Crystal structure of the feruloyl esterase from Lentilactobacillus buchneri reveals a novel homodimeric state

Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan

Enhancing methane production from lignocellulosic biomass by combined steam‑explosion pretreatment and bioaugmentation with cellulolytic bacterium Caldicellulosiruptor bescii

Background: Biogas production from lignocellulosic biomass is generally considered to be challenging due to the recalcitrant nature of this biomass. In this study, the recalcitrance of birch was reduced by applying steam-explosion (SE) pretreatment (210 °C and 10 min). Moreover, bioaugmentation with the cellulolytic bacterium Caldicellulosiruptor bescii was applied to possibly enhance the methane production from steam-exploded birch in an anaerobic digestion (AD) process under thermophilic conditions (62 °C). Results: Overall, the combined SE and bioaugmentation enhanced the methane yield up to 140% compared to untreated birch, while SE alone contributed to the major share of methane enhancement by 118%. The best methane improvement of 140% on day 50 was observed in bottles fed with pretreated birch and bioaugmentation with lower dosages of C. bescii (2 and 5% of inoculum volume). The maximum methane production rate also increased from 4-mL CH4/ g VS (volatile solids)/day for untreated birch to 9-14-mL CH4/ g VS/day for steam-exploded birch with applied bioaugmentation. Bioaugmentation was particularly effective for increasing the initial methane production rate of the pretreated birch yielding 21-44% more methane than the pretreated birch without applied bioaugmentation. The extent of solubilization of the organic matter was increased by more than twofold when combined SE pretreatment and bioaugmentation was used in comparison with the methane production from untreated birch. The beneficial effects of SE and bioaugmentation on methane yield indicated that biomass recalcitrance and hydrolysis step are the limiting factors for efficient AD of lignocellulosic biomass. Microbial community analysis by 16S rRNA amplicon sequencing showed that the microbial community composition was altered by the pretreatment and bioaugmentation processes. Notably, the enhanced methane production by pretreatment and bioaugmentation was well correlated with the increase in abundance of key bacterial and archaeal communities, particularly the hydrolytic bacterium Caldicoprobacter, several members of syntrophic acetate oxidizing bacteria and the hydrogenotrophic Methanothermobacter. Conclusion: Our findings demonstrate the potential of combined SE and bioaugmentation for enhancing methane production from lignocellulosic biomass

Data_Sheet_1_Crystal structure of the feruloyl esterase from Lentilactobacillus buchneri reveals a novel homodimeric state.PDF

Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan.</p

A Highly Efficient Recombinant Laccase from the Yeast <i>Yarrowia lipolytica</i> and Its Application in the Hydrolysis of Biomass

<div><p>A modified thermal asymmetric interlaced polymerase chain reaction was performed to obtain the first yeast laccase gene (YlLac) from the isolated yeast <i>Yarrowia lipolytica</i>. The 1557-bp full-length cDNA of YlLac encoded a mature laccase protein containing 519 amino acids preceded by a signal peptide of 19 amino acids, and the YlLac gene was expressed in the yeast <i>Pichia pastoris</i>. YlLac is a monomeric glycoprotein with a molecular mass of ~55 kDa as determined by polyacrylamide-gel electrophoresis. It showed a higher catalytic efficiency towards 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (<i>k_cat/K_m</i> = 17.5 s^-1 μM^-1) and 2,6-dimethoxyphenol (<i>k_cat/K_m</i> = 16.1 s^-1 μM^-1) than other reported laccases. The standard redox potential of the T1 site of the enzyme was found to be 772 mV. The highest catalytic efficiency of the yeast recombinant laccase, YlLac, makes it a good candidate for industrial applications: it removes phenolic compounds in acid-pretreated woody biomass (<i>Populus balsamifera</i>) and enhanced saccharification.</p></div

Strains, plasmids, and oligonucleotide primers used in this study.

<p>^<i>a</i>The XhoI site is in small letters.</p><p>^<i>b</i>The XbaI site is in small letters.</p><p>^<i>c</i>The KpnI site is in small letters.</p><p>W = A / T, S = G / C, N = A/T / G / C</p><p>Strains, plasmids, and oligonucleotide primers used in this study.</p

Substrate docking of YlLac with 2,6-DMP in the active site pocket.

<p>(a) 2,6-DMP docking into the active site of YlLac. The hydroxyl group of C1 of 2,6-DMP was bound in the active site through H-bonds (green dotted lines) with the oxygen in the carboxyl groups of D226 (3.4 Å) and H477 (3.0 and 3.8 Å). (b) Docking of 2,6-DMP into the active site of 3KW7 (crystal structure). Distances of 3.3 and 4.2 Å have been observed between C1 of 2,6-DMP and nitrogen in D206, respectively. The amino acid residues are shown in a stick model. Catalytic residues are labeled in red, and the residue labels refer to those of the laccase active site.</p

Overall structure of YlLac with its template.

<p>(a) Putative catalytic and copper binding domains of YlLac (yellow color carbon) superimposed on <i>Trametes</i> sp. AH28–2(PDB entry 3KW7, gray color carbon). The residues are shown in the stick model and are labeled with YlLac amino acid residue numbers. Copper ions for YlLac and <i>Trametes</i> sp. AH28–2 are shown in black and metallic sphere, respectively. (b) Ribbon diagram of the superimposed YlLac (green color) and <i>Trametes</i> sp. AH28–2 (grey color) structures with the catalytic residues, represented as a stick model. Amino acid numbers are based on the YlLac sequence. The YlLac catalytic site residues (His 477 and Asp 226) corresponding to the <i>Trametes</i> sp. AH28–2 catalytic residues have similar orientation. The figure was generated using DS 3.1</p

Time course of phenolic content of <i>P</i>. <i>balsamifera</i> prehydrolysate during pretreatment with YlLac at different pHs, (a) 3, (b) 4, (c) 5.

<p>Untreated control (without laccase) is also shown, (d) Reducing sugar production from acid-pretreated <i>P</i>. <i>balsamifera</i> by Celluclast 1.5L: without laccase pretreatment (black bar), and with YlLac pretreatment (gray bar). Error bars indicate standard deviations from mean values.</p

Dependence of the catalytic current on pH for YlLac-coated glassy carbon electrode with ABTS (○) and 2,6-DMP (■), as substrates.

<p>Dependence of the catalytic current on pH for YlLac-coated glassy carbon electrode with ABTS (○) and 2,6-DMP (■), as substrates.</p