Extent of Enzyme Inhibition by Phenolics Derived from Pretreated

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The extent of enzyme inhibition by phenolics derived from pretreated biomass is significantly influenced by the size and carbonyl group content of the phenolics Rui Zhai, Jinguang Hu, and John (Jack) N Saddler ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04178 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Sustainable Chemistry & Engineering

The extent of enzyme inhibition by phenolics derived from pretreated biomass is significantly influenced by the size and carbonyl group content of the phenolics Rui Zhai†‡, Jinguang Hu‡* and Jack (John) N. Saddler‡* †

School of Environmental and Biological Engineering, Nanjing University of Science and

Technology, 200 Xiaolingwei Street, Nanjing 210094, China ‡

Forest Products Biotechnology and Bioenergy Group, Department of Wood Science, Faculty of

Forestry, The University of British Columbia, 2424 Main Mall, Vancouver BC, Canada.

Corresponding Authors Jack (John) N. Saddler, Email: [email protected] Jinguang Hu, Email: [email protected]

ABSTRACT In the current biorefinery concept, hydrothermal based pretreatments such as steam pretreatment are often used to, first open up the plant cell wall structure and, increase the accessibility of the cellulose component to cellulase enzymes. However, this pretreatment process also generates water-soluble phenolics that are strongly inhibitory to the cellulase

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enzymes, therefore limiting the efficiency of the enzymatic hydrolysis of the pretreated biomass. Although some earlier work assessed the influence of phenolics on cellulose hydrolysis, the characteristics of the phenolics that most influenced inhibition have not yet been elucidated. As biomass derived phenolics are heterogeneous, monomer/oligomeric/polymeric aromatic compounds that have diverse functional groups and varied molecular size, the current work investigated the influence of the major structural properties of the pretreatment derived phenolics, such as their molecular weight, the nature of their hydroxyl and carbonyl groups, on enzyme inhibition. It was apparent that phenolics derived from steam pretreated softwood were more inhibitory than the phenolics derived from steam pretreated hardwoods with phenolics with a smaller molecular size and greater carbonyl content being more inhibitory to enzyme-catalyzed cellulose hydrolysis.

KEYWORDS: Cellulase; Phenolics inhibition; Enzymatic hydrolysis; Steam pretreatment; Lignocellulose

INTRODUCTION

Although lignocellulosic biomass have been considered as promising sustainable feedstocks for the production of a wide range of bio-based fuels, chemicals and materials, it has remained challenging to develop an economical feasible production process due to the recalcitrant characteristics of the nature biomass 1. Therefore, a physicochemical pretreatment process such as steam explosion has been commonly used to firstly open up the biomass structure to partially overcome its recalcitrance before any further application 1. Steam pretreatment has been considered as a promising industrial relevant method as it could greatly increase the accessibility of cellulose to cellulase enzymes and can be used to treat a wide range of biomass feedstocks


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with short time and limited chemical loading 2. However, one of the problems of steam pretreatment (similar with other types of acid catalyzed pretreatment) is the generation of watersoluble inhibitory compounds such as sugars, phenolics, furans and organic acid, which have been shown to strongly inhibit the following enzymatic hydrolysis and fermentation processes 3. Among them, the water-soluble phenolics derived from lignin degradation are one of the major groups of inhibitors which, not only inhibit, but also deactivate commercial cellulase preparations 4. We have recently shown that the partial phenolics removal (by activated carbon) could alleviate this phenolics-mediated inhibition, which indicated that only certain phenolics were strongly inhibitory to the enzymatic hydrolysis 5. However, it was not clear which aspect of the phenolics resulted in this inhibition and which of the major characteristics of the various phenolics predominated in causing this inhibition of cellulose hydrolysis.

During the acid catalyzed steam pretreatment process, the lignin of the biomass can be either depolymerized or repolymerized, generating soluble phenolics or more condensed lignin respectively. Although the structure of lignin and their effect on cellulose hydrolysis has been extensively studied

6–10

, the major characteristics of the soluble phenolics that might inhibit the

hydrolytic performance of cellulase enzyme have remained unclear. One of the potential structural properties of phenolics that might relate to its inhibitory effect on cellulases is the molecular size of the compound. For example, some studies have found that polymeric phenolics are more inhibitory than monomeric phenolics

11,12

. However, other work has shown that the

dehydrogenative polymers from ferulic acid are actually less inhibitory than monomeric ferulic acid

13

. These contradictory results indicated that the effects of phenolics on cellulase enzymes

might be more complicated rather than just a correlation with the molecular weight of the phenolics. For example, some studies have reported that “Hibbert's ketones” type phenolics (e.g.



phenylpropanoid ketones) can strongly inhibit microorganisms because of their strong binding affinity to cell membrane proteins

14,15

. Thus, these ketone-type phenolics might also be highly

inhibitory to cellulose hydrolysis by strongly binding to cellulase enzymes. In addition, hydroxyl groups on model phenolic compounds have also been shown to be critical components in contributing to the inhibitory effects of lignin on cellulase enzymes 7. However, whether these hydroxyl groups on the pretreatment-derived water-soluble phenolics are also inhibitory towards cellulase enzymes has yet to be determined.

To try to better understand the roles and function of pretreatment derived soluble phenolics on cellulase enzymes, we extracted the phenolics from the water-soluble fractions of steam pretreated softwood (lodgepole pine) and hardwood (poplar) and assessed their influence on the hydrolytic performance of the recent cellulase preparation (CTec 3). The extracted phenolics were further fractionated into several fractions according to their molecular size to assess the effects of phenolic size on the cellulose hydrolysis. In addition, the effects of essential phenolic functional groups such as carbonyl groups and phenol hydroxyl groups on the cellulase enzymes were also systematically evaluated, to try to better understand the main properties of phenolics that induced enzyme inhibition.

MATERIALS AND METHODS

Chemicals and enzyme preparation. The mountain pine beetle killed lodgepole pine (Pinus contorta) (LPP) and hybrid poplar (POP) were provided by the British Columbia Forest service and FP Innovations, respectively. Commercial cellulase (Cellic® CTec3) was provided by Novozymes, North America Inc. (Franklinton, NC). The total protein content of CTec3 (245 mg protein/mL enzyme cocktail) was


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measured according to the ninhydrin method with bovine serum albumin as the protein standard 16

. The chemicals, acid and base were obtained from Sigma Aldrich.

Pretreatment of lodgepole pine and poplar

Softwood (lodgepole pine) and hardwood (poplar) chips were pretreated by SO2-catalyzed steam pretreatment using 4% and 3% w/w SO2 (based on the dry weight of the biomass), respectively in a 2-L StakeTech II steam gun (Stake Technology, Norval, Ontario) at 200 °C for 5 min, which have previously been found to be the optimal conditions 17. After pretreatment, the slurry was fractionated by filtration into water insoluble fraction and water-soluble (WS) fraction.

Extraction of soluble phenolics from pretreatment derived water-soluble fractions

Activated carbon was used to selectively adsorb the potential inhibitory phenolics in the WS fractions from steam pretreated lodgepole pine (SPLP) and poplar (SPP) 5. Firstly, the WS fractions were incubated with an optimized dosage of 5 % w/w activated carbon (based on the weight of the WS fractions) overnight at 30 oC 5. After 16 h incubation, the activated carbon was filtered through Whatman No.1 filter paper. The collected activated carbon was washed with water to removed unabsorbed components and then desorbed with 50%-100% acetone at 40 oC. The obtained acetone/water mixture containing desorbed phenolics were air dried and redissolved 0.05 M sodium acetate buffer (pH 4.8) to make phenolics stock solution.

Enzymatic hydrolysis

To study the inhibitory effect of the extracted phenolics on the enzymatic hydrolysis of cellulosic substrates, dissolving pulp (DP), a delignified, high purity cellulose pulp (94% cellulose) from Tembec (Temiscaming, Canada), was used as the model cellulosic substrate. The



enzyme mediated hydrolysis of the pure cellulosic substrate, dissolving pulp (DP), was performed using CTec3 (10-30 mg protein/g cellulose) at 50 °C on a rotary incubator for 72 h in 0.05 M sodium acetate buffer (pH 4.8) with and without addition of phenolics. Samples were taken at different time and the glucose concentration in the supernatants was measured using HPLC (Dionex DX-3000, Sunnyvale, CA). Substrate and enzyme blanks were performed at the same time by incubating the substrates in buffer without enzymes and by incubating the enzymes alone. The cellulose hydrolysis yields (%) of DP were determined based on the amount of released glucose divided by the theoretical cellulose content of the substrates.

Determination of the content of phenolics

The total phenolics in the stock solution were measured using the Folin-Ciocalteu method and calculated as the equivalent vanillin amount, as proposed by Singleton and Rossi (1965). Briefly, the concentration of total phenolics in WS fraction was analyzed using Folin-Ciocalteu reagent with vanillin as the standard. 100 µL of the diluted sample was mixed with 250 µL of the FolinCiocalteu reagent. After 5 minutes, 750 µL of 20% (w/v) Na2CO3 was added into the mixture. The total volume was adjusted to 5 mL using deionized water and the mixture was then incubated for 2 hours at 22 °C. Finally, the absorbance of reaction mixture was read at 760 nm spectrophotometrically. The reactions were carried out in triplicate, and the value reported was the average of three analyses. To analyze the composition of the phenolics stock, the concentration of sugars (arabinose, galactose, glucose, xylose, and mannose) and the degradation products (5-hydroxymethyl furfural (HMF), furfural and acetic acid) were determined using High Performance Liquid Chromatography (HPLC) system according to a previous study 5.

Fractionation of soluble phenolics according to molecular size


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In order to study the effect of the molecular size of phenolics on the inhibitory effect of phenolics on enzymatic hydrolysis of cellulose. The extracted phenolics was fractionated by filtering a phenolics solution through 1, 3 and 10 kDa ultrafiltration membranes (Millipore, Bedford, MA) by using an Amicon ultrafiltration system (Amicon, Beverly, MA) under 30 psi nitrogen gas pressure. Before use, the membranes were washed twice with nanopure water and incubated in 50% ethanol solution. The phenolics solution was then transferred into the Amicon container and filtered through until 10% of the original volume remained. After filtration, the filtrates and retentates were stored at 4 oC for further use.

Hydroxypropylation of phenolics

To test the effect of the contribution of the phenolic hydroxyl group to enzyme inhibition, the phenolics were modified by hydroxypropylation to block phenolic hydroxyl group according to the method described previously 18. 3 mL of phenolics stock (~20 g/L) was mixed with 180 mg NaOH and vortexed for 1 min. Then 2 mL propylene (PO) was added into the mixture and stirred at room temperature (∼22 °C) for 24 h. Subsequently, the reaction mixture was heated to 60 °C to remove potentially unreacted PO. Then the HCl (6 M) was added to adjust the pH to 4.8.

Chemical reduction of phenolics

The chemical reduction of the extracted phenolics was performed as follows. Firstly, the pH of the phenolics solution was adjusted to 7.0 with a 5 M solution of sodium hydroxide. Then the sodium borohydride (Sigma–Aldrich) was added directly to the phenolics solution in different concentrations. The reaction was carried out at room temperature for 24 h. After the reaction, the



pH of the phenolics solution was adjusted to 4.8 with 6 M HCl to decompose the unreacted NaBH4. Then, the NaBH4 treated phenolics solution were stored at 4 oC for further analysis.

Characterization of phenolics using Fourier Transform Infrared Spectroscopy

The extracted phenolics from pretreatment water-soluble fraction were analyzed by fourier transform infrared (FTIR) spectroscopy. The analysis was carried out by adding several drops of the phenolics stock solution onto a KBr plate and evaporating the solvent. The solid residual on the KBr plate was subjected to FTIR analysis. The FTIR spectra were the then obtained on a Varian 3100 (Varian Inc. Palo Alto, CA) with MIRacle Accessory (Pike technologies, Madison, WI). The absorbance was recorded between 400-4000 cm-1 at a resolution of 4 cm-1 with 32 scans.

RESULTS AND DISCUSSION

Effect of phenolics derived from pretreatment on cellulose hydrolysis

The pretreatment derived phenolics were fractionated from the water-soluble fractions of steam pretreated lodgepole pine (SPLP-WS-Phe) and poplar (SPP-WS-Phe), respectively 19, and their effects on the cellulose hydrolysis were firstly assessed on a “model/pure” cellulosic substrate dissolving pulp (DP) with various CTec3 enzyme loadings (10-30 mg protein/g cellulose). Although the phenolics stock solution (20 g/L) derived from SPLP and SPP also contains tiny amount of monomeric sugars and other non-phenolic degradation products such as furans and acetic acid (with concentrations within 0-0.5 g/L), these components at the low concentrations have no effect on the cellulose hydrolysis catalyzed by CTec 3 (data not shown).


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Similarly, other researchers have also reported that these small amount of sugars and their degradation products had no inhibitory effects on the enzymatic hydrolysis of cellulose 20,21.

To study the effect of phenolics on cellulose hydrolysis, a phenolics concentration of 3 g/L was selected since this was the typical phenolic concentration found in the water-soluble fractions derived from pretreatment

20,22–24

. It was apparent that both types of phenolics

significantly inhibited cellulose hydrolysis, where the softwood phenolics (SPLP-WS-Phe) were more inhibitory than the hardwood phenolics (SPP-WS-Phe), especially at high enzyme loading (Figure 1a). It also showed that higher enzyme loadings could alleviate (to some degree) the inhibitory effects of the SPP-WS-Phe but not the SPLP-WS-Phe, which was likely due to the different phenolic structure generated from hardwood and softwood lignin 25.

The effect of phenolics on the kinetic of cellulose hydrolysis was next assessed at relatively low enzyme loadings (10 mg protein/g cellulose), since it is preferred in the industrial application. As expected, both SPLP-WS-Phe and SPP-WS-Phe fractions significantly decreased both the rate of cellulose hydrolysis (cellulose conversion after 12 h) and the extent of hydrolysis (cellulose conversion after 72 h), with the extent of the hydrolysis more severely inhibited (Figure 1b). When different phenolics concentrations (from 1 to 6 g/L) were added in the cellulose hydrolysis, it appeared that the inhibitory effects of phenolics were levelled off when the phenolic concentration reached 3 g/L, and the extent of cellulose hydrolysis was more strongly inhibited than the rate of hydrolysis in all the conditions tested (see the Supporting Information, Figure S1). All these observations indicated that phenolics derived from steam pretreated biomass would be more problematic in longer term cellulose hydrolysis, and their inhibitory effects were highly dependent on the concentration as well as the type of phenolics: It



was apparent that the inhibitory effect of steam pretreated softwood derived phenolics was much stronger than the phenolics derived from steam pretreated hardwoods.

Possible influence of the phenolic size on cellulose hydrolysis It has been acknowledged that the size and the chemical structure of inhibitors are usually the determining factors for their inhibitory effects 26. To study the size effects, the isolated phenolics were further fractionated into four fractions according to their molecular size distribution, namely WS-PheA (>10 kDa), WS-PheB (10 kDa -3 kDa), WS-PheC (3 kDa-1 kDa) and WSPheD (

Extent of Enzyme Inhibition by Phenolics Derived from Pretreated

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