Hydrolysis of different chain length xylooliogmers by cellulase and hemicellulase

Commercial cellulase complexes produced by cellulolytic fungi contain enzyme activities that are capable of hydrolyzing non-cellulosic polysaccharides in biomass, primarily hemicellulose and pectins, in addition to cellulose. However, xylanase activities detected in most commercial enzyme preparations have been shown to be insufficient to completely hydrolyze xylan, resulting in high xylooligomer concentrations remaining in the hydrolysis broth. Our recent research showed that these xylooligomers are stronger inhibitors of cellulase activity than others have previously established for glucose and cellobiose, making their removal of great importance. In this study, a HPLC system that can measure xylooligomers with degrees of polymerization (DP) up to 30 was applied to assess how Spezyme CP cellulase, Novozyme 188 β-glucosidase, Multifect xylanase, and non-commercial β-xylosidase enzymes hydrolyze different chain length xylooligomers derived from birchwood xylan. Spezyme CP cellulase and Multifect xylanase partially hydrolyzed high DP xylooligomers to lower DP species and monomeric xylose, while β-xylosidase showed the strongest ability to degrade both high and low DP xylooligomers. However, about 10-30% of the higher DP xylooligomers were difficult to be breakdown by cellulase or xylanase and about 5% of low DP xylooligomers (mainly xylobiose) proved resistant to hydrolysis by cellulase or β-glucosidase, possibly due to low β-xylosidase activity in these enzymes and/or the precipitation of high DP xylooligomers.     

Xylanase production by endophytic Aspergillus niger using pentose-rich hydrothermal liquor from sugarcane bagasse

Fungal xylanases have been widely studied and various production methods have been proposed using submerged and solid-state fermentation. This class of enzyme is used to supplement cellulolytic enzyme cocktails in order to enhance the enzymatic hydrolysis of plant cell walls. The present work investigates the production of xylanase and other accessory enzymes by a recently isolated endophytic Aspergillus niger DR02 strain, using the pentose-rich liquor from hydrothermal pretreatment of sugarcane bagasse as carbon source. Batch and fed-batch submerged cultivation approaches were developed in order to minimize the toxicity of the liquor and increase enzyme production. Maximum xylanase activities obtained were 458.1 U/mL for constant fed-batch, 428.1 U/mL for exponential fed-batch, and 264.37 U/mL for pulsed fed-batch modes. The results indicated that carbon-limited fed-batch cultivation can reduce fungal catabolite repression, as well as overcome possible negative effects of toxic compounds present in the pentose-rich liquor. Enzymatic panel and mass spectrometric analyses of the fed-batch A. niger secretome showed high levels of xylanolytic enzymes (GH10, GH11, and GH62 Cazy families), together with cellobiohydrolase (G6 and GH7), β-glucosidase, β-xylosidase (GH3), and feruloyl esterase (CE1) accessory enzyme activities. The yields of glucose and xylose from enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse increased by 43.7 and 65.3%, respectively, when a commercial cellulase preparation was supplemented with the A. niger DR02 constant fed-batch enzyme complex.     

β-甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达

【目的】β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域。构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价。【方法】通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl。【结果】菌株诱导21 h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍。【结论】SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外。此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好。菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础。     

Production of β-xylosidase from <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.     

A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—Factors affecting enzymes,conversion and synergy

Lignocellulose is a complex substrate which requires a variety of enzymes, acting in synergy, for its complete hydrolysis. These synergistic interactions between different enzymes have been investigated in order to design optimal combinations and ratios of enzymes for different lignocellulosic substrates that have been subjected to different pretreatments. This review examines the enzymes required to degrade various components of lignocellulose and the impact of pretreatments on the lignocellulose components and the enzymes required for degradation. Many factors affect the enzymes and the optimisation of the hydrolysis process, such as enzyme ratios, substrate loadings, enzyme loadings, inhibitors, adsorption and surfactants. Consideration is also given to the calculation of degrees of synergy and yield. A model is further proposed for the optimisation of enzyme combinations based on a selection of individual or commercial enzyme mixtures. The main area for further study is the effect of and interaction between different hemicellulases on complex substrates.     

Cellulase and Xylanase Production by the Mexican Strain <Emphasis Type="Italic">Talaromyces stollii</Emphasis> LV186 and Its Application in the Saccharification of Pretreated Corn and Sorghum Stover

A Mexican strain of Talaromyces stollii LV186 was isolated from decaying pretreated corn stover. The production of cellulase and xylanase enzyme cocktails was evaluated with corn and sorghum stover used as inducers in a mineral medium. The volumetric and specific activities of T. stollii LV186 were compared with the values produced by Trichoderma reesei ATCC 26921 in a time-course experiment. After the submerged culture and a posterior ultrafiltration stage, the enzyme complexes were evaluated over acid-pretreated corn or sorghum stover in baffled flasks under controlled temperature and agitation conditions, and hydrolysis levels of 30 and 39 % of the theoretical maximum were obtained after only 72-h reactions, for each substrate. A side-by-side comparison showed a better ratio of endoglucanase to cellobiohydrolase to β-glucosidase and of xylanase to β-xylosidase enzymes in T. stollii than in T. reesei ATCC 26921. Furthermore, the hydrolysis of pretreated corn and sorghum stover achieved by T. stollii is significantly higher compared with that of a commercial cocktail from T. reesei ATCC 26921 (Celluclast). Therefore, the T. stollii LV186 strain is a good candidate for the hydrolysis of complex lignocellulose substrates. To the authors’ knowledge, this study is the first to describe the cellulolytic and hemicellulolytic activities produced by a T. stollii strain.     

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

Optimization of enzyme complexes for lignocellulose hydrolysis

The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and beta-glucosidase activity. Although the well-documented relief of product inhibition by beta-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a approximately twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis.     

Production of xylanase and β-xylosidase from autohydrolysis liquor of corncob using two fungal strains

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and β-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of β-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of β-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of β-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of β-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.     

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential. Received: 5 May 1999 / Received revision: 31 May 2000 / Accepted: 3 June 2000     

Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance

Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-β-xylanase fused to a GH43 β-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the β-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis.     

Evaluation of different microbial xylanolytic systems

The xylanolytic enzymes produced by Trichoderma reesei QM 9414, Aspergillus awamori VTT-D-75028, Fusarium oxysporum VTT-D-80134, Bacillus subtilis ATCC 12711 and Streptomyces olivochromogenes ATCC 21713 differed with respect to β-xylosidase activity and side-group cleaving activities. The highest xylanase activity was produced by T. reesei. All the fungi produced β-xylosidase, whereas in the bacterial culture filtrates β-xylosidase activity was negligible. T. reesei culture filtrate contained all the side-group cleaving activities assayed (acetyl esterase, α-glucuronidase and α-arabinosidase) and those of F. oxysporum and S. olivochromogenes contained esterase. All the side-group cleaving activities were low in the culture filtrates of A. awamori and B. subtilis.The differences between the xylanolytic systems were reflected in the hydrolysis of steamed birchwood hemicellulose. The xylose yields obtained ranged from 0 (with B. subtilis) to 90% (with T. reesei) of the theoretical maximum. The best enzyme for complete hemicellulose hydrolysis was therefore that of T. reesei. However, in some applications in which complete hydrolysis is not needed or in which hydrolysis of cellulose is to be avoided, one of the other xylanases may be more suitable than that of T. reesei.     

Assessment of commercial hemicellulases for saccharification of alkaline pretreated perennial biomass

The objective of this research was to measure the effects of different cellulase and hemicellulase mixtures on fermentable sugar production from two different perennial biomasses--switchgrass and a low-impact, high-diversity prairie biomass mixture (LIHD). Each was subjected to NaOH pretreatment, followed by hydrolysis with a commercial cellulase and β-glucosidase mixture [CB] supplemented with either of two hemicellulases. For both biomasses, there was little gain in sugar yield when using CB alone beyond 20-25 mg/g TS; further gain in yield was possible only through hemicellulase supplementation. An equation that modeled CB and hemicellulase effects as occurring independently fit the data reasonably well, except at the lowest of cellulase loadings with hemicellulase, where synergistic interactions were evident. Examination of the marginal effectiveness of enzyme loadings (incremental grams sugar per incremental mg enzyme) over a broad range of loadings suggests that there is no need to customize enzymatic hydrolysis for NaOH-pretreated switchgrass and LIHD.     

Screening of filamentous fungi to produce xylanase and xylooligosaccharides in submerged and solid-state cultivations on rice husk,soybean hull,and spent malt as substrates

We investigated the enzymatic complex produced by selected fungi strains isolated from the environment using the agro-industrial residues rice husk, soybean hull, and spent malt as substrates. Microbial growth was carried out in solid-state cultivation (SSC) and in submerged cultivations (SC) and the enzymatic activities of xylanase, cellulase, β-xylosidase, and β-glucosidase were determined. All substrates were effective in inducing enzymatic activities, with one strain of Aspergillus brasiliensis BLf1 showing maximum activities for all enzymes, except for cellulases. Using this fungus, the enzymatic activities of xylanase, cellulase, and β-glucosidase were generally higher in SSC compared to SC, producing maxima activities of 120.5, 25.3 and 47.4 U g^?1 of dry substrate, respectively. β-xylosidase activity of 28.1 U g^?1 of dry substrate was highest in SC. Experimental design was carried out to optimize xylanase activity by A. brasiliensis BLf1 in SSC using rice husk as substrate, producing maximum xylanase activity 183.5 U g^?1 dry substrate, and xylooligosaccharides were produced and characterized. These results suggest A. brasiliensis BLf1 can be used to produce important lytic enzymes to be applied in the preparation of xylooligosaccharides.     

Production and properties of hemicellulases by a Thermomyces lanuginosus strain

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.     

Assessment of the biomass hydrolysis potential in bacterial isolates from a volcanic environment: biosynthesis of the corresponding activities

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80?% of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99?% 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, β-glucosidase and β-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the β-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of β-glucosidase. Xylanase and β-xylosidase production was practically unaffected by aeration levels.     

食用菌半纤维素酶系研究进展

半纤维素是一类取之不尽而又亟待开发利用的碳水化合物。对食用菌半纤维素酶系的研究已经积累了诸多资料,主要包括β-1,4-内切木聚糖酶、β-木糖苷酶、α-阿拉伯呋喃糖苷酶、乙酰木聚糖酯酶、α-葡萄糖醛酸酶的研究。主要对半纤维素酶系,尤其是木聚糖酶的生物化学与分子生物学研究进行了简要概括,包括结构、酶学性质、基因的克隆与表达等,并综述了在食用菌生产中半纤维素酶活性的变化,最后展望了食用菌半纤维素酶系今后的主要研究方向。     

Hydrolytic potential of Trichoderma sp. strains evaluated by microplate-based screening followed by switchgrass saccharification

Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. Large-scale screening of different microbial strains would provide optimal enzyme cocktails for any target feedstock. The aim of this study was to screen a large collection of Trichoderma sp. strains for the hydrolytic potential towards switchgrass (Panicum virgatum L.). Strains were cultivated in a small-scale system and assayed in micro-plates for xylanase and cellulase activities. The population distributions of these traits are reported after growth on switchgrass in comparison with cellulose. The distribution profiles suggest that the growth on switchgrass strongly promotes xylanase production. The IK4 strain displayed the highest xylanase activity after growth on switchgrass (133U/mL). Enzymes (10FPU/g substrate) from IK4 were compared with those from 2 cellulolytic Trichoderma strains and a commercial enzyme in saccharification time-course experiments on untreated and pretreated switchgrass and on an artificial substrate. Samples were analysed by DNS assay and by an oxygraphic method for sugar equivalent or glucose concentration. On the untreated substrate, IK4 enzymes even outperformed a 5-fold load of commercial enzyme, suggesting that xylanase or accessory enzymes are a limiting factor on this type of recalcitrant substrate. On the other substrates, IK4 preparations showed intermediate behaviour if compared with the commercial enzyme at 10FPU/g substrate and at 5-fold load. IK4 also nearly halved the time to release 50% of the hydrolysable sugar equivalents (T(50%)), with respect to the other preparations at the same enzymatic load. DNS assay and oxygraphic method gave highly correlated results for the 3 saccharified substrates. The study suggests that accessory enzymes like xylanase play a key role in improving the performance of cellulase preparations on herbaceous lignocellulosic feedstocks like switchgrass.     

Use of Ascomycete Extracts in Enzymatic Cocktail Formulations Increases Sugar Cane Bagasse Hydrolysis

Hemicellulases and accessory enzymes are essential for supplementation of cellulolytic enzyme extracts, and combinations of these enzymes can lead to high performance in plant biomass hydrolysis. In this work, enzyme extracts rich in hemicellulases and β-glucosidase, produced by the unique ascomycete strains Annulohypoxylon stygium DR47 and Aspergillus niger DR02, were tested for use in formulations with Celluclast 1.5 L. Statistical analysis showed that a mixture based on these enzymes was able to increase the hydrolysis of hydrothermally pretreated sugar cane bagasse. The two A. stygium extracts only effectively increased glucose release when they were combined. These extracts had no positive effect when used together with the A. niger extract, and the findings suggested that a blend based on the commercial cellulose preparation and the xylanase-rich extract from A. niger provided the best carbohydrate solubilization. Supplementation at low cellulolytic loading resulted in 120 and 238 % increases in cellulose and hemicellulose hydrolysis yields.     

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.