yielding a ‘total’ meal fraction (before air classification), and then air classification-derived ‘fine’ and ‘course’ meal fractions.

BLC23O fermentative scale production

An optimized version of the BLC23O coding region was cloned into the pET28B + vector such that the expressed fusion protein included an N-terminal 6 x His tag as previously described (Adewale et al., 2021). Echerichia coli BL21 cells containing the expression vector encoding the BLC23O gene was used to inoculate an overnight culture containing 50 mL of Terrific Broth (TB) medium (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) with 50 µg/mL kanamycin. The culture was incubated overnight at 37°C in a MaxQ 6000 shaker (Thermo Scientific) at 150 rpm. The overnight culture was used to inoculate a BioFlo/CelliGen 115 benchtop fermentor & bioreactor (New Brunswick Scientific) containing 4 L of TB medium, 50 mg/L kanamycin, and 0.5 mL of antifoam. The bioreactor had an agitation of 250 rpm, its water jacket temperature was set to 37°C, and the dissolved oxygen setpoint was 30%. The culture was grown to an OD600 between 0.6 and 0.7 at which time protein expression was induced with the addition of 0.7 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was left growing in the bioreactor overnight at 18°C. The media was centrifuged in a Sorvall Lynx 4000 centrifuge (Thermo scientific) at 3250 x g for 30 min at 4°C.

Purification of recombinantly produced BLC23O

The obtained pellet was resuspended in 500 mL of 1x lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 3U/mL benzonase, 1 mg/mL lysozyme, pH 8.0). Proteins were extracted by high pressure homogenization using an EmulsiFlex-C5 (Avestin) according to the manufacturer’s protocol. The lysate was centrifuged (Thermo scientific) at 12,000 rpm for 15 min at 4°C. The supernatant was collected and applied to nickel-nitriloacetic acid (Ni-NTA) resin (Qiagen) with binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) by a batch method, where resin was mixed with the cleared lysate in a MaxQ 6000 shaker (Thermo Scientific) set to 200 rpm at 4°C for 2 hrs. The sample was centrifuged in a Sorvall Legend X1R centrifuge at 1000 x g for 3 min at 4°C (Thermo scientific) and the supernatant discarded. The remaining beads were washed three times using this same process with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and then BLC23O was eluted using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Obtained elution supernatants containing the BLC23O enzymes were pooled and concentrated using an Amicon Ultra-15 centrifugal filter with a 10 kDa cutoff (MilliporeSigma). The final concentration of the obtained BLC23O sample was determined using a Quick Start Bradford protein assay (Bio-Rad) according to the manufacturer’s protocol. Proteins samples were visualized by 12% SDS-PAGE.

A small portion (approx. 4% of the total sample) of the obtained enriched enzyme was further subjected to Fast Performance Liquid Chromatography (FPLC; Amersham Pharmacia Biotech) size-exclusion chromatography (SEC) with a HiLoad 16/600 Superdex 200 (Cytvia) column. Size exclusion buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) was degassed and the FPLC flowrate was set to 1 mL/min. Elution fractions were collected in increments of 1.4 mL. Fractions containing BLC23O were combined and concentrated with an Amicon Ultra-15 centrifugal filter with a 10 kDa cutoff (MilliporeSigma). The final concentrations of BLC23O were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. Proteins samples were visualized by 12% SDS-PAGE.

BLC23O kinetic analysis

Reaction solutions with total volumes of 250 µL were prepared in triplicate in a 96-well microplate (Greiner Bio-One) with 50 mM phosphate buffer pH 7.5, 18.7 µg/mL of BLC23O purified by Ni-NTA and SEC, 10 mM Mn²⁺, and substrate (3-methylcatechol) concentrations varying from 0.05 mM to 3 mM. Plates were pre-incubated for 5 min at 32.5°C and reactions were initiated with the addition of substrate and samples left at 32.5°C. The absorbance was read at 388 nm for 60 min in 30 sec intervals using a SpectraMax M5e spectrophotometer (Molecular Devices). The initial reaction rate, in µM of product formed per second, was plotted and the data fitted to the Michaelis-Menten equation using Microsoft Excel’s Solver add-in.

Determination of phenolic content by F-C reagent.

Based on previous reports (Blainski, Lopes, & de Mello, 2013; Everette et al., 2010), a calibration curve for the quantification of phenolic content was created where reaction solutions with a volume of 1 mL were prepared with 0.125 N F-C reagent, 0.125 g/mL of sodium carbonate, and tannic acid concentrations ranging from 0 to 30 µg/mL. The solutions were left to react for 45 min and then 250 µL of each was transferred into a 96-well microplate (Greiner Bio-One) and the absorbance was read at 725 nm using a SpectraMax M5e spectrophotometer (Molecular Devices). This was repeated in triplicate. Tannic acid reaction kinetics were assessed in a similar manner, but with reaction solution volumes of 250 µL and tannic acid concentrations ranging from 0 to 14 µg/mL. In this case, as soon as the tannic acid was added, the absorbance at 725 nm was monitored for 180 min in 15 min intervals using an Epoch 2 microplate spectrophotometer (Biotek).

Initial phenolic content in the three faba bean meal fractions was determined using the same procedure as described above, but with 0.1, 0.2, and 0.3 mg/mL of each type of faba bean sample instead of tannic acid, and incubation with the F-C reagent for 60 min. Results were compared to the tannic acid calibration curve for quantification, based on the assumption that a µg of commercial tannic acid is equivalent to a µg of phenolic content in the meal.

BLC23O-mediated phenolic reduction in faba bean flour

BLC23O reaction solutions with volumes of 4 mL were prepared for each of the faba bean meal fractions with 1.2 mg/mL of each fraction type and reactions initiated with the addition of 0.06 mg/mL of BLC23O enzyme. A blank solution with no meal or enzyme, as well negative and background controls of meal only and enzyme only were also prepared. Samples from the reaction mixtures were taken at 15 min intervals for 3 hrs, vortexed into 0.125 N F-C reagent and 0.125 g/mL of sodium carbonate and then left to react for 60 min at room temperature. Subsequently, 250 µL from each mixture was transferred to a 96-well microplate (Greiner Bio-One) and the absorbance was read at 725 nm using Epoch 2 microplate spectrophotometer (Biotek). This was performed in triplicate. The % phenolic reduction was calculated using the following equation: (phenolic content of meal in reaction mixture (%) = (R_abs (t) - E_abs (t)) / (F_abs (t)) * 100%, where Rabs(t) is the absorbance of the reaction mixture at time t, Eabs(t) is the absorbance of the enzyme sample at time t, and Fabs(t) is the absorbance of the specific flour (fine, meal or coarse) at time t. Additional assays were left to react for 24 h before being added to the F-C and sodium carbonate.

Statistical analysis and graphing

Statistical analyses and graphing of data were performed using Microsoft Excel. Unless otherwise stated, statistical analyses were done using a t-test and a p-value of 0.05 or lower was considered significant.

Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O); anti-nutritional factors (ANFs); Folin-Ciocalteu (F-C);

Ethics approval and consent to participate – Not Applicable

Consent for publication – Not Applicable

Availability of data and materials - All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests - The authors declare that they have no competing interests

Funding - This work was funded by the Natural Sciences and Engineering Research Council - Alliance Program [Project # ALLRP 550057-20] to M.E.L and M.C.L. This funding body had no input into the design of the study or collection, analysis, or interpretation of data or in writing the manuscript.

Authors' contributions - RM designed, conducted and interpreted all experiments, wrote first draft of manuscript. FH trained RM, designed experiments, interpreted results. MEL conceived of some ideas, conducted faba bean meal de-hulling, grinding and air-classification. TCY conceived of some ideas, interpreted the data. MCL conceived of some ideas, supervised FH and RM, designed experiments, interpreted the data and finalized the manuscript. All authors read and approved the manuscript.

Acknowledgements - This manuscript represents National Research Council of Canada Communication NRCC# 58344.

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Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

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Abstract

Figures

Introduction

Results And Discussion

Large-scale (4 L fermentative) production and Ni-NTA enrichment of BLC23O

Measurement of phenolic content in faba bean meal by BLC23O

Biocatalytic reduction of phenolics in faba bean meal

Conclusions

Methods And Materials

BLC23O fermentative scale production

Purification of recombinantly produced BLC23O

BLC23O kinetic analysis

BLC23O-mediated phenolic reduction in faba bean flour

Statistical analysis and graphing

Abbreviations

Declarations

References

Supplementary Files

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Version 1