Low-Cost Cellulase-Hemicellulase Mixture Secreted by ...

Fermentable sugars are important intermediate products in the conversion of lignocellulosic biomass to biofuels and other value-added bio-products. The main bottlenecks limiting the production of fermentable sugars from lignocellulosic biomass are the high cost and the low saccharification efficiency of degradation enzymes. Herein, we report the secretome of Trichoderma harzianum EM under induction of lignocellulose. Numerously and quantitatively balanced cellulases and hemicellulases, especially high levels of glycosidases, could be secreted by T. harzianum EM. Compared with the commercial enzyme preparations, the T. harzianum EM enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomass. Moreover, 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding and alkali pretreated corn stover. These findings demonstrate a natural cellulases and hemicellulases mixture for complete conversion of biomass polysaccharide, suggesting T. harzianum EM enzymes have great potential for industrial applications.

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Production of extracellular enzymes with high cellulase activity has been proved in members of Trichoderma [ 17 ]. T. reesei Rut-C30 mainly expresses cellulases including cellobiohydrolase and endoglucanase with a total abundance of 90&#;95% of the extracellular proteins [ 18 ]. Compared with the industrial cellulase producer T. reesei, Trichoderma harzianum harbors more comprehensive lignocellulosic degrading enzyme encoding genes in its genome [ 19 ]. To be specific, a total of 42 cellulase genes and 24 hemicellulase genes were annotated in genome of T. harzianum T, which were 1.5 and 1.7-fold of those of T. reesei Rut-C30 [ 20 ]. Meanwhile, the expression levels of xylanase, mannase, and various glycosidases of T. harzianum was significantly higher than that in T. reesei [ 19 , 21 ]. Compared with T. reesei, some T. harzianum strains produced a cellulolytic complex with higher β-glucosidase and endoglucanases activities, and the xylanase activity of some T. harzianum strains was higher than that of T. reesei [ 22 , 23 , 24 ]. However, efficiency of simultaneous cellulose and hemicellulose hydrolysis by the secretome of T. harzianum was still low [ 23 , 25 ]. Lignocellulose degrading enzymes in microbes are induced by substrates, and the low-cost carbon source and culture conditions had a great influence on the enzyme secretion and composition [ 26 ]. Nevertheless, the mechanism of lignocellulolytic enzyme inducing was still unclear [ 27 ]. So, searching and exploring highly effective enzyme systems for efficiently converting the whole component of plant cell wall polysaccharides is expected, and the comprehensive understanding of secretomes in the related microbes could help the development of efficiently tailor-made lignocellulolytic enzyme cocktails in vitro [ 28 ]. Isolated in our laboratory, T. harzianum EM could secret high levels of cellulase and hemicellulase simultaneously, and its extracellular enzyme cocktail showed strong ability of lignocellulose degradation. The enzyme cocktail of T. harzianum EM contained a great amount of complete lignocellulolytic enzymes as revealed by proteome analysis under the optimal inducing condition. Aiming at analyzing the degradation mechanism of T. harzianum EM, we performed the present study. The results provide a basis for tailor-made preparation of low-cost enzymes to effectively degrade the whole component of plant cell wall polysaccharides.

Currently, Trichoderma reesei has been commonly used as a cellulase producer. Although two exoglucanases (CBH I and CBH II), eight endoglucanases (EGI-EGVIII), and seven β-glucosidase (BG I-BG VII) are produced by this fungus, the fairly low protein abundance (<1%) of β-glucosidase in the extracellular proteome limited the effective conversion of cellubiose to glucose [ 5 ]. The total cellulase activities of commercial preparations Viscozyme L and Celluclast 1.5L were as high as 33 and 95.2 FPU/mL, but the β-glucosidase activity was only 0.2 and 0.3 U/mL, respectively [ 6 ]. When β-glucosidase preparation Novezyme 188 was supplied to Celluclast 1.5L, glucose yield from sugarcane bagasse increased 1.5-fold than that of Celluclast 1.5L alone [ 7 ]. In addition, higher content of cellobiohydrolase in Trichoderma reesei extracellular proteome was detected, which caused the accumulation of cellobiose on account of the deficient amount of β-glucosidase, and then gave rise to severe product feedback inhibition of endoglucanase and cellobiohydrolase [ 8 ]. Some researchers found that hemicellulases, oxidoreductases, non-hydrolytic proteins and other auxiliary enzymes synergize to achieve an efficient enzymatic hydrolysis of cellulose [ 2 ]. Hemicellulase and pectinase could degrade the non-cellulose polysaccharide that covered cellulose and in turn increase the cellulose hydrolysis efficiency, and this synergistic effect could give rise to the cost reduction in deconstruction of lignocellulose [ 9 ]. Goldbeck et al. [ 10 ] found that when xylanase was added to the commercial cellulase preparation Accellerase , the glucose yield of dilute acid pretreated sugarcane bagasse presented a 1.4-fold increase than that of Accellerase alone. Furthermore, hemicellulose hydrolysis products (xylose, arabinose, mannose, and galactose etc.) also have great potential in food and feed industry applications. Therefore, in terms of the whole component utilization of plant cell wall polysaccharides, not only lack of β-glucosidase restricted the efficient conversion of cellulose, the lack of hemicellulase in Trichoderma reesei extracellular proteome also made it difficult to utilize hemicellulose [ 11 ]. Hemicellulase activity of commercial cellulase production model strains T. reesei QM6a and T. reesei QM has been determined by Li et al. [ 12 ], in which the xylanase activity was 5.42 and 1.27 U/mL, while xylosidase activity was only 0.4 and 0.005 U/mL, respectively. Recent studies showed that extracellular enzymes from Penicillium species perform better than cellulases from Trichoderma sp. On account of the higher hemicellulases activities of enzyme mixture secreted by Penicillium sp., greater fermentable sugar yield was obtained [ 13 , 14 ]. Yang et al. [ 15 ] found that when 50% of the commercial cellulase was replaced by Penicillium chrysogenum enzyme cocktail, release of reducing sugars was 78.6% higher than that with the cellulase alone, while the glucan and xylan conversion was increased by 37% and 106%, respectively. Additionally, the hydrolysis efficiency of cellulase increased more with the addition of multi-component hemicellulases cocktail than with xylanase alone. The artificial enzyme cocktail of pectinase, xylanase, arabinofuranosidase, acetyl xylan esterase, and ferulic acid esterase presented higher efficiency of bagasse degradation than the single component [ 16 ]. The appropriate dosage of auxiliary enzymes for efficient conversion of different lignocellulosic biomass is still uncertain. Therefore, exploring novel lignocellulosic degrading strains with complete and balanced enzyme cocktail for efficiently saccharification of lignocellulose is necessary for industrial enzyme preparation production.

Recently, bio-based industries have developed rapidly, and potential bio-based products including ethanol, aldehydes, organic acids, polyhydric alcohols, and other bio-chemicals and biomaterials have attracted a lot of attention. As the most important intermediate compounds of biological and chemical transformation from biomass, fermentable sugars (mainly glucose and xylose) are crucial for the production of downstream products [ 1 , 2 ]. Due to the structural complexity of lignocellulose, complete enzymatic deconstruction requires the synergistic action of cellulases, hemicellulases, and ligninases [ 3 ]. In order to obtain high yield of glucose from cellulose, endoglucanase, cellobiohydrolase, and β-glucosidase are required to work together. Endoglucanases randomly cleave the internal O-glycosidic bonds and produce glucan chains with different lengths, cellobiohydrolases attack the reducing and non-reducing ends of cellulose to release β-cellobiose, and β-glucosidases hydrolyze the terminal non-reducing β-d-glucosyl residues into glucose. These enzymes can be produced by many fungi (like Aspergillus and Trichoderma) and bacteria (like Cellulomonas and Clostridium) [ 2 , 3 ]. In any case, the rapid and complete conversion of cellulose is the limiting step during the process of bio-refinery [ 4 ].

To compare the saccharification efficiency of EM with those of two commercial enzyme preparations, an enzyme loading of 5, 10, and 30 FPU/g substrate was selected as the low, moderate and high enzyme dosage, respectively. Glucose and xylose yields from UGCS and ALKCS were measured after 72 h of hydrolysis reaction. Cellulose of UGCS was completely converted to glucose by EM at low enzyme dosage, and a xylan conversion of 72.35% was obtained simultaneously. Meanwhile, only 45.68% of the cellulose and 57.15% of the xylan were converted by Celluclast 1.5L. The xylan conversion gradually increased with increasing enzyme dosages, xylose yield was 100% when 30 FPU EM enzymes/g substrate was used. Meanwhile, only 85.59% of the cellulose and 66.18% of the xylan were converted by Celluclast 1.5L even at the highest enzyme dosage ( a,b). When ALKCS was used as substrate, 63.08% cellulose conversion and 70.47% xylan conversion were obtained at low dosage of EM, which were 1.55 and 1.40-fold of the same conversions by Celluclast 1.5L. The yields of glucose and xylose were all gradually improved with increasing enzyme dosages of the two preparations. 100% glucose and xylose were released when 30 FPU/g EM was used, and only 73.78% of the cellulose and 81.28% of the xylan were converted by Celluclast 1.5L at the highest enzyme dosage ( c,d).

The structural carbohydrate and lignin contents of differently pre-treated fractions of corn stover were determined ( a). As expected, chlorite/acetic acid and NaOH pretreatment resulted a significant decrease in lignin content: only 2% of the initial lignin content remained in ALKCS, whereas the relative cellulose content was highly increased up to 60.7%, which was 1.4-fold of that in NTCS. Dilute acid and steam explosion treatment significantly decreased the content of hemicellulose, which decreased from 26.1% to 5% in DACS, and the highest cellulose content (62.5%) was observed at the same time ( a). At the enzyme dosage of 5 mg proteins/g substrate, hydrolysis with EM generated 14.62, 19.00, 13.22, 12.43, and 8.83 mg reducing sugars per 20 mg of ultrafine grinding corn stover (UGCS), sodium hydroxide treated corn stover (ALKCS), sodium chlorite treated corn stover (DLCS), dilute acid treated corn stover (DACS), and stream explosion treated corn stover (SECS) after 72 h reaction, respectively. In contrast, only 4.31 mg reducing sugars were released from 20 mg of NTCS. Reducing sugar yield of UGCS and ALKCS was 96.6% and 100%, respectively. Furthermore, rapid saccharification was observed with these two substrates, which resulted in a sugar yield of 81.9% and 74.2% after 24 h of hydrolysis ( b).

The optimal pH of total cellulases, endoglucanases and xylanases was 4.5. The activity was above 75% of the highest activity for total cellulase at pH 3.0&#;6.0, above 70% for endoglucanase at pH 3.5&#;5.0, and above 90% for xylanase at pH 2.5&#;5.0. In addition, favorable pH stability was also observed for the enzymes: 75% total cellulase activity was maintained after 1 h of incubation at pH-values between 2.0 and 12.0, in which 90% activity was maintained at pH 3.0 to 9.0. In addition, about 98% endoglucanase activity and 80% xylanase activity were maintained at the pH range of 2.0&#;12.0 after 1 h incubation, which 95% xylanase activity was maintained at the pH range of 2.0&#;9.0 ( ).

The quantitative proteomic data showed high ratios in the protein abundance of a core set of glycoside hydrolases secreted by T. harzianum EM, including total cellulase abundance of 31.5% and total hemicellulase abundance of 32.2% ( c). Cellulases, including endoglucanases, cellobiohydrolases and glucosidases accounted for 11.85%, 8.8%, and 9.76% of the total proteins, respectively ( c). Among the secreted cellulases and hemicellulases, some proteins in these sets took up a great proportion. For instance, the two GH5 endogluananses accounted for 4.54% and 5.30% of the total proteins, respectively, and both contained a CBM domain at each of the C and N ends of the protein. Cellobiohydrolases I (GH7) and cellobiohydrolases II (GH6) accounted for 7.30% and 1.50% of total protein abundance, respectively. Two GH3 β-glucosidases accounted for an outstanding proportion among the multiple glucosidases, with abundances of 6.00% and 1.76% ( a). Multiple abundant hemicellulases were also detected, including xylanase (21.01%), xylosidases (0.24%), arabinofuranosidases (6.66%), galactosidases (2.1%), mannosidases (1.18%), and carboxylesterase (1.01%). It was noteworthy that two highly expressed xylanases were relatively abundant in the secretome, with an abundance index of 14.69% and 4.51%, and accounted for 91.4% of the total xylanase abundance. A lytic polysaccharide monooxygenase (LPMO, AA9) secreted by T. harzianum EM accounted for 3.23% of the total quantified CAZymes ( c). Moreover, the main glycosidases (glucosidases, xylosidases, arabinofuranosidases, and mannosidases) accounted for 17.2% of the total extracellular degradation enzymes secreted by T. harzianum EM ( ).

Enzyme activities of commercial preparations (C and Celluclast 1.5L) and EM (Enzyme preparation of T. harzianum EM) were measured by using the model substrates. The three enzyme preparations showed significant differences in their specific activities of cellulases, including endoglucanase, cellobiohydrolase, and β-glucosidase. The endoglucanase activity of EM was only 56.9% of C , and the cellobiohydrolase activity showed no significant difference with that of Celluclast 1.5L. Most notably, EM displayed the highest level of β-glucosidase activity, which was 311 and 2.9 folds of that in C and Celluclast 1.5L, respectively. In addition, EM displayed significantly higher hemicellulases activities than the two commercial preparations. The xylanase specific activitiy of EM was 310.70 U/mg, which was 29.1 folds higher than that of Celluclast 1.5L. Xylosidase and arabinofuranosidase activities were as high as 203.60 and 11.72 U/mg, which was 333.8 and 37.8 folds higher than that of commercial enzyme preparations C and Celluclast 1.5L, respectively. Furthermore, amylase activities of EM and C were 3.92 and 0.3 U/mg, respectively, and it was not detected in Celluclast 1.5L. EM contained a more complete lignocellulosic enzyme system than the commercial enzyme preparations. It presented high levels of glycosidases that were necessary for cellulose and hemicellulose degradation, and showed a broader prospect in fermentable monosaccharide production from complex polysaccharide compounds ( ).

In order to prepare low-cost lignocellulolytic enzymes cocktails, different kinds of lignocellulosic substrates were used separately as the carbon source for T. harzianum EM, including wheat bran, sunchoke (Jerusalem artichoke) stalks, corncob, miscanthus, giant juncao grass, switchgrass, corn stover, sugarcane bagasse, and straw of Triarrhena lutarioriparia. Measurement of the extracellular cellulase and xylanase revealed that the highest filter paper activity (1.54 U/mL) was induced by corn stover after seven days of cultivation ( ). Simultaneously, high levels of endoglucanase activity (8.61 U/mL) and xylanase activity (54 U/mL) were induced ( ), in which the xylanase activity was 91% of the maximum value (59.9 U/mL) detected in the culture with corncob after seven days of cultivation. In addition, when corn stover was used, rapid enzyme production capacity was observed in T. harzianum EM. The filter paper activity reached more than 70% of the maximum value, while the endoglucanase and xylanase activities reached more than 80% of their maximum values after two days of fermentation. Therefore, corn stover was selected as the optimal substrate for T. harzianum EM to prepare the lignocellulolytic enzyme cocktail (EM) in further study.

3. Discussion

Saprophytic fungi secrete a series of enzymes to degrade the plant cell walls in order to obtain nutrition for their growth. The main drawback of biofuels and other value-added bio-products generated from biomass are the high cost and the low saccharification efficiency of lignocellulolytic enzymes. In addition, the global market for industrial enzymes was expected to increase from nearly $5.0 billion in to $6.3 billion in . Hence, lignocellulolytic enzymes which can be produced at low-cost and are capable of producing high yields of fermentable sugars require investigation. Using the agricultural residue corn stover as the sole carbon source, T. harzianum EM secreted a quantitatively balanced enzyme system, which provides a low-cost enzyme preparation for biomass hydrolysis. High levels of endoglucanases and β-glucosidases could be secreted by T. harzianum, which has a high potential for application in industrial settings. According to Li et al. [12], the filter paper activity of the enzyme cocktail secreted by T. harzianum K (CGMCC ) was 1.62 U/mL using sugarcane bagasse as the substrate supplied with 20 g/L glucose as carbon source. We observed a comparable activity for T. harzianum EM when grown on corn stover without supplementation. These facts illustrate the possibility to develop low-cost degradation enzyme cocktails with high efficiency [2,26]. The high levels of lignocellulolytic enzymes and rapid enzyme production capacity of T. harzianum EM made it a suitable candidate for enzyme cocktail production, since most of the previous studies on developing cost-effective cell wall degrading enzymes have focused on improving the activity of cellulase by adopting genetic operation and screening natural strains so as to obtain high yield of glucose [4,35]. Meng et al. [36] overexpressed the endoglucanase gene in T. reesei Rut-C30, which showed 90.0% and 132.7% increases in the activities of total cellulases and endoglucanases under flask culture conditions, respectively. Furthermore, hemicellulose is the second most abundant component of lignocellulose and is mainly composed of xylan, mannan, and xyloglucan. Hemicelluloses can be hydrolyzed into pentoses (xylose and arabinose) and hexoses (glucose, mannose and galactose) at high yield which is important for bio-refinery applications [37,38]. Besides the enzymes degrading the main chains of the structural polysaccharides in plant cell walls, various glycosidases have been found to be essential for obtaining fermentable sugars from lignocellulose [39]. Glucosidase, xylosidase, and arabinofuranosidase played key roles in the production of glucose, xylose, and arabinose, respectively. In addition, mannosidase, galactosidase, and esterase were also necessary to deconstruct the complex lignocellulose [40]. It was reported that T. harzianum LZ117 could secrete a more complete enzyme cocktail than T. reesei. This strain produced 29.24 U/mg xylanase after 144 h of fermentation, but its CMCase, pNPCase, and pNPGase were only 4.30, 0.19, and 1.81 U/mg, respectively, much lower than the other cellulase producers [12]. In our present study, CMCase, pNPCase, and pNPGase of EM enzyme cocktail was 11.20, 33.76, and 84.97 U/mg, respectively. In addition, high levels of pNPXase and pNPAFase activities up to 203.60 and 11.72 U/mg were detected. Specific activities of glycosidases of EM was the highest compared to the two extensively used inartificial enzyme cocktails under the conditions tested ( ). When using natural lignocellulose substrate as the sole carbon source, T. harzianum that secret cellulase and hemicellulase simultaneously, especially high levels of glycosidases has not been reported [27]. Therefore, the patterns of enzyme activities observed for T. harzianum EM highlight its potential as efficient enzyme producer for industrial applications.

Different induction conditions have been shown to greatly influence the secretion of cellulolytic enzymes by fungi as revealed by Filho et al. [20], at the transcriptional level. In the transcriptome of T. harzianum, the GH7 endoglucanase gene was the highest expressed one when grown on cellulose, while expression level of GH10 xylanase was significantly increased when grown on the natural bagasse [20]. Secretome analysis of T. harzianum EM demonstrated the multiple functions and the synergistic effects of the lignocellulolytic enzymes. The balanced abundance of three kinds of cellulases play a crucial role for their synergistic degradation effects [41]. The abundant endoglucanases increased the frequency for randomly degrading the main chain of cellulose, which provided more access points for cellobiohydrolases. The high abundance of glucosidases could quickly remove the cellobiose and release glucose efficiently. In addition, much higher abundance of glycosidases responsible for side chain degradation could also effectively release series of fermentable sugars from complex polysaccharides [42]. A total of 44 glycosidases among the 81 CAZymes were found in the secretome of EM, with GH3, GH5, GH11, GH30, and GH55 as the dominant components, which may play crucial role in lignocellulose degradation (Figure S1). Vanessa et al. [43] also reported that the glycoside hydrolases were the most abundant class of proteins secreted by Trichoderma harzianum IOC , which represent 67% of the total proteins; however, pretreated (partially delignified) cellulolignin from sugarcane bagasse was used in their study as the inducer for lignocellulosic enzyme secretion. In addition, the most abundant groups in the secretome of T. harzianum IOC were GH3 (17%), GH5 (11%), GH 7 (10%), GH6 (6%), and GH55 (6%), which was slightly different from those verified for T. harzianum EM in our present study and demonstrated the possible effects of the secretome.

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For lignocellulose degradation, the amount of cellulases is not the most critical factor affecting sugar yield, since the synergistic effects between the cellulases and hemicellulases and between the enzymes for main- and side-chain degrading enzymes of the polysaccharides complex might be more important. Therefore, the high conversion efficiency of the secretome of T. harzianum EM might be achieved from its numerously and quantitatively balanced cellulases and hemicellulases. Previously, partial replacement of cellulases with auxiliary enzymes has reduced the required amount of commercial cellulases to achieve high hydrolysis yields [11,44]. Yang et al. [45] reported a degree of synergy of 1.35 for the hydrolysis of delignified corn stover when studying the synergistic effect between a commercial enzyme preparation and a bifunctional enzyme consisting of an acetyl xylan esterase and a α-l-arabinofuranosidase. For glucan conversion of delignified corn stover, Zhu et al. [46] found a synergy of 1.96 between a hemicellulase preparation EMSD5 (including xylanases, β-xylosidases, α-l-arabinofuranosidases, α-glucuronidases, and acetyl xylan esterases) and the commercial cellulase from T. reesei. These findings revealed the significant role of glycosidases for efficient lignocellulose degradation. Moreover, 9 carbohydrate binding modules (CBMs) were also observed, which could help hydrolyze substrate more effectively [46]. In T. harzianum EM enzyme enzyme cocktail, a LPMO with abundance of 3.23% was found. It was discovered that LPMOs promote degradation of the most recalcitrant crystalline cellulose by carrying out oxidative cleavage of polysaccharides [47]. Several studies showed synergetic effects between LPMOs and the lignocellulolytic enzymes during the saccharification process of cellulose and hemicellulose components [9,47]. All CAZymes and non-hydrolyzed protein detected in the secreted proteins of T. harzianum EM are important for fermentable sugars production. In addition, some glycoside hydrolases such as GH30 xylanase and GH26 mannase secreted by T. harzianum EM have been rarely studied in Trichoderma. Synergy between cellulolytic enzymes and these enzymes from Acremonium alcalophilum and Aspergillus nidulans has been described, which deserves more attention in the future [48,49]. The currently reported lignocellulosic degrading bacteria and fungi with industrial application prospects presented distinct optimal conditions for different enzymes even in the same induced enzyme system, and the stabilities against temperature and pH were also different, so it is necessary to determine the optimal conditions for enzymatic hydrolysis and saccharification with overall consideration [1,50,51]. In the present study, similar optimal temperature and pH for various functional enzymes in T. harzianum EM enzyme cocktail were observed, which gave rise to better synergistic effects in efficient deconstruction of lignocellulose complex. Pretreatments could make the macroscopic and microscopic deconstruction and change chemical composition of lignocellulose, which improve the accessibility of enzymes and facilitate release of fermentable sugars. Among many pretreatment methods, dilute alkali treatment is widely used because of its remarkable effect, and physical crushing pretreatment without any pollution has been widely used as well [52].

When EM was used, reducing sugar yields of 96.6% and 100% were obtained from UGCS and ALKCS, respectively, after 72 h of saccharification. The efficacy after 24 h of saccharification was 81.9% and 74.2%, respectively. ( b). So, the enzyme cocktail EM was much more effective than the current commercial products under the conditions tested, since only 46.9% sugar yield was acquired from UGCS by Celluclast 1.5 L after 72 h hydrolysis [33] (Table S1). In general, saccharification efficiency of lignocellulose biomass by EM was better than that in any other literature reported under the conditions tested [29,30,31,32,33,34]. To be specific, in our study, the maximum 85.59% glucose yield and 66.18% xylose yield were obtained from UGCS hydrolyzed with 30 FPU/g of Celluclast 1.5 L used; while 100% glucose yield and 100% xylose yield were obtained from UGCS and ALKCS with the same enzyme dosage of T. harzianum EM. To some extent, the glucose and xylose yields by EM were lower than the total sugar yields when the same substrates were used, which indicated that not only monosaccharides but also oligosaccharides were obtained from UGCS and ALKCS. From these results, it could be estimated that various auxiliary enzyme including hemicellulase and amylase played important role to completely and effectively deconstruct the complex lignocellulose substrates [11].

Some accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugars for use in a biorefinery setting in order to improve total utilization of biomass. Supplementation of a commercial enzyme preparation with 30% crude enzyme complex from Aspergillus oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%, which demonstrated the potential to use the supplementary enzymes in the total lignocellulose degradation, although 51.2% of cellulose conversion and 78.1% of xylan conversion were obtained [53]. With supplementary of β-glucosidase produced by Aspergillus niger and endoglucanase produced by Talaromyces emersonii to the currently used commercial enzyme cocktails, greater saccharification of alkaline-pretreated bagasse (87% glucose yield and 94% of xylose yield) was obtained, but its enzyme dosage was as high as 500 FPU/g substrate [54]. In the present study, only enzyme dosage of 5 mg EM/g substrate (13.85 FPU/g) was required for complete saccharification of lignocellulose, which provided sufficient sugars for the downstream fermentation of downstream products. Therefore, EM presented great potential in industrial applications. To the best of our knowledge, it was the first example that enzyme cocktail from T. harzianum completely and simultaneously converted cellulose and xylan in natural biomaterials into fermentable sugars at the same time. To date, induction and saccharification mechanisms of lignocellulosic enzymes secreted by T. harzianum are still unknown. Differences in the composition of the T. harzianum secretome in response to different substrates was reported, which revealed the diversity of the fungus enzymatic toolbox [55]. Nevertheless, enzyme cocktails with excellent efficiency of complete saccharification of lignocellulose should be tailor-made, and degradation mechanisms remain to be elucidated in more detail. The expression of total extracellular proteins of T. harzianum EM also could be optimized at the molecular level, in order to further expand the application scope of T. harzianum EM.

Related Reading

Introductions

Hemicellulase is an enzyme capable of degrading the hemicellulose present in the cell wall of plants. Hemicellulose is a glucose polymer linked to a monosaccharide which can be a five-carbon polymer such as xylose or a six-carbon polymer such as galactose, mannose or rhamnose. Hemicellulase belongs to the enzymatic family of glycoside hydrolases. Its action consists in breaking the bond engaging glucose and polymers present in plant fibres with water molecules. The enzymatic reaction leads to the hydrolysis of hemicellulose.

Figure 1. Structure of Hemicellulase.

How Does Hemicellulase Work?

Hemicellulase possesses the distinct ability to boost this prebiotic activity. Different types of this enzyme have been used for different purposes in food technologies, particularly for its ability to enhance the quality of dough, as well as produce fruit juices and alcoholic beverages. In fact, it is a commonly-added enzyme in the production of wines, as the enzyme helps strip away the unwanted compounds from the skins of the grapes that might change the taste of the wine. Although plants make hemicellulases for growth and development, most of the commercial interest is in the enzymes produced by microorganisms.

The Health Benefits of Hemicellulase

• Improve overall health

Animal studies have shown that supplementing feed with hemicellulases not only helps improve nutrient digestibility, but also improves performance and increases food conversion. Hemicellulases can promote overall health. Chickens were given hemicellulase supplements and subsequently analyzed for nutrient utilization, performance and digestibility.

• Better Digestive Capacity with Aging

Taking digestive enzymes such as hemicellulase can offset the net loss of enzymes that occurs with age, whether due to loss of enzyme production from the pancreas or changes in beneficial flora in the gut. Research supports this. In Japan, scientists have demonstrated that intestinal enzyme output shows a gradual decrease with age. People aged 65 and older were tested, especially women, and found to have the greatest decline in enzyme health.

• May reduce Candida

Some studies have shown that increasing this enzyme may help prevent and reduce yeast infestations such as Candida. This may be related to the fact that the cell wall of Candida is made up of hemicellulose. Again, since hemicellulase digests hemicellulose, it may help reduce Candida.

• Supporting the immune system

In addition, hemicellulase has been shown to support a healthy immune system due to its glucan degrading ability. It works like this: glucanase activity prompts the release of glucan from the cell wall of Candida. This then triggers an immune system response that sends specific substances such as cytokines and interleukins to the area of concern where they begin to destroy Candida.

Conclusions

There are many different types of hemicellulases that break down a wide range of hemicelluloses. This provides countless benefits in terms of optimal digestion, including fiber-rich plant foods such as grains, fruits, vegetables and cereals. This enzyme also supports the aging digestive system by replenishing the body's natural digestive enzymes when supplies are low. Finally, hemicellulase has the powerful ability to target Candida by breaking down its cell walls and the slimy anti-microbial layer called biofilm. This supports a healthy immune system by reducing the likelihood of long-term inflammation caused by the body's response to yeast overgrowth.

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