Characterization of hemicellulase and cellulase from the ...

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Both of the extracellular (extra-) and intracellular (intra-) enzymes of C. owensensis cultivated on corncob xylan or xylose had cellulase (including endoglucanase, cellobiohydrolase and β-glucosidase) and hemicellulase (including xylanase, xylosidase, arabinofuranosidase and acetyl xylan esterase) activities. The enzymes of C. owensensis had high ability for degrading hemicellulose of native corn stover and corncob with the conversion rates of xylan 16.7 % and araban 60.0 %. Moreover, they had remarkable synergetic function with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). When the native corn stover and corncob were respectively, sequentially hydrolyzed by the extra-enzymes of C. owensensis and CTec2, the glucan conversion rates were 31.2 and 37.9 %,which were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 alone), whereas the glucan conversion rates of the steam-exploded corn stover and corncob hydrolyzed by CTec2 alone on the same loading rate were 38.2 and 39.6 %, respectively. These results show that hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment on degradation of native lignocellulosic biomass. A new process for saccharification of lignocellulosic biomass by sequential hydrolysis is demonstrated in the present research, namely hyperthermal enzymolysis (70&#;80 °C) by enzymes of C. owensensis followed with mesothermal enzymolysis (50&#;55 °C) by commercial cellulase. This process has the advantages of no sugar loss, few inhibitors generation and consolidated with sterilization.

Pretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars.

In this work, the characteristics of hemicellulase and cellulase of C. owensensis cultivated on different carbon sources were assayed. The extra-enzymes and intra-enzymes of C. owensensis were applied to deconstruct lignocellulosic biomasses by themselves or synergetic hydrolysis with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). The aims are to comprehensively understand the lignocellulolytic enzymes of C. owensensis and develop new enzyme cocktails and enzymatic hydrolysis processes for bioconversion of lignocellulosic biomass.

The hemicellulose is much easier to be enzymatically hydrolyzed than cellulose in the native lignocellulose because the hemicellulose is amorphous. Removing hemicellulose can increase the surface area and porosity of lignocellulose hence improving the access for cellulase to touch with cellulose. The two-step hydrolysis by first using lignocellulolytic enzymes focusing on hemicelluloses deconstruction then hydrolysis by cellulase may be an efficient strategy for avoiding the current pretreatment process for biofuels production. Here we hope that the enzymes of C. owensensis which has the high ability of deconstructing native hemicellulose would be competent to support the two-step hydrolysis strategy.

Many extreme thermophiles are able to utilize a variety of carbohydrates pertinent to the conversion of lignocellulosic biomass to biofuels. Characterization of the enzymes from these extremely thermophilic bacteria is likely to generate new opportunities for the use of renewable resources as biofuels [ 16 , 17 ]. Among them, the genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile has recently attracted high interest for it can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass [ 18 &#; 20 ]. It was reported [ 19 ] that the open Caldicellulosiruptor pangenome encoded 106 glycoside hydrolases (GHs) from 43 GH families. The gene clusters that encode multidomain cellulases or hemicellulases were found in the genome of Caldicellulosiruptor. Many novel heat-stable extracellular enzymes for biomass degradation had been heterogeneously expressed [ 21 &#; 26 ]. Especially, some enzymes are not only multimodular, but possess catalytic domains with different activities (multifunctional) [ 19 , 27 ]. They differ from the two general cellulolytic enzymes systems: one with free cellulases and hemicellulases produced by fungi and most bacteria [ 28 ], and the other in which glycosidases self-assemble onto a common protein scaffold to form large macromolecular assemblies called cellulosomes [ 29 , 30 ]. For example, the cellulase CelA produced from C. bescii, comprises a GH 9 and a GH 48 catalytic domain, could hydrolyze the microcrystalline cellulose not only from the surface as common cellulases done but also by excavating extensive cavities into the surface of the substrate [ 31 ]. The major commercial cellulolytic enzymes are currently produced by fungi with free noncomplexed cellulases and hemicellulases [ 32 ]. The cellulolytic enzymes from genus Caldicellulosiruptor with different characteristics may be complementary with fungal cellulolytic enzymes on hydrolysis of lignocellulosic biomass, therefore showing potential commercial application value. C. owensensis grows on a wide variety of carbon sources including pentose, hexose, oligosaccharide, and polysaccharide [ 33 ]. Comparing of the growth of the seven species of Caldicellulosiruptor (C. bescii, C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, C. saccharolyticus, C. owensensis) on xylose, the cell density of C. owensensis was only slightly lower than C. saccharolyticus and higher than the other five species [ 18 ]. Moreover, the results of analyzing of the diversity of biomass deconstruction-related glycoside hydrolases in Caldicellulosiruptor showed that C. owensensis owned abundant xylan deconstruction-related glycoside hydrolases including the 5, 10, 11, 39, 43, 51 and 67 GH families, which were the total xylan deconstruction-related glycoside hydrolase families in Caldicellulosiruptor [ 18 ]. The diversity of the xylan deconstruction-related glycoside hydrolases and the physiological characteristics of C. owensensis showed that it is a promising candidate for hemicelluloses deconstruction.

Therefore, after pretreatment the detoxification step is essential for improving the fermentation efficiency. The methods of detoxification can be divided into three main groups: biological, physical and chemical [ 8 ], such as using microorganisms or enzymes to change the inhibitors&#; chemical structures [ 9 , 10 ], adsorbing the inhibitors by using activated charcoal [ 11 ] and ion exchange resins [ 12 ], and adding reductive substances [ 13 ] or pH modification [ 14 , 15 ]. The detoxification process is also costly and many of the detoxification methods result in sugar losses [ 8 ]. Can the pretreatment and detoxification be removed from the bioconversion process of lignocellulosic biomass?

Currently, the high cost of enzymolysis is a major obstacle for production of biofuels at an industrial scale [ 3 ]. Exploring the highly efficient cellulase and hemicellulase is attached much attention for reducing the cost of biofuels production. Before enzymatic hydrolysis, pretreatment process is required to break down the rigid association of lignocelluloses, so that the enzymes can easily access the cellulose to hydrolyze into monomers [ 4 ]. Pretreatment, such as steam-explosion pretreatment, hydrothermal pretreatment, and acid or alkali pretreatment, allows to change the structure of the lignocellulose, such as increasing the surface area and porosity of biomass, partially removing the hemicelluloses and lignin, and reducing the crystallinity of cellulose [ 5 ]. Although pretreatment is efficient for improving the enzymatic hydrolysis of lignocelluloses, it has been viewed as one of the most expensive processing steps in biomass-to-biofuel conversion [ 6 ]. Moreover, during pretreatment process some sugars are damaged and converted into furan derivatives (furfural and HMF) and carboxylic acids, which, together with phenol derivatives (from lignin), will inhibit the fermentation process [ 7 ].

Production of biofuels from the renewable lignocellulosic biomass is gradually considered as a promising way to replacement of fossil fuels. However, its bioconversion has been limited by its hydrolysis because the main components of the lignocellulosic biomass (cellulose, hemicellulose and lignin) are tightly held together and form lignin-carbohydrate complexes (LCC). The lignin-carbohydrate complexes create a barrier for microbial conversion [ 1 ]. Conversion of lignocellulosic biomass to fermentable sugars represents a major challenge in global efforts to utilize renewable resources in place of fossil fuels to meet the rising energy demands [ 2 ]. Enzymatic hydrolysis is the most common process to degrade the cellulose and hemicellulose into fermentable sugars such as glucose and xylose.

Results and discussion

Growth and xylanase secretion lines

Selecting a suitable incubation time for C. owensensis is important for assaying its enzymes. The growth and xylanase secretion lines were therefore analyzed at the beginning of this work. Figure  shows that regardless of xylose or corncob xylan as carbon source the cell quantity reached the highest after 24 h cultivation. The xylanase activities in the culture supernatant also increased to the peak after 24 h cultivation. The cell quantity by xylose was higher than that by xylan while the xylanase activity was reverse. It seems that xylose is more benefit for biomass accumulation while xylan can induce a higher xylanase secretion. The enzymes produced after 24 h cultivation were used in the following experiments.

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Characteristic of cellulase and hemicellulase from C. owensensis

The culture supernatants and cells were separated by centrifugation after 24 h cultivation. The extra-enzyme and intra-enzyme C. owensensis were respectively obtained after precipitation of the protein in supernatant by ammonia sulfate and breaking the cell wall by sonication. The hemicellulase (xylanase/Xyan, beta-xylosidase/pNPX, arabinofuranosidase/pNPAF, xylan esterase/pNPAC) and cellulase (endoglucanase/CMC, cellobiohydrolase/pNPC, beta-glucosidase/pNPG, filter paper activity/FPA) activities were measured.

Table  shows that no matter cultivated on xylose or corncob xylan both the extra-enzyme and intra-enzyme of C. owensensis had hemicellulase and cellulase activities. Hemicellulase and cellulase are not essential for cell growth and usually considered as the induced enzymes. However, the result of this experiment indicates that substrate inducing is not necessary for C. owensensis on hemicellulase and cellulase secretion. Maybe some genes for such enzymes share the promoters of the constitutive enzyme and are expressed during cell growth. On the whole, the hemicellulase activities cultivated on corncob xylan were higher than those on xylose. For example, the xylanase activities of extra-enzyme and intra-enzyme on corncob xylan were, respectively, 4.72 and 1.57 U/mg, while those on xylose were, respectively, 1.93 and 0.19 U/mg. However, the cellulase activities of the enzymes on corncob xylan and on xylose were varied slightly. This is possibly because both xylose and xylan are not the inducing substrates for cellulase secretion.

Table 1

Microorganism and substrateHemicellulase activity (U/mg)Cellulase activity (mU/mg)ReferencesHydrolysis substrateXyanpNPXpNPAFpNPACCMCpNPCpNPGFP C. owensensis on corncob xylanThis work Extra-enzyme4.72 ± 0.650.77 ± 0.010.12 ± 0.010.27 ± 0..9 ± 7.225.1 ± 1.94.2 ± 0.310.7 ± 1.3 Intra-enzyme1.57 ± 0..14 ± 0.231.54 ± 0.070.73 ± 0..1 ± 0.813.0 ± 1..4 ± 37.35.8 ± 0.5 C. owensensis on xylose Extra-enzyme1.93 ± 0.150.04 ± 0..10 ± 0..29 ± 0..5 ± 5.87.8 ± 0.537.8 ± 0.46.4 ± 1.3 Intra-enzyme0.19 ± 0..22 ± 0..15 ± 0..24 ± 0..6 ± 1.644.6 ± 5..4 ± 10.65.3 ± 0.5 Thermoascus aurantiacus on switchgrass or microcrystalline cellulose (extracellular proteins)0.1&#;1.90.007&#;0.012&#;&#;20&#;.8&#;34.445.6&#;177.3&#;[34] Thielavia terrestris on switchgrass or microcrystalline cellulose (extracellular proteins)0.66&#;3.270.001&#;0.006&#;&#;230&#;.7&#;16.25.7&#;26.1&#;[34]Open in a separate window

Table  also shows that the activities of extra-enzyme and intra-enzyme were different. The extra-enzyme had higher xylanase and endoglucanase activities, while the intra-enzyme had higher β-d-xylosidase, β-d-glucosidase and arabinofuranosidase activities. Especially, the β-d-glucosidase activity of intra-enzyme on corncob xylan was about 125-fold higher than that of the extra-enzyme (532.4 mU/mg VS 4.2 mU/mg). This indicates that for degrading the lignocellulosic biomass by C. owensensis the main function of the extra-enzyme is to cleave the polysaccharides to oligosaccharides, while further hydrolysis of xylobiose and cellobiose takes place mainly in cell by intra-enzyme.

Comparing with the thermophilic fungi Thermoascus aurantiacus and Thielavia terrestris which were recently reported to be high cellulase producers [34], the hemicellulase activities of C. owensensis were higher than those of fungi (Table  , the highest activities of each enzyme were used as the results for discussing). With regard to the cellulase, it showed that although the endoglucanase activity of C. owensensis was much lower than those of fungi, the cellobiohydrolase activity was almost the same as those of the fungi and the β-d-glucosidase activity was much higher than those of fungi. Three enzymes, endoglucanases, exoglucanases and β-d-glucosidases, compose the cellulase system functions in a coordinated manner for degradation of cellulose into glucose units [35]. Since most glucanases are inhibited by cellobiose and short cellooligosaccharides, β-d-glucosidases catalyze the rate limiting step of the cellulose hydrolysis process as a whole [36]. Filamentous fungi are the major source of commercial cellulases. Commercial cellulase preparations are mainly based on mutant strains of T. reesei which have usually been characterized by a low secretion of β-glucosidase [37]. Thus, T. reesei cellulase preparations had to be supplemented with added β-glucosidase to provide the more efficient saccharification of cellulosic substrates [32, 37]. The enzyme system of C. owensensis with high hemicellulases and β-d-glucosidase activities may complement with the fungi cellulase for deconstruction of native lignocellulose.

To identify the protein components in extra- and intra-enzymes of C. owensensis cultivated on corncob xylan, the proteins were analyzed with HPLC/MS. More than 100 and 150 kinds of proteins were identified in the extra- and intra-enzymes respectively. Enzymes related to polysaccharide degradation were shown in Table  . The extra-enzymes include β-xylanase (Calow_, Calow_), β-galactosidase (Calow_), α-N-arabinofuranosidase (Calow_), polysaccharide deacetylase (Calow_), pectate disaccharide-lyase (Calow_), esterase (Calow_), α-l-fucosidase (Calow_), α-amylase (Calow_ and Calow_), pullulanase, type I (Calow_) and a glycoside hydrolase belong to family 28 (Calow_). The intra-enzymes include the glycoside hydrolases belong to family 18 (Calow _), family 31 (Calow_), family 43 (Calow_, Calow_), family 4 (Calow_) and family 20 (Calow_), pullulanase, type I (Calow_, Calow_), arabinogalactan endo-β-1,4-galactanase (Calow_) and α-l-fucosidase (Calow_). Although the cellulase were not identified, the enzymes belong to different GH families might have multi-activity, including cellulolytic activity. Besides these enzymes related to polysaccharide degradation, the extra-enzymes related to carbohydrate metabolism were also identified and shown in Additional file 1, including glycosyltransferase, extracellular solute-binding protein, ATP-binding cassette (ABC) transporter-related protein, and S-layer domain-containing protein. They gave useful information for further research on carbohydrate hydrolysis and metabolism of C. owensensis.

Table 2

Entry nameDetected sequenceGene nameEnzymeMW kDaSignal peptide (aa)a Transmembrane domain (aa)b Extra-enzymes cultivated on corncob xylan E4Q6K1_CALOWE.PVVIEF.LCalow_β-galactosidase118.37NoNo E4Q2A1_CALOWG.VGGNNHHQ.LCalow_β-xylanase152.48No13&#;35 E4Q5G9_CALOWQ.AYEGSY.SCalow_β-xylanase187.021&#;335&#;27 E4Q4M1_CALOWE.DAILVGCM.LCalow_α-N-arabinofuranosidase57.89NoNo E4Q281_CALOWP.EIAKLY.VCalow_α-amylase catalytic region66.17NoNo E4Q359_CALOWG.YDPHDYYDLGQ.YCalow_α-amylase catalytic region53.671&#;&#;35 E4Q4A4_CALOWL.VAPISMFVAYKSD.ECalow_Pullulanase, type I127.94No13&#;32 E4Q6L6_CALOWG.VRISNC.YCalow_Glycoside hydrolase family .05NoNo E4Q2L6_CALOWT.IVGGY.KCalow_Glycoside hydrolase 15-related protein70.76NoNo E4Q1R5_CALOWT.KFALPIIL.SCalow_Polysaccharide deacetylase29.98NoNo E4Q4M2_CALOWI.QVISALF.ECalow_α-

l

-fucosidase86.88NoNo E4Q6L2_CALOWT.PGDSSV.FCalow_Pectate disaccharide-lyase188.76No28&#;50 E4Q5E4_CALOWE.NPDPVL.VCalow_Esterase/lipase-like protein30.17NoNoIntra-enzymes cultivated on corncob xylan E4Q6Z2_CALOWT.YEEVMALVGHHLSLN.ICalow_Glycoside hydrolase family .441&#;245&#;27 E4Q4N4_CALOWV.VLVEK.GCalow_Glycosidase-related protein35.84NoNo E4Q2L6_CALOWT.IVGGY.KCalow_Glycoside hydrolase 15-related protein70.76NoNo E4Q4J8_CALOWW.SHDIAGFE.SCalow_Glycoside hydrolase family .91NoNo E4Q6K9_CALOWI.WAPAIRYHNGRFYIY.FCalow_Glycoside hydrolase family .87NoNo E4Q4G6_CALOWG.TVRLYDIDFEAAKTNEV
IGNKLSS.KCalow_Glycoside hydrolase family 452.72NoNo E4Q6J2_CALOWP.VVSPER.YCalow_Glycoside hydrolase family .051&#;267&#;29 E4Q1W6_CALOWK.NWIF.ECalow_Glycoside hydrolase, family .68NoNo E4Q347_CALOWN.YDEDEGF.ICalow_Pullulanase, type I96.51NoNo E4Q4A4_CALOWY.VSGTMN.DCalow_Pullulanase, type I127.94No13&#;32 E4Q281_CALOWI.MYKWYLALKDKGWN.SCalow_α-amylase catalytic region66.17NoNo E4Q4A2_CALOWV.AKVKVANLIQNSGF.ECalow_Arabinogalactan endo-β-1,4-galactanase90.88NoNo E4Q4L3_CALOWP.QWHMK.WCalow_α-

l

-fucosidase56.75NoNoOpen in a separate window

Hydrolysis of lignocellulosic biomass by enzymes of C. owensensis

The enzymes of C. owensensis were used to hydrolyze native corn stover, native corncob and steam-exploded corn stover with the loading rate of 15 mg enzyme per gram dry substrate at 70 °C for 48 h. The experiment was performed in three groups respectively using extra-enzyme, intra-enzyme and extra-enzyme mixed with intra-enzyme at the ratio of 1:1. The data in Table  show that the enzyme of C. owensensis had high ability of degrading hemicellulose. The highest conversion rates of xylan on native corn stover, native corncob and steam-exploded corn stover were respectively 14.7, 16.8 and 59.1 %. Moreover, the conversion rates of araban on native corn stover and native corncob reached 53.5 and 60.0 %, respectively. However, the enzyme of C. owensensis was not such perfect at degrading cellulose. As Table  shows the glucose can not be detected after hydrolysis of both native corncob and steam-exploded corn stover for 48 h. This may because the endoglucanase in the enzyme of C. owensensis was weak (shown in Table  ). Blumer&#;Schuette et al. [19] analyzed the core genomes, pangenomes, and individual genomes and predicted that the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicelluloses. The results in this experiment were in accord with the prediction.

Table 3

SubstrateEnzymes (15 mg/g ds)Sugar releasedXylose (% of xylan)Arabinose (% of araban)Glucose (% of glucan)Reducing sugar (% of carbohydrate)a Native corn strawExtra-enzyme9.6 ± 0..9 ± 0.783.9 ± 0..4 ± 0.56Extra-/Intra- = 1:114.7 ± 0..5 ± 1.233.7 ± 0..3 ± 0.67Intra-enzyme7.9 ± 0..5 ± 2.813.1 ± 0..5 ± 0.43Native corncobExtra-enzyme11.7 ± 0..9 ± 1.12ND14.5 ± 0.61Extra-/Intra- = 1:116.8 ± 0..7 ± 1.73ND13.7 ± 0.57Intra-enzyme10.4 ± 0..0 ± 2.85ND10.9 ± 0.49SE corn stoverExtra-enzyme50.0 ± 2.23NDND4.6 ± 0.17Extra-/Intra- = 1:159.1 ± 2.54NDND3.8 ± 0.16Intra-enzyme36.4 ± 1.67NDND2.5 ± 0.12Open in a separate window

As described in the section of characteristic of cellulase and hemicellulase, the xylanase was mainly existed in the extra-enzyme of C. owensensis while the β-d-xylosidase and arabinofuranosidase were mainly existed in the intra-enzyme of C. owensensis. The extra-enzyme and the intra-enzyme may have synergetic function for hemicellulose hydrolysis. For each substrate, extra-enzyme mixed with intra-enzyme contributed higher levels of xylose releasing than those by extra-enzyme and intra-enzyme respective hydrolysis. However, the extra-enzyme led to the highest reducing sugar releasing, indicating that the xylanase, with the function of cleaving the xylan to xylo-oligosaccharides and xylose, is the most important enzyme for xylan degradation.

The morphology changes induced by hydrolysis with the extra-enzyme of C. owensensis were examined by SEM to provide direct insight into the structure modification in the native corn stover. Before hydrolysis, the vascular bundle and the epidermis (with stoma and epidermal hair, Fig.  a, b) of the samples were intact. After hydrolysis, the residual corn stover was changed dramatically (Fig.  c, d); the initial structure was destroyed and replaced by a collapsed and distorted cell wall structure. The cuticle waxy layer appeared to be almost desquamated, and the microfibrils were exposed to the surface. Clearly, the structure of the native corn stover was greatly changed in appearance after hydrolysis. Figure  e, f shows the images of the corn stover after incubation in the acetate buffer at 70 °C for 48 h as control. When comparing with the initial corn stover (Fig.  a, b), the acetate-buffer-incubated corn stover was slightly changed with some fissures in the sample. The acetate-buffer incubation cannot make much change for the biomass structure was proved by the hydrolysis experiment: The acetate-buffer-incubated corn stover and corncob and the samples without incubation were hydrolyzed by CTec2 (Novoyzmes) at 50 °C for 72 h. As a result, the sugar yields of the buffer-incubated samples and the corresponding samples without incubation were almost the same. The glucan conversion rates (%) were as follows: incubated corn stover 18.1 ± 1.7, corn stover without incubation 17.9 ± 1.5, incubated corncob 20.1 ± 1.6, corncob without incubation 20.4 ± 1.9.

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Synergetic hydrolysis by the enzymes of C. owensensis and CTec2

Two trials were performed for synergetic hydrolysis by the enzymes of C. owensensis cultivated on corncob xylan and the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). One was that the native corn stover and corncob were sequentially hydrolyzed (SH) by the enzymes of C. owensensis at 70 °C for 48 h then added CTec2 and incubated at 50 °C for 72 h. The other was that these lignocellulosic biomasses were co-hydrolyzed (CH) by the enzyme of C. owensensis and CTec2 at 50 °C for 72 h. The loading rates of CTec2 (http://www.bioenergy.novozymes.com/) for synergetic hydrolysis were 30 mg/g glucan (High loading). These lignocellulosic biomasses were hydrolyzed by CTec2 only at 50 °C for 72 h as controls.

Figure  shows that after sequential hydrolysis (SH) on native corn stover and native corncob by extra-enzyme and CTec2, the conversion rates of glucan were 31.2 and 37.9 %, which respectively were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 only). Using the same loading (high loading, 30 mg enzyme/g glucan) of CTec2 for hydrolysis of the steam-exploded (SE) corn stover and SE corncob the glucan conversion rates were 38.2 and 39.6 %, respectively (Fig.  a), which were not much higher than glucan conversion rates of the native corn stover and corncob sequentially hydrolyzed (SH) by the enzyme of C. owensensis and CTec2. The glucan conversion rates of native corn stover and corncob by SH were respectively 81.7 % and 95.7 % of those of the SE corn stover and SE corncob hydrolyzed by CTec2 (Fig.  b). It seems that in this experiment, the hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment for glucan degradation from native lignocellulosic biomass. Sequential hydrolysis by the extra-enzyme of C. owensensis and CTec2 could greatly increase the hydrolysis rate for native lignocellulosic biomass possibly due to the hemicelluloses degraded by the hemicellulase in extra-enzyme of C. owensensis hence increasing the accessibility of cellulose to CTec2. The cellulases, especially the endoglucanase in the extra-enzyme (shown in Tables  , ) would also contribute to improve the cellulose hydrolysis. This is made sure by what Brunecky et al. [38] have recently reported that pre-digestion of biomass with the cellulases (CelA and endocellulase E1) from extremely thermophilic bacterium C. becsii, and Acidothermus cellulolyticus at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone.

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Co-hydrolyzed (CH) by the extra-enzyme of C. owensensis and CTec2 the conversion rates of glucan from native corn stover and corncob were 21.4 and 23.1 % which were respectively 1.19 and 1.13 times of each control (Fig.  ). The increased extents of glucan conversion were not high as those of SH. This is because the optimum temperature for the enzymes of C. owensensis was 70&#;80 °C [33]. When co-hydrolyzed with CTec2 at 50 °C the enzyme activity was decreased.

Figure  also shows the xylan conversion rates from native corn stover and corncob synergetically hydrolyzed by the extra-enzyme of C. owensensis and CTec2. Totally, the xylan conversion rates of native corncob were higher than those of native corn stover. Especially, the xylan conversion rate of native corncob by sequential hydrolysis (SH) reached 34.8 %. The possible reason is that the enzyme of C. owensensis used in this experiment was produced using corncob as inducing substrate; hence, the ratio of the compositions in this enzyme was more fitted for corncob hydrolysis. It is believed that deconstructing of hemicellulose in lignocellulose will benefit cellulose degradation. This was proved by the results in Fig.  . Namely, the higher xylose releasing (34.8 % from native corncob vs 11.8 % from native corn stover) led to a higher glucose releasing (37.9 % from native corncob vs 31.2 % from native corn stover).

Figure  shows the sugar conversion rates from native corn stover and corncob by synergetic hydrolysis using intra-enzyme of C. owensensis and CTec2. It can be see that the conversion rates of glucan and xylan were lower than those counterparts of synergetic hydrolysis with extra-enzyme of C. owensensis and CTec2 (Fig.  ). This is possibly due to the higher xylanase and endoglucanase activities in the extra-enzyme.

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Inhibitors in the hydrolysate

The furfural and 5-hydroxymethyl furfural (HMF) were not detected in the hydrolysates from the native corn stover and corncob by sequential hydrolysis (SH). It is not surprising since at the temperature of 70 and 50 °C in the hydrolysis buffer the sugars are stable.

Kataeva et al. [39] found that C. bescii could solubilize all components of switchgrass, including lignin. Therefore, the phenolics may be released from lignin during hydrolysis by the extra-enzyme of C. owensensis. Really, the phenolics concentrations of the hydrolysates form native corn stover and corncob by SH were respectively 35.8 ± 3.2 and 34.3 ± 2.7 mg/l, which were slightly higher than these of the hydrolysates form native corn stover and corncob by CTec2 with 24.4 ± 1.8 and 25.1 ± 2.3 mg/l respectively. The phenolics concentrations of the hydrolysis buffer soaked (at 70 °C for 48 h) with native corn stover and corncob were, respectively, 4.2 ± 0.3 and 3.7 ± 0.3 mg/l. While the phenolics concentrations of the hydrolysates form the stream exploded corn stover and corncob by CTec2 were much high as 232 ± 17.6 and 219 ± 20.5 mg/l, respectively. These results show that only few phenolics from lignin can be released by the extra-enzyme of C. owensensis and CTec2. Among the biofuel-production microorganisms, Clostridium is very sensitive to phenolics which are lethal to Clostridium even at low concentrations [40, 41]. Even so, the research by Lee et al. [42] showed that the cell growth and metabolite production of Clostridium tyrobutyricum and Clostridium beijerinckii were not or slight inhibited when the phenolics concentrations were less than 100 mg/l. Wang and Chen [43] used the detoxified hydrolysate from steam-exploded rice straw to produce butanol by Clostridium acetobutylicum ATCC 824, and found that fermentation was improved when the phenolics concentration of the hydrolysate was less than 890 mg/l. The phenolics concentration of the hydrolysate by SH in this work was below 40 mg/l. This suggests that the hydrolysate by SH may be used for biofuels production without detoxification.

5.3c Hemicellulases and Lignin-degrading Enzymes

Hemicellulases work on the hemicellulose polymer backbone and are similar to endoglucanases. Because of the side chain, &#;accessory enzymes&#; are included for side-chain activities. An example of hemicellulase activity on arabinoxylan and the places where bonds are broken by enzymes are shown (blue) in the first figure below. The second figure shows another example of how hemicellulose breaks down hemicellulose, a complex mixture of enzymes in order to degrade hemicellulose. The example depicted is cross-linked glucurono arabinoxylan.

The complex composition and structure of hemicellulose require multiple enzymes to break down the polymer into sugar monomers&#;primarily xylose, but other pentose and hexose sugars also are present in hemicelluloses. A variety of debranching enzymes (red) act on diverse side chains hanging off the xylan backbone (blue). These debranching enzymes include arabinofuranosidase, feruloyl esterase, acetylxylan esterase, and alpha-glucuronidase [Table 6.4 shows enzyme families for degrading the hemicellulose]...As the side chains are released, the xylan backbone is exposed and made more accessible to cleavage by xylanase. Beta-xylosidase cleaves xylobiose into two xylose monomers; this enzyme also can release xylose from the end of the xylan backbone or a xylo-oligosaccharide. (U.S. DOE, )

Example of hemicellulase activity on arabinoxylan, showing bonds that are broken. The hemicellulases are shown in blue.

Credit: U.S. DOE. . Breaking the Biologic Barriers to Cellulosic Ethanol

Complex mixture of enzymes for degrading hemicelluloses. Instead of using chemical structures, this example uses abbreviations for different parts of the glucurono arabinoxylan so the connections can be observed more easily. The backbone is shown in blue, while the hemicellulases are shown in red.

Credit: U.S. DOE. . Breaking the Biologic Barriers to Cellulosic Ethanol

Enzyme families for degraded hemicelluloses, i.e., glycoside hydrolase (GH) and carbohydrate esterase (CE). Enzyme Enzyme Families Endoxylanase GH5, 8, 10, 11, 43 Beta-xylosidase GH3, 39, 43, 52, 54 Alpha-L-arabinofuranosidase GH3, 43, 51, 54, 62 Alpha-glucurondiase GH4, 67 Alpha-galatosidase GH4, 36 Acetylxylan esterase CE1, 2, 3, 4, 5, 6, 7 Feruloyl esterase CE1

Lignin-degrading enzymes are different from hemicellulases and cellulases. They are known, as a group, as oxidoreductases. Lignin degradation is an enzyme-mediated oxidation, involving the initial transfer of single electrons to the intact lignin (this would be a type of redox reaction or reduction-oxidation reaction). Electrons are transferred to other parts of the molecule in uncontrolled chain reactions, leading to the breakdown of the polymer. It is different from carbohydrate hydrolysis because it is an oxidation reaction, and it requires oxidizing power (e.g., hydrogen peroxide, H2O2) to break the lignin down. In general, it is a significantly slower reaction than the hydrolysis of carbohydrates.

Examples of lignin-degrading enzymes include lignin peroxidase (aka ligninase), manganese peroxidase, and laccase, which contain metal ions involved in electron transfer. Lignin peroxidase (previously known as ligninase) is an iron-containing enzyme, that accepts two electrons from hydrogen peroxide (H2O2), and then passes them as single electrons to the lignin molecule. Manganese peroxidase acts in a similar way to lignin peroxidase but oxidizes manganese (from H2O2) as an intermediate in the transfer of electrons to lignin. Laccase is a phenol oxidase, which directly oxidizes the lignin molecule (contains copper). There are also several hydrogen-peroxide generating enzymes (e.g., glucose oxidase), which generate H2O2 from glucose. (The Microbial World website)

If you are interested in learning about the mechanisms of these enzymes, then visit this website from the Department of Chemistry, University of Maine. There are several pages that discuss how each of the different types of enzymes works mechanistically.

Lesson 6 will discuss the process of ethanol production after the use of cellulases on cellulose.

If you want to learn more, please visit our website Hemicellulase Enzymes.