Recovering Activities of Inactivated Cellulases by the Use of

Research Article pubs.acs.org/journal/ascecg

Recovering Activities of Inactivated Cellulases by the Use of Mannanase in Spruce Hydrolysis Donglin Xin, Ming Yang, Xiang Chen, Li Ma, and Junhua Zhang* College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi China S Supporting Information *

ABSTRACT: Softwood materials have gained considerable attention because of their abundance and high contents of carbohydrates that can be converted into high value products, such as alternative biofuels. However, during the enzymatic conversion of softwood to biofuels, there is a decrease of cellulase activity that significantly limits the conversion efficiency. This study examined the role of mannanase in recovering lost cellulase activity by relieving the inhibition of mannan on cellulases. Kinetic experiments indicated that mannan competitively inhibited Thermoascus aurantiacus cellobiohydrolase (Ta Cel7A) activity and irreversibly inhibited T. aurantiacus endoglucanase (Ta Cel5A) but had no inhibitory effect on Acremonium thermophilum β-glucosidase (At Cel3A). In spruce hydrolysis (100 g/L biomass) by cellulases, further supplementation of mannanase suppressed the inhibition of residual mannan on Ta Cel5A and Ta Cel7A, and the activities of Ta Cel5A and Ta Cel7A increased by 14.3 and 10.9%, respectively. The increase of Ta Cel5A and Ta Cel7A activity enhanced cellulases hydrolytic action and may benefit the subsequent cellulases recovery process. These results may help to characterize the role of mannanase in the production of alternative biofuels from softwood. KEYWORDS: Cellobiohydrolase, Mannan, Enzymatic hydrolysis, Cellulase activity, Mannanase, Spruce

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loadings and final biomass conversion yield were also reported by other researchers.9,10 To a large degree, this phenomenon offsets the benefits of hydrolysis at high solid loadings. Therefore, it is necessary to explore the reason behind this negative effect and overcome hydrolysis resistance at high solid loadings. Several theories have been offered to explain the reason for the low hydrolysis efficiency at high solid loadings: (1) High concentrations of substrate and products in the hydrolysate increase its viscosity and decrease the availability of water, thus limit mass transfer.11 (2) Products in the hydrolysate, such as glucose, cellobiose, hemicellulose oligomers, lignin, and its derivatives, inhibit cellulases.2,9,12−14 (3) Nonproductive adsorption of cellulases to lignin and high concentrations of products block the adsorption of cellulases to cellulose.15−17 According to these reports, it appears that hydrolysate products play a key role in limiting the biomass saccharification at high solid loadings. In lignocellulosic biomass, especially in biomass after pretreatment with alkali solutions, there is a considerable amount of hemicellulose that remains. To use biomass material effectively, the hemicelluloses should be maintained and saccharified in parallel with cellulose. However, hemicellulose

INTRODUCTION During the last two decades, there has been intensive research on the enzymatic conversion of lignocellulosic biomass to biofuels.1 However, the conversion process is not currently economical, and many factors limit the large-scale biological production of biofuels at a high efficiency. The solids loading is a potentially significant limitation. For most laboratory-scale reactions, the solids loading is less than 10%. Although low solids loading routinely results in a higher cellulose conversion, low solid hydrolysis in industrial-scale reactions requires larger processing volumes and higher operating costs due to lower product concentrations.2 According to previous reports, the ethanol concentration in the fermentation broth should be more than 4% (w/w) in order to make the following distillation process economically feasible.3,4 Theoretically, for most lignocellulosic biomass, the solid loadings in the enzymatic hydrolysis process should be more than 15% to meet this requirement.4 Several studies reported the feasibility of using fed-batch and simultaneous saccharification and fermentation mode to hydrolyze chemically pretreated spruce, corn stover, and coniferous wood at high substrate loading (>15%). 5−7 However, the biomass hydrolysis yield was low when the solid concentration increased. It was observed that the conversion yield of olive tree after pretreatment with liquid hot water and steam explosion were as low as 49.9 and 39.6% at 30% substrate loading.8 The negative correlation of solid © 2017 American Chemical Society

Received: February 26, 2017 Revised: April 13, 2017 Published: May 2, 2017 5265

DOI: 10.1021/acssuschemeng.7b00605 ACS Sustainable Chem. Eng. 2017, 5, 5265−5272

Research Article

ACS Sustainable Chemistry & Engineering

Trichoderma reesei strain21 (for updates of CAZy, see http://www. cazy.org). The cbh1, cbh2, egl1, and egl2 genes encoding Tr Cel7A, Tr Cel6A, Tr Cel7B, and Tr Cel5A, respectively, were deleted as described elsewhere.22−24 All enzyme preparations were adjusted to pH 6.0 and treated at 60 °C for 2 h to inactivate the background T. reesei enzymes. These thermostable enzyme preparations were kindly provided by RoalOy (Rajamäki, Finland). Ta Cel7A activity was determined by pNP release from 1 mg/mL pnitrophenol-D-cellobioside (pNPC) in 50 mM sodium citrate buffer at 50 °C for 30 min, as described in Deshpande et al.25 The reaction was stopped by adding 0.5 mL of 2% Na2CO3. After incubation, the amount of pNP was determined by measuring the absorbance at 410 nm. Ta Cel5A activity was measured in 50 mM sodium citrate buffer at 50 °C for 10 min, using 10 mg/mL hydroxyethyl cellulose (HEC) as substrate, as previously described.26 To stop the reaction, 1.5 mL of DNS was added. After boiling for 5 min and cooling, the amount of reducing sugars was determined by measuring the absorbance at 540 nm. The commercial cellulase preparations Celluclast 1.5L and Novozyme 188 (Novozymes A/S, Bagsværd, Denmark) were used to provide a complete mixture of cellulases. The FPU activity of Celluclast 1.5L was 74.7 FPU/mL (169.6 mg protein/mL), measured according to the IUPAC standard assay26 based on the amount of reducing sugars released from Whatman number 1 filter paper using 2,4-dinitrosalicylic acid (DNS). The activity of Novozyme 188 was determined to be 5121 nkat/mL (187.9 mg protein/mL) by measuring the amount of p-nitrophenol (PNP) released from p-nitrophenyl-β-Dglucopyranoside (PNPG), as described by Bailey and Nevalainen.27 Endo-1,4-β-mannanase from Aspergillus niger was purchased from Megazyme (Bray, Wicklow, Ireland), with an activity of 10 mkat/mL (9.8 mg protein/mL) based on the supplier’s data. The protein contents of these enzymes were quantified by the Lowry method28 using bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as a standard. Biomass Pretreatment. Dilute acid pretreatment was carried out with 2% (w/w) dilute acid and a solid to liquid ratio of 1:25 at 121 °C for 2 h in an autoclave, as described in our previous report.29 After incubation, the suspension was centrifuged at 3040 g for 10 min to collect the solid residue. The solid material was subsequently washed with distilled water to neutralize it and was air-dried. Na2SO3 pretreatment was carried out in an autoclave with 8% (w/ w) Na2SO3 and 0.5% NaOH and a solid to liquid ratio of 1:10 at 121 °C for 2 h, according to method modified from that of Li et al.30 After pretreatment, the liquor and solid material were separated by filtration. The solid material was washed with distilled water to neutralize it and air-dried. The pretreated material was further pretreated with NaclO2 (0.6 g NaclO2/g raw biomass, 0.2 mL acetic acid/g raw biomass, solid to liquid ratio 1:33, as described previously)31,32 to remove lignin. After pretreatment, the solid material was washed and air-dried as described above. All of the dried materials (DA-SP, NA-SP, and DL-NA-SP) were ground to pass through an 80 mesh screen and stored at room temperature for further use. The chemical compositions of the materials were determined by the procedure published by the National Renewable Energy Laboratory (NREL).33 The corresponding data are shown in Figure S1. Enzymatic Hydrolysis. The hydrolysis of DA-SP, NA-SP, and DLNA-SP (100 g/L biomass) by the cellulase preparations was performed in tubes with a working volume of 2 mL of 50 mM sodium citrate buffer (pH 5.0) containing 0.02% NaN3 at 50 °C.34 Commercial cellulases were dosed at 20 FPU of Celluclast 1.5L/g DM (dry matter) and 500 nkat Novozyme 188/g DM. Samples were withdrawn after 48 h of hydrolysis. The samples were centrifuged at 10 000 g for 10 min, and the supernatants were analyzed for glucose, xylose, mannose. The cellulase activities were determined (including Cel5A, Cel7A, and Cel3A) in the hydrolysates. The hydrolysis of Avicel, Cellulose fiber, and cellobiose by the cellulase preparations were performed at a working volume of 2 mL of 50 mM sodium citrate buffer (pH 5.0) containing 0.02% NaN3 at 50

can be difficult to completely hydrolyze to xylose or mannose in the absence of powerful hydrolytic enzymes, such as endoxylanase, β-xylosidase, debranching enzymes (such as arabinofuranosidases and glucuronidase, depending on the residual substituent groups of xylan in pretreated biomass), or the corresponding mannan hydrolytic enzymes in commercial cellulase preparations.18 Although most commercial cellulase preparations contain a considerable amount of hemicellulases and could convert most oligosaccharides to monosaccharides, some oligomeric sugars are found to not be converted, leading to an accumulation of oligosaccharides in the hydrolysate.19 The hemicelluloses and their derivatives could not be directly utilized because of the lack of corresponding metabolizing yeast and bacteria in the conversion process. Even worse, the hemicelluloses (both xylan and mannan) and oligosaccharides are found to be inhibitors of cellulases.12,20 It is known that hemicelluloses in original bound form could limit the access of cellulases to cellulose therefore limiting cellulose hydrolysis. Kumar and Wyman12 previously reported that even low amounts of added mannan (2.5 mg/mL) dropped the conversion yield of simulated substrate, Avicel, from approximately 90 to 70%. The results indicated that mannan even in its free form has a negative effect on cellulose hydrolysis; however, the inhibition mechanism has not been fully investigated. In addition, the most commonly used commercial cellulases (from Trichoderma reesei) are composed mainly of endoglucanases (Cel5A, Cel7B, Cel12A, Cel45A, Cel61A, and Cel74A), cellobiohydrolases (Cel6A and Cel7A), and βglucosidase (Cel3A); however, the effect of mannan on the action of individual components of cellulases has not been evaluated. In contrast to our previous works, we measured the cellulase activities (including Cel5A, Cel7A, and Cel3A) in the hydrolysates of spruce after pretreatment with dilute acid (DA-SP), Na2SO3 (NA-SP), and NA-SP after delignification (DL-NA-SP) to evaluate the behavior of cellulase during the hydrolysis of mannan-containing materials. The presence of mannan and manno-oligosaccharides (MOS) in the hydrolysates was analyzed by FTIR and ESI-MS. This research further analyzed the effects of different variations of mannan on the hydrolytic action of commercial cellulases, pure T. aurantiacus endoglucanase (Ta Cel5A), Thermoascus aurantiacus cellobiohydrolase (Ta Cel7A), and Acremonium thermophilum β-glucosidase (At Cel3A). To investigate the inhibition mechanism, mannan-induced inhibition of Ta Cel5A and Ta Cel7A was studied using mannan with different structural properties. Additionally, the role of mannanase (MAN) in reducing mannan-induced inhibition was evaluated.

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EXPERIMENTAL SECTION

Materials. Microcrystalline cellulose (Avicel PH-101), cellulose fiber (medium), and cellobiose were purchased from Sigma Chemical Co. (St. Louis, MO). Spruce (SP) was purchased from Shanghai Tingzhong Industrial Co., Ltd. (Shanghai, China). Linear mannan (LMan, Lot131001a), low-viscosity carob galactomannan (GalM-L, mannose to galactose ratio: 3.76:1, viscosity 3 dL/g, Lot 10501b), and high-viscosity carob galactomannan (GalM-H, mannose to galactose ratio: 3.76:1, viscosity 11.2 dL/g, Lot 60305b) were purchased from Megazyme (Bray, Wicklow, Ireland). All other chemicals used in this work were analytical-grade and purchased from Sigma. Enzymes. Glycosyl hydrolase (GH) 5 family endoglucanase Ta Cel5A, GH 7 family cellobiohydrolase Ta Cel7A, and GH 3 family βglucosidase At Cel3A were produced in a genetically modified 5266


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Figure 1. Inhibition of mannan on cellulase. Hydrolysis of Avicel and cellulose (1%) by Celluclast 1.5L (10 FPU/g DM) (A), Ta Cel7A (8 mg/g DM) and At Cel3A (0.2 mg/g DM) (B), and Ta Cel5A (2 mg/g DM) and At Cel3A (0.2 mg/g DM) (C) with the addition of mannan (2.5 mg/mL) at 50 °C for 48 h. Hydrolysis of cellobiose (1%) by At Cel3A (0.2 mg/g DM) with the addition of mannan (2.5 mg/mL) (D) at 50 °C for 0.5, 2, 6 h. The error bars represent the standard error of three independent experiments. °C.34 The DM content of substrate was 20 g/L. Commercial cellulase preparation was dosed at 10 FPU Celluclast 1.5L/g DM. For the hydrolysis of Avicel and cellulose fiber, Ta Cel7A was dosed at 8 mg/g DM, Ta Cel5A was dosed at 2 mg/g DM, and At Cel3A was dosed at 0.2 mg/g DM. Cellobiose was hydrolyzed by At Cel3A at 0.2 mg/g DM. L-Man, GalM-L, and GalM-H were added to the reaction at the beginning of the enzymatic hydrolysis. Samples were removed and boiled for 10 min to stop the enzymatic hydrolysis. After cooling, the samples were centrifuged at 10 000 g for 10 min, and the supernatants were analyzed for glucose. Kinetics of Inhibition on Ta Cel5A and Ta Cel7A. Kinetic of inhibition of Ta Cel5A (HEC as substrate) and Ta Cel7A (pNPC as substrate) was performed, and the corresponding kinetic parameters were determined as previously described.35 FTIR Analysis. CEL-processed DL-NA-SP hydrolysate was freezedried. The dried samples were pressed into a disc with KBr and analyzed on a Thermo Nicolet 470 FTIR spectrophotometer (Thermo Nicolet Corporation, VA). The background measurement was subtracted from each sample readout. A total of 32 scans were collected for each measurement over the wavelength range of 4000− 400 cm−1. ESI-MS Analysis. For oligosaccharides compositional analysis in the DL-NA-SP hydrolysate, electrospray ionization-mass spectrometry (ESI-MS) analysis was conducted with a Thermo LCQ Fleet instrument (Thermo Fisher Scientific, USA) equipped with an electrospray ionization source. Ionization was obtained in the positive ion mode. The operating temperature was maintained at 275 °C, and the source and spray voltages were set at 35 V and 4.0 kV, respectively. Data analyses were performed with Xcalibur software (Thermo Fisher Scientific, USA). For full-scan MS analysis, the spectra were recorded in the range of m/z 80−2000. Carbohydrate Analysis. The concentrations of glucose, mannose, and xylose in the supernatants were determined using the Agilent 1260 infinity HPLC system (Agilent Technologies, USA). The system was equipped with a refractive index detector and a standard autosampler. Ion-moderated partition chromatography column (Aminex column HPX-87P) with cation-P microguard cartridge was used. The column was maintained at 85 °C with ultrapure grade water as the eluent at a flow rate of 0.6 mL/min. Before injection, 20 μL of each sample was

filtered through a 0.22 μm MicroPES filters. Peaks were detected by the refractive index and identified and quantified by comparison to the retention times of authentic standards (D-glucose, D-mannose, and Dxylose). The glucose yield in the hydrolysis of biomass materials was calculated according to NREL LAP-009.36 The degree of inhibition was evaluated with eq 1 below: Degree of inhibition (%) =

Y0 − Yi × 100 Y0

(1)

where Y0 is the glucose yield (%) without the addition of the inhibitors, and Yi is the glucose yield (%) with the addition of inhibitors.

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RESULTS Inhibition of Cellulases by Mannan. Mannan from different types of lignocellulosic materials has different structural properties. In order to fully understand the inhibitory effects of mannan on cellulases, mannans with different chain lengths and side groups were used in this work. The weightaverage molecular weights (Mw) of the GalM-L and GalM-H have been evaluated by size exclusion chromatography, as described by the supplier. The Mw molecular weights of GalMH and GalM-L were 556 and 107 kDa, respectively. However, Mw of L-Man is difficult to quantify because it contains lowmolecular-weight and high-molecular-weight fractions.37 The hydrolytic capacity of commercial cellulases decreased after supplementation with mannans (Figure 1A). This corroborated the previous results of Kumar and Wyman.12 Cellulases used in this work are mainly composed of cellobiohydrolase, endoglucanase, and β-glucosidase. Ta Cel5A, Ta Cel7A, and At Cel3A responses to mannan were further studied. Both Ta Cel5A and Ta Cel7A showed a negative response to mannan; however, the action of At Cel3A remained stable before and after mannan supplementation (Figure 1B−D). Mannan with high viscosity and Mw showed a 5267


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Figure 2. Inhibition of mannan on Ta Cel7A and Ta Cel5A activities. Hydrolysis of p-nitrophenol-D-cellobioside (5 mM) by Ta Cel7A (8 mg/g DM) (A) and HEC (10 mg/mL) by Ta Cel5A (2 mg/g DM) (B) with the addition of mannans (0.8, 1.6, and 3.2 mg/mL) at 50 °C for 2 h. The error bars represent the standard error of three independent experiments.

Figure 3. . Inhibition mechanism of mannans on Ta Cel7A and Ta Cel5A. Lineweaver−Burk plots of Ta Cel7A activities at different L-Man (A), GalM-L (B), and GalM-H (C) concentrations (0.1, 0.5, and 1 mg/mL). The measurements were performed at 50 °C and pH 5.0 using pnitrophenol-D-cellobioside as a substrate. Effects of L-Man, GalM-L, and GalM-H (0.2 mg/mL) on Ta Cel5A activity at different substrate concentrations (D). The measurements were performed in 50 mM sodium citrate buffer (pH 5.0) and 50 °C.

degree of inhibitory (Table S2). Higher Km values indicated a stronger competitive inhibition. Km values of 1 mg/mL for LMan, GalM-L, and GalM-H on Ta Cel7A were 0.63, 0.65, and 0.68, indicating a higher inhibitory capacity of GalM-H on Ta Cel7A activity, followed by GalM-L and L-Man. This phenomenon could explain the strongest inhibitory effect of GalM-H on Ta Cel7A cellulose hydrolysis (Figure 1B). Somewhat surprisingly, the negative effect of mannans on Ta Cel5A did not follow Michaelis−Menten kinetics. The corresponding results are shown as Ta Cel5A activity versus [S] (Figure 3D). These results indicated that the inhibition of mannan on Ta Cel5A was irreversible under our experimental conditions (pH 5.0, 50 °C, using HEC as substrate). Additionally, it was observed that the inhibitory capacities of

stronger inhibitory effect on Ta Cel5A and Ta Cel7A. Additionally, the inhibitory degree of GalM-H on Ta Cel7A was 89.2%, which was higher than inhibition on Ta Cel5A (54.8%) (Table S1), indicating a stronger inhibitory effect of mannan on Ta Cel7A. To further investigate the responses of Ta Cel7A and Ta Cel5A to mannans, experiments were performed using their substrates pNPC and HEC, respectively. By inhibiting cellulose hydrolysis, mannans reduced the amount of pNP and reducing sugars released (Figure 2). These results indicated a negative effect of mannan on Ta Cel7A and Ta Cel5A activities. Kinetic experiment on Ta Cel7A further proved that mannans competitively inhibited Ta Cel7A activity (Figure 3A−C). Michaelis constant (Km) values were calculated to quantify the 5268


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Figure 4. Role of MAN in the hydrolysis of mannan-containing materials. Hydrolysis of Avicel (10 mg/mL) by Ta Cel5A (2 mg/g DM), Ta Cel7A (8 mg/g DM), and At Cel3A (0.2 mg/g DM) with the addition of mannan (2.5 mg/mL), MOS (2.5 mg/mL), mannose (2.5 mg/mL), and MAN (500 nkat/g DM) at 50 °C for 48 h (A). Hydrolysis of spruce after pretreatment with dilute acid (DA-SP), Na2SO3 (NA-SP), and NA-SP after delignification (DL-NA-SP) by CEL (10 FPU/g DM Celluclast 1.5L and 500 nkat/g DM Novozyme 188) and MAN (500 nkat/g DM) at 50 °C and pH 5.0 for 48 h (B). The error bars represent the standard error of three independent experiments.

Table 1. Activity of Cellulases in Hydrolysatesa substrates none

DA-SP

NA-SP

DL-NA-SP

enzymes MAN CEL CEL+MAN MAN CEL CEL+MAN MAN CEL CEL+MAN MAN CEL CEL+MAN

Cel5A (nakt/mL) 15.2 327.45 361.31 1.02 45.47 49.22 1.87 281.36 307.92 2.07 257.56 294.06

± ± ± ± ± ± ± ± ± ± ± ±

Cel7A (nkat/mL)

0.4 18.1 14.3 0.9 5.1 7.3 0.7 2.1 2.5 0.8 10.4 7.1

0.13 5.42 5.71 0.07 1.28 1.54 0.07 4.77 5.22 0.03 4.01 4.45

± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.1 0.2 0.0 0.1 0.3 0.0 0.2 0.1 0.0 0.4 0.2

Cel3A (nkat/mL) 1.01 30.9 32.6 0.05 26.09 27.05 0.03 26.52 26.84 0.02 28.6 26.9

± ± ± ± ± ± ± ± ± ± ± ±

0.4 1.0 0.9 0.0 2.1 0.8 0.0 0.3 1.4 0.0 1.2 0.5

a

Cel5A, Cel7A, and Cel3A activity in the hydrolysates of the pretreated spruce materials after 48 h of hydrolysis by CEL and/or MAN. The error bars represent the standard error of three independent experiments.

GalM-H, GalM-L, and L-Man on Ta Cel5A were similar to those on Ta Cel7A. The results further revealed that the inhibitory action of mannan exhibited a close relationship with its viscosity and molecular weight. Therefore, mannans with a high viscosity and molecular weight should be hydrolyzed to low chain length to suppress this inhibition. Suppressing Mannan Inhibition by Mannanase (MAN). Since part of mannan in softwood could be hydrolyzed to MOS and mannose by corresponding hydrolytic enzymes in commercial cellulase preparations during the saccharification process, inhibitory effects of mannan, MOS, and mannose on cellulase hydrolysis were compared. MOS was prepared by hydrolyzing L-Man with endo-1,4-β-mannanase for 6 h. The composition of the MOS preparation was analyzed by ESI-MS (Figure S3B). As expected, the main compositions were mannobiose (M2) and mannotriose (M3). In Figure 4A, MAN showed an efficient role in suppressing L-Man inhibition on cellulose hydrolysis. After supplementation of MAN, L-Man was mainly hydrolyzed to mannan oligosaccharides, and the inhibitory degree of L-Man on cellulose hydrolysis clearly decreased from 39.9 to 11.1%, which was similar to the degree of inhibition caused by MOS (14.2%). After addition of an equal amount of mannose (2.5 mg/mL), the glucose yield was 40.2%, which was similar to the control (42.1%). These results showed that end-product mannose has negligible inhibition on cellulose hydrolysis, revealing a

potential method to suppress mannan inhibition by hydrolyzing mannan to a less inhibitory form of mannose. These results show the negative effect of mannan on cellulose hydrolysis and the positive effect of mannan hydrolytic enzymes in suppressing this inhibition. However, in real softwood biomasses, the location and structure of mannan could be different than added mannans. To study the effect of mannan on real softwood hydrolysis, hydrolysis by cellulases and MAN of DA-SP, NA-SP, and DL-NA-SP at solid loadings of 100 g/L was investigated. During spruce hydrolysis by cellulases, further addition of MAN resulted in an increase of glucose released from cellulose in NA-SP and DL-NA-SP (Figure 4B). It was worth noting that MAN did not promote the conversion of cellulose in DA-SP by CEL. Low contents of mannan and high contents of lignin in the DA-SP material (Figure S1) could provide an explanation for the phenomenon. In addition, considerable amounts of mannose were released from spruce materials by CEL (Table S3), indicating that the commercial cellulases used in this work contain some mannan hydrolytic enzymes. These mannan hydrolytic enzymes inevitably offset the role of added MAN. Furthermore, we used FTIR and ES-MS qualitatively to analyze free mannan and MOS in the hydrolysates derived from DL-NA-SP by cellulases (Figure S2). The weak bands at 810 and 874 cm−1 indicated the low contents of mannan in the hydrolysate.38,39 ESI-MS further confirmed the presence of MOS, especially M2, in the hydrolysate (Figure S3A). As 5269


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ACS Sustainable Chemistry & Engineering confirmed by the above results, further addition of MAN could hydrolyze the mannan and MOS to mannose, thus suppressing the inhibition of them on cellulases. To confirm this phenomenon in industrial real item softwood material, we further investigated the activity of cellulases (Cel5A, Cel7A, and Cel3A) in the hydrolysates of the spruce materials before and after supplementation of MAN. Incubation of CEL and/or MAN in the absence of solid material was performed as a control. After 48 h of hydrolysis by CEL, the cellulase activities, including those of Cel5A, Cel7A, and Cel3A, clearly decreased compared with the control. This phenomenon is probably caused by the nonspecific adsorption of cellulases onto lignin and the inhibition by the products in the hydrolysates. It is worth noting that the cellulase activity was positively correlated with the amount of glucose released, indicating that cellulase activity in the hydrolysates has a decisive effect on the conversion of cellulose to glucose. After supplementation with MAN, the cellulase activities, especially those of Cel5A and Cel7A, modestly increased by 14.3 and 10.9%, respectively (Table 1). At the same time, the amount of mannose and xylose increased, which could be due to the hydrolysis of mannan both in the original-bound form and free form. The degradation of mannan and MOS to end-product mannose thus suppressed their inhibition on cellulases, especially Cel5A and Cel7A.

Figure 5. Inhibition mechanism of mannan on cellulases. During the hydrolysis of mannan-containing biomass materials, mannan decreases cellulases efficiency by competitively inhibiting cellobiohydrolase (Cel7A) activity and irreversibly inhibiting endoglucanase (Cel5A) activity.

low concentration of mannan in the hydrolysates could not be ignored because even low amounts of added mannan (0.1 mg/ mL) could significantly decrease the hydrolysis yield of cellulose.12 In our previous results, MOS, especially M2, was found to be a strong inhibitors of Ta Cel7A activity.35 We thus believe that mannan and MOS potential caused the reduction of Cel5A and Cel7A activity during the hydrolysis process (Table 1). The results in Figure 4 and Table S3 indicated that mannan degradation by MAN to end-product mannose benefited the release of glucose from cellulose in spruce materials. This promoting effect could be explained by the following reasons: (1) MAN solubilized the original bound mannan to free-form mannan and soluble sugars, thus increasing the access of cellulose to cellulose. (2) MAN further hydrolyzed the free mannan to less inhibitory MOS and mannose, relieving the mannan inhibition on cellulases, especially Cel5A and Cel7A. Increasing the access of cellulases to cellulose by solubilizing the original hemicelluloses has been previously reported in several publications.46−48 However, suppressing the inhibition of cellulases by free mannan and MOS in the hydrolysates has not been studied. After supplementation with MAN, the free mannan and MOS could be partly converted into mannose, thereby reducing inhibition of Cel5A and Cel7A. The results in Table 1 support the proposed mechanism, based on the levels of Cel5A and Cel7A activity after supplementation with MAN. We believe that MAN promoted hydrolysis of mannancontaining biomass material by suppressing mannan’s ability to inhibit cellulase activities.

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DISCUSSION Cellulases binding to cellulose is the key step of enzymatic hydrolysis. In our previous results, we found that all of the tested mannans showed a strong tendency to precipitate on the surfaces of cellulose.40 The interaction of mannan with cellulose surfaces inevitably inhibited the access of cellulases to cellulose, leading to cellulases inhibition. However, aside from this possibility, in this work, we found that mannan could directly affect cellulase activity, especially Ta Cel5A and Ta Cel7A. It seems that cellobiohydrolase and endoglucanase were sensitive to their reaction surroundings. In previous results, xylan, xylo-oligosaccharides, cello-oligosaccharides, phenolic compounds, furan derivatives, and organic acids were found to be their inhibitors.13,20,41−44 Additionally, the positive response of surfactants to cellobiohydrolase has been reported.45 Cellobiohydrolase and endoglucanase play key roles in converting polysaccharide to soluble oligosaccharides, which could be further converted to fermentable monosaccharides by β-glucosidase. Therefore, it was necessary to investigate the mechanism of cellulase inhibition and to find an effective way to suppress it. On the basis of the kinetic experiments (Figure 3), we suggested a model in which mannan competitively inhibits Cel7A and irreversibly inhibits Cel5A as shown in Figure 5. Similar β-1,4-linked backbones of mannan and cellulose could cause the binding of mannan into the active site of Cel7A, forming a steric hindrance and preventing the cellulose chain to move into the tunnel of Cel7A. Unlike competitive inhibition of mannan on Cel7A, mannan may react with amino acid side chains of Cel5A to form covalent adducts and therefore inhibit the activity of the enzyme. The inhibition cannot be reversed by ultrafiltration, dialysis, and other physical methods but could be reversed by specific chemical reactions. However, the detailed mechanism describing the irreversible inhibition of Cel5A needs to be further investigated. In spruce hydrolysis by cellulases, low contents of free-form mannan and MOS were found in the hydrolysates. The relative

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CONCLUSIONS During softwood hydrolysis, it is known that the accumulation of products in hydrolysis supernatant may impede the conversion efficiency of softwood by limiting the access of cellulases to cellulose or decreasing the activity of cellulases. The results in this work confirmed that mannan weakens the hydrolytic action of cellulases by competitively inhibiting the Cel7A enzyme and irreversibly inhibiting Cel5A enzyme. The degradation of mannan to end-product mannose by mannanase can help to suppress the mannan-induced inhibition and 5270


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facilitate recovery the inactivated cellulases, especially Cel5A and Cel7A. Therefore, special enzyme cocktails consisting of powerful mannan hydrolytic enzymes should be utilized in softwood conversion.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00605. Degree of inhibition of mannan on Ta Cel5A and Ta Cel7A, Kapp m values of mannan on Ta Cel7A, formation of mannose and xylose in spruce hydrolysis, chemical compositions of spruce materials, FTIR spectra of the hydrolysates, ESI-MS analysis of the hydrolysate, and MOS preparation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Donglin Xin: 0000-0003-0983-6595 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (31670598 and 31270622) and the Science Foundation for Distinguished Young Scholars of Northwest A&F University (2452015098). We are grateful to Prof. Liisa Viikari (University of Helsinki, Finland) and Roal Oy (Rajamäki, Finland) for providing Ta Cel5A, Ta Cel7A, and At Cel3A.

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ABBREVIATIONS At Cel3A, Acremonium thermophilum β-glucosidase; DA-SP, spruce after pretreatment with dilute acid; DL-NA-SP, delignification of spruce after pretreatment with Na2SO3; DM, dry matter; ESI-MS, electrospray ionization-mass spectrometry; GalM-L, low viscosity carob galactomannan; GalM-H, high viscosity carob galactomannan; GH, glycosyl hydrolase; Km, Michaelis constant; L-Man, linear mannan; HEC, hydroxyethyl cellulose; MAN, mannanase; MOS, mannooligosaccharides; Mw, weight-average molecular weight; NA-SP, spruce after pretreatment with Na2SO3; PNP, p-nitrophenol; PNPG, p-nitrophenyl-β-D-glucopyranoside; SP, spruce; Ta Cel5A, Thermoascus aurantiacus endoglucanase; Ta Cel7A, Thermoascus aurantiacus cellobiohydrolase

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