Blending of cellulolytic enzyme preparations from different fungal sources for improved cellulose hydrolysis by increasing synergism (original) (raw)
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Enzyme and Microbial Technology, 2003
The production of cellulose and hemicellulose-degrading enzymes by cultivation of Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, Schizophyllum commune ATCC 38548, and Trichoderma reesei Rut-C30 was studied. Wet-oxidised wheat straw suspension supplemented with NH 4 NO 3 , MgSO 4 , and KH 2 PO 4 was used as cultivation medium aiming to obtain an enzyme mixture optimal for enzymatic hydrolysis of wet-oxidised wheat straw. The cultivations with B. cinerea and P. brasilianum gave the highest endoglucanase (EC 3.2.1.4) and -glucosidase (EC 3.2.1.21) activities, in contrast to the other fungi where lower activities were found. The culture filtrates were concentrated by ammonium sulphate precipitation. After enzyme concentration, the highest enzyme activities (1.34 FPU/ml) were found in the culture broth originating from P. brasilianum. Enzymatic hydrolysis of filter cake from wet-oxidised wheat straw for 48 h with an enzyme loading of 5 FPU/g biomass resulted in glucose yields from cellulose of 58% (w/w) and 39% (w/w) using enzymes produced by P. brasilianum and a commercial enzyme mixture, respectively. At higher enzyme loading (25 FPU/g biomass) using either enzyme mixtures the glucose yield from cellulose was in the range of 77-79% (w/w).
Comparative study of saccharification of biomass by various cellulolytic enzymes
In this paper a comparative study of saccharification of cellulose-rich switchgrass biomass by various enzyme preparations (Accelerase 1500, GC 220, NZ50013 and CTec 2) has been carried out. Furthermore, an effect of supplementary β-glucosidase on activity of enzymes and yield of sugars has been studied. It was found that adding of β-glucosidase to enzyme preparations improves cellulolytic activity and increases the yield of glucose after enzymatic hydrolysis of the cellulosic biomass. In order to evaluate the yield of glucose (Y) from cellulosic substrate after its cleavage by different enzymatic preparations, a combined parameter of cellulolytic activity, P=L2/D, has been proposed; where L is used level of cellulolytic activity expressed in FPU per 1g substrate; and D is used doze of the enzyme expressed in mg proteins per 1g substrate. There is a directly proportional dependence between yield of glucose and combined parameter of cellulolytic activity. Due to high squared correlation coefficient (R2 = 0.934), the regression equation Y = k P can be used to predict the yield of glucose from the biomass hydrolyzed by different enzyme preparations.
Applied Biochemistry and Biotechnology
We evaluated various agricultural lignocellulosic biomass and variety of fungi to produce cellulolytic enzymes cocktail to yield high amount of reducing sugars. Solid-state fermentation was performed using water hyacinth, paddy straw, corn straw, soybean husk/tops, wheat straw, and sugarcane bagasse using fungi like Nocardiopsis sp. KNU, Trichoderma reesei, Trichoderma viride, Aspergillus flavus, and Phanerochaete chrysosporium alone and in combination to produce cellulolytic enzymes. Water hyacinth produced (U ml −1) endoglucanase (51.13) and filter paperase (0.55), and corn straw produced (U ml −1) β-glucosidase (4.65), xylanase (113.32), and glucoamylase (41.27) after 7-day incubation using Nocardiopsis sp. KNU. Production of cellulolytic enzymes was altered due to addition of various nitrogen sources, metal ions, vitamins, and amino acids. The maximum cellulolytic enzymes were produced by P. chrysosporium (endoglucanase; 166.32 U ml −1 and exoglucanase; 12.20 U ml −1), and by T. viride (filter paperase; 1.57 U ml −1). Among all, co-culture of T. reesei, T. viride, A. flavus, and P. chrysosporium showed highest β-glucosidase (17.05 U ml −1). The highest xylanase (1129 U ml −1) was observed in T. viride + P. chrysosporium co-culture. This study revealed the dependency on substrate and microorganism to produce good quality enzyme cocktail to obtain maximum reducing sugars.
2016
Production of cellulolytic enzymes like CMCase (endoglucanase), FPase, and xylanase by Aspergillus fumigatus SKH2 under solid state fermentation was carried out employing wheat bran as low cost substrate. Fermentation time, medium pH and incubation temperature were optimized at 48 h, pH 5.0 and 35 °C, respectively. At optimized state, CMCase (endoglucanase), FPase and xylanase of 826, 102 and 1130 U/gds yield was noticed, respectively. Crude enzyme cocktail was assayed at varied pH and temperature, and pH 5.0 and 35 °C were proved to be optimal for the studied enzyme activities. Fourier transform infrared spectroscopic FTIR analysis attested that NaOH was a good delignifying agent for sugarcane bagasse and grass Aristida sp., which enhanced subsequent saccharification efficiency of cellulolytic enzyme cocktail. By correlating FTIR analysis with saccharification profile it was found that highest saccharification was achieved after 16 h and 48 h after treating with 1M and 3M NaOH for ...
Journal of the Taiwan Institute of Chemical Engineers, 2017
In-house production of saccharifying enzymes using lignocellulosic biomass as inducer of enzyme production can yield (hemi)cellulolytic enzymes with more specificity and efficiency for hydrolyzing the same lignocellulosic substrates. In the present study, production of (hemi)cellulolytic enzymes was carried out using microwave assisted alkali (MAA) treated wheat straw and the crude enzyme was evaluated for hydrolysis of the same substrate. Co-production of cellulolytic and hemicellulolytic enzymes by Aspergillus niger ADH-11 was optimized using MAA treated wheat straw as a substrate and corn steep liquor (CSL) as moistening medium under solid state fermentation employing response surface methodology. Under optimized conditions viz. inoculum 30% (v/v of moistening agent), CSL 7.1% and incubation time of 4.99 days, 2.34 U/g of FP activity, 308.16 U/g endo-glucanase, 96.61 U/g of β-glucosidase, 3815.96 U/g of xylanase and 174.42 U/g of β-xylosidase activity were produced. By statistical optimization, FP activity and xylanase yield were enhanced by 2.0 and 14.22 fold respectively and time for production was reduced significantly. It was found that supplementation of in-house produced enzyme to commercial cellulase can improve the levels of xylanase, β-glucosidase and β-xylosidase significantly. Enzyme cocktail containing 5 FPU/g of SIGMA cellulase and 5 FPU/g in-house produced enzyme yielded 610.35 mg/g of reducing sugars in 72 h with 68.41% saccharification and released more glucose as FP activity: β-glucosidase ratio was enhanced. The cocktail was also assessed for its efficacy at high substrate loading and lower temperature for its use in simultaneous saccharification and fermentation (SSF) process for bioethanol production.
Journal of the Japan Petroleum Institute, 2017
Bioethanol is currently employed as a renewable energy to replace gasoline and addresses the issue of fossil fuel depletion. The main feedstocks used to produce bioethanol are sugar and starchy materials, with corn, sugarcane, and cassava typically being employed in the USA, Brazil, and Indonesia, respectively, as these three materials contain sugar or starch compounds that can be fermented to produce ethanol after hydrolysis when necessary. However, the use of such sugary and starchy materials competes with the demand for food supply, resulting in increased prices and the threatening of food security. In this context, lignocellulosic materials from agricultural and forest residues, such as rice straw, sawdust, and palm oil empty fruit bunch, have attracted growing attention as alternative feedstocks for bioethanol production, as they are both abundant and unutilized 1). In the case of lignocellulosic materials, the cellulose present in these materials can be hydrolyzed to give glucose, which can then be employed as a substrate for ethanol fermentation. However, due to the presence of lignin and hemicellulose in the lignocellulosic structure, this potential feedstock must go through an appropriate pretreatment method prior to hydrolysis 2). For example, methods such as ammonia explosion, dilute acid treatment, lime pretreatment, and hydrothermal pretreatment are among a number of techniques reported to date 3). Following such pretreatment, hydrolysis must then be carried out to convert cellulose to glucose, and so this stage is particularly important when considering the production costs of bioethanol 2). For example, hydrolysis by enzymes has been identified as a "green" and environmentally friendly process 4) in which the cellulose polymer is degraded by cellulase to give the monomer glucose, which can in turn be naturally fermented by the yeast Saccharomyces cerevisiae to yield ethanol 5). As such, a number of synergistic studies have been conducted to decrease the enzyme costs for the commercial production of ethanol 6). One potential strategy is low-cost enzyme production and subsequent recovery and reuse of the enzyme. For example, Nojiri et al. 7) reported that alkaline-treated woody biomass can be used as a low-cost material for an enzyme production medium. In addition, Kobayashi et al. 8) employed the functional lignin-based material lignocresol, which was synthesized from hinoki wood meal, to produce immobilized cellulase. An alternative strategy involves the optimization of an enzyme cocktail for saccharification. Currently, several commercial cellulases have been identified to produce cellobiose and glucose as its main products. The composition of these cellulases can be divided into three categories of enzyme, namely endo-1,4-β-g l u c a n a s e s , c e l l o b i o h y d r o l a s e s , a n d 322
Research and reviews in biosciences, 2014
The crude enzyme from Trichoderma reesei and Aspergillus niger have different activities. The use of two crude enzyme separately only able to produce a low levels of glucose, but after the two crude enzyme being mixed, it is able to improve the yield of glucose on cellulose hydrolysis process by using rice straw as substrate which has undergone pretreatment by alkali microwave. The combination of crude enzyme from Trichoderma reesei and Aspergillus niger (2:1) has the FP-ase activity of 1,002 IU/ml, CMC-ase activity of 2.23 IU/ml and â-glucosidase activity of 0.168 IU/ml. The activity of the crude enzyme cellulose combination are able to generate sugar at 12.89 mg/ml in the process of hydrolysis for 72 hours. 2014 Trade Science Inc. - INDIA
Environmental Progress & Sustainable Energy, 2015
The cost effective production of cellulolytic enzymes and their use for generating higher reducing sugars from lignocellulosic biomass is critical for attaining the commercial viability of lignocellulosic biofuels production. Optimizing the use of locally available agroresidues and natural nutritional alternatives for fungal growth and enzyme production can reduce the cost of enzyme production. In the present work, maximum cellulolytic activity of 30.85 IU/gds was obtained from Trichoderma reesei NCIM 1186 using wheat bran as the substrate and coconut water as the nutritional supplement. The produced cellualse was used for enzymatic saccharification of phosphoric acid pretreated wheat straw. The enzymatic sachharification was optimized through central composite design (CCD) based response surface methodology (RSM). Maximum reducing sugar yields of 371.44 mg/ g dry substrate were obtained at 18% (w/v) substrate concentration, 50 C and 24 h of incubation time. Further, the saccharified sugar hydrolysate was fermented using S. cerevisiae NCIM 3215. Maximum ethanol production (2.58% w/v) obtained after 24 h of fermentation at 30 C.
AMB Express, 2016
Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery.
Fungal Biology and Biotechnology, 2017
Background: The industrial applications of cellulases are mostly limited by the costs associated with their production. Optimized production pathways are therefore desirable. Based on their enzyme inducing capacity, celluloses are commonly used in fermentation media. However, the influence of their physiochemical characteristics on the production process is not well understood. In this study, we examined how physical, structural and chemical properties of celluloses influence cellulase and hemicellulase production in an industrially-optimized and a non-engineered filamentous fungus: Trichoderma reesei RUT-C30 and Neurospora crassa. The performance was evaluated by quantifying gene induction, protein secretion and enzymatic activities. Results: Among the three investigated substrates, the powdered cellulose was found to be the most impure, and the residual hemicellulosic content was efficiently perceived by the fungi. It was furthermore found to be the least crystalline substrate and consequently was the most readily digested cellulose in vitro. In vivo however, only RUT-C30 was able to take full advantage of these factors. When comparing carbon catabolite repressed and de-repressed strains of T. reesei and N. crassa, we found that cre1/cre-1 is at least partially responsible for this observation, but that the different wiring of the molecular signaling networks is also relevant. Conclusions: Our findings indicate that crystallinity and hemicellulose content are major determinants of performance. Moreover, the genetic background between WT and modified strains greatly affects the ability to utilize the cellulosic substrate. By highlighting key factors to consider when choosing the optimal cellulosic product for enzyme production, this study has relevance for the optimization of a critical step in the biotechnological (hemi-) cellulase production process.