Chapter 1: Principle of the Assay Procedure
Chapter 2: Substrate & Kit Description
Chapter 3: Dissolution of Azo-CM-Cellulose
Chapter 4: Precipitant Solution
Chapter 5: Preparation of Buffer Solution
Chapter 6: Assay Procedure
Chapter 7: Calculation
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|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-Cellulase|
|Assay Format:||Spectrophotometer, Petri-dish (Qualitative)|
|Reproducibility (%):||~ 7%|
High purity dyed, soluble Azo-CM-Cellulose for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.
Substrate for the specific measurement of endo-1,4-β-D-glucanase (cellulase).
Please note the video above shows the protocol for assay of endo-cellulase using Azo-CM cellulose. The procedure for the assay of endo-Cellulase using Azo-CM-Cellulose (Powder) is equivalent to this.
View other soluble chromogenic enzyme substrates.
(Aspergillus niger) E-CELBA - Cellulase (endo-1,4-β-D-glucanase)
(Bacillus amyloliquefaciens) E-CELTE - Cellulase (endo-1,4-β-D-glucanase)
(Talaromyces emersonii) E-CELTH - Cellulase (endo-1,4-β-D-glucanase)
(Thermobifida halotolerans) E-CELTR - Cellulase (endo-1,4-β-D-glucanase)
(Trichoderma longibrachiatum) E-CELTM - Cellulase (endo-1,4-β-D-glucanase)
McCleary, B. V. (1980). Carbohydrate Research, 86(1), 97-104.
New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.Hide Abstract
Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting.
Benedetti, M., Vecchi, V., Guardini, Z., Dall’Osto, L. & Bassi, R. (2020). Plants, 9(12), 1799.
Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW−1 (1.67 Units g DW−1) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.Hide Abstract
An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing.
Guo, Z. P., Borsenberger, V., Croux, C., Duquesne, S., Truan, G., Marty, A. & Bordes, F. (2020). Communications Biology, 3(1), 1-10.
The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, independent and linear supplementary chromosome with a yield over 90%. Additionally, our results reveal that ylAC can be genetically maintained over multiple generations either under selective conditions or, without selective pressure, using an essential gene as the selection marker. Overall, the ylACs reported herein are game-changing technology for Y. lipolytica, opening myriad possibilities, including enzyme screening, genome studies and the use of this yeast as a previous unutilized bio-manufacturing platform.Hide Abstract
A processive endoglucanase with multi-substrate specificity is characterized from porcine gut microbiota.
Wang, W., Archbold, T., Lam, J. S., Kimber, M. S. & Fan, M. Z. (2019). Scientific Reports, 9(1), 1-13.
Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50°C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc®, and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.
Enzymatic potential and biosurfactant production by endophytic fungi from mangrove forest in Southeastern Brazil.
Martinho, V., dos Santos Lima, L. M., Barros, C. A., Ferrari, V. B., Passarini, M. R. Z., Santos, L. A., de Souza Debastianes, F. L., Lacava, P. T. & de Vasconcellos, S. P. (2019). AMB Express, 9(1), 1-8.
Microbial activity is the main route for cycling mangrove nutrients. In general, microorganisms have abilities to degrade lignocellulosic compounds. Among the biotechnological potential of the microbiota from mangroves, it is noteworthy about endophytic fungi, which can be considered as effective sources of different bioactive compounds. In this sense, thirty (30) endophytic fungi were isolated from mangrove forest sampling Cananeia, SP, Brazil. These microorganisms were analyzed about their enzymatic activities including: lignin peroxidase EC 126.96.36.199, manganese peroxidase EC 188.8.131.52 and laccase EC 184.108.40.206, as well endo-cellulase EC 220.127.116.11 and endo-xylanase EC 18.104.22.168. Besides that, production of bioactive secondary metabolites like biosurfactant and/or bioemulsifier was also investigated. As results, nineteen (19) isolates were selected about their ligninolytic abilities, nine (9) of them about cellulase activity and thirteen (13) showed xylanase abilities. The fungal isolate named as 3(3), characterized as Fusarium sambucinum, showed a prominent lignin peroxidase (42.4 U L−1) and manganese peroxidase (23.6 U L−1) activities. The isolate 63.1, also related to Fusarium sp. genera, was selected about its laccase activity (41.5 U L−1). From all the investigated fungi, the isolate 47(4) Trichoderma camerunense was selected about its cellulolytic and xylanolytic activities, showing 45.23 and 26.09 U mL−1, respectively. The same fungi also showed biosurfactant ability demonstrated by superficial tension decreasing to 38 mN/m. In addition, fifteen (15) fungi exhibited bioemulsifier activity, with E24 values up to 62.8%.Hide Abstract
Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues.
Davison, S. A., Keller, N. T., van Zyl, W. H. & den Haan, R. (2019). Enzyme and Microbial Technology, 131, 109382.
In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, β-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants produced up to 1.34 fold higher glucose concentrations (g/L) than a control composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted ratio of 1:15 (cellulase activity unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments and yielded 4.05 g/L ethanol in fermentations. We demonstrate that the choice of optimal genetic background and cellulase activity secretion ratio can improve cellulosic ethanol production by consolidated bioprocessing yeast strains.Hide Abstract
Innovative microscale workflow from fungi cultures to Cell Wall‐Degrading Enzyme screening.
Raulo, R., Heuson, E., Siah, A., Phalip, V. & Froidevaux, R. (2019). Microbial Biotechnology, 12(6), 1286-1292.
This study aimed at developing a complete miniaturized high‐throughput screening workflow for the evaluation of the Cell Wall‐Degrading Enzyme (CWDE) activities produced by any fungal strain directly cultivated on raw feedstock in a submerged manner. In this study, wheat straw was selected as model substrate as it represents an important carbon source but yet poorly valorised to yield high added value products. Fungi were grown in a microbioreactor in a high‐throughput (HT) way to replace the fastidious shaking flask cultivations. Both approaches were compared in order to validate our new methodology. The range of CWDE activities produced from the cultures was assayed using AZO‐died and pNP‐linked substrates in an SBS plate format using a Biomek FXp pipetting platform. As highlighted in this study, it was shown that the CWDE activities gathered from the microbioreactor cultivations were similar or higher to those obtained from shake flasks cultures, with a lower standard deviation on the measured values, making this new method much faster than the traditional one and suitable for HT CWDE production thanks to its pipetting platform compatibility. Also, the results showed that the enzymatic activities measured were the same when doing the assay manually or using the automated method.Hide Abstract
Improved production of succinic acid from Basfia succiniciproducens growing on A. donax and process evaluation through material flow analysis.
Cimini, D., Zaccariello, L., D’Ambrosio, S., Lama, L., Ruoppolo, G., Pepe, O., Faraco, V. & Schiraldi, C. (2019). Biotechnology for Biofuels, 12(1), 22.
Background: Due to its wide range of applications in the food, pharmaceutical and chemical fields, microbial synthesis of succinic acid is receiving growing attention, generating already relevant industrial results, as well as fueling constant research for improvements. In order to develop a sustainable process, a special focus is now set on the exploitation and conversion of lignocellulosic biomasses into platform chemicals. Results: In the present work we used Basfia succiniciproducens BPP7 in separated hydrolysis and fermentation experiments with Arundo donax as starting material. Fed-batch strategies showed a maximal production of about 37 g/L of succinic acid after 43 h of growth and a productivity of 0.9 g/L h on the pilot scale. Global mass balance calculations demonstrated a hydrolysis and fermentation efficiency of about 75%. Moreover, the application of a material flow analysis showed the obtainment of 88.5 and 52 % of succinic acid, per kg of virgin biomass and on the total generated output, respectively. Conclusions: The use of fed-batch strategies for the growth of B. succiniciproducens on A. donax improved the titer and productivity of succinic acid on pre-pilot scale. Process evaluation through material flow analysis showed successful results and predicted a yield of succinic acid of about 30% in a fed-batch process that uses A. donax as only carbon source also in the feed. Preliminary considerations on the possibility to achieve an energetic valorization of the residual solid coming from the fermentation process were also carried out.Hide Abstract
Cellulose-binding activity of a 21-kDa endo-β-1,4-glucanase lacking cellulose-binding domain and its synergy with other cellulases in the digestive fluid of Aplysia kurodai.
Tsuji, A., Yuasa, K. & Asada, C. (2018). PloS One, 13(11), e0205915.
Endo-β-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo β-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another β-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.Hide Abstract
Young, D., Dollhofer, V., Callaghan, T. M., Reitberger, S., Lebuhn, M. & Benz, J. P. (2018). Bioresource Technology, 268, 470-479.
Aerobic and anaerobic fungi are among the most effective plant biomass degraders known and have high potential to increase the efficiency of lignocellulosic biomass utilization, such as for biogas generation. However, limited information is available on their contribution to such industrial processes. Therefore, the presence of fungi along the biogas production chain of one-phase and two-phase biogas plants in Germany was analyzed. Seventeen aerobic species of Zygomycota, Ascomycota and Basidiomycota were identified, including efficient producers of lignocellulases, such as Trichoderma capillare isolated from a hydrolysis tank and Coprinopsis cinerea from fibers separated from pressed digestate. Five anaerobic fungal species of the phylum Neocallimastigomycota (comprising two novel clades) were present in an acidic fermenter of a biogas plant fed with cow manure displaying endoglucanase transcriptional activity. The broad fungal presence demonstrated in this study can serve developing bioaugmentation systems with relevant lignocellulolytic fungi to improve biogas production from recalcitrant fiber material.Hide Abstract
Developing cellulolytic Yarrowia lipolytica as a platform for the production of valuable products in consolidated bioprocessing of cellulose.
Guo, Z. P., Robin, J., Duquesne, S., O’Donohue, M. J., Marty, A. & Bordes, F. (2018). Biotechnology for Biofuels, 11(1), 141.
Background: Both industrial biotechnology and the use of cellulosic biomass as feedstock for the manufacture of various commercial goods are prominent features of the bioeconomy. In previous work, with the aim of developing a consolidated bioprocess for cellulose bioconversion, we conferred cellulolytic activity of Yarrowia lipolytica, one of the most widely studied “nonconventional” oleaginous yeast species. However, further engineering this strain often leads to the loss of previously introduced heterologous genes due to the presence of multiple LoxP sites when using Cre-recombinase to remove previously employed selection markers. Results: In the present study, we first optimized the strategy of expression of multiple cellulases and rescued selection makers to obtain an auxotrophic cellulolytic Y. lipolytica strain. Then we pursued the quest, exemplifying how this cellulolytic Y. lipolytica strain can be used as a CBP platform for the production of target products. Our results reveal that overexpression of SCD1 gene, encoding stearoyl-CoA desaturase, and DGA1, encoding acyl-CoA:diacylglycerol acyltransferase, confers the obese phenotype to the cellulolytic Y. lipolytica. When grown in batch conditions and minimal medium, the resulting strain consumed 12 g/L cellulose and accumulated 14% (dry cell weight) lipids. Further enhancement of lipid production was achieved either by the addition of glucose or by enhancing cellulose consumption using a commercial cellulase cocktail. Regarding the latter option, although the addition of external cellulases is contrary to the concept of CBP, the amount of commercial cocktail used remained 50% lower than that used in a conventional process (i.e., without internalized production of cellulases). The introduction of the LIP2 gene into cellulolytic Y. lipolytica led to the production of a strain capable of producing lipase 2 while growing on cellulose. Remarkably, when the strain was grown on glucose, the expression of six cellulases did not alter the level of lipase production. When grown in batch conditions on cellulose, the engineered strain consumed 16 g/L cellulose and produced 9.0 U/mL lipase over a 96-h period. The lipase yield was 562 U lipase/g cellulose, which represents 60% of that obtained on glucose. Finally, expression of the hydroxylase from Claviceps purpurea (CpFAH12) in cellulolytic Y. lipolytica procured a strain that can produce ricinoleic acid (RA). Using this strain in batch cultures revealed that the consumption of 11 g/L cellulose sustained the production of 2.2 g/L RA in the decane phase, 69% of what was obtained on glucose. Conclusions: In summary, this study has further demonstrated the potential of cellulolytic Y. lipolytica as a microbial platform for the bioconversion of cellulose into target products. Its ability to be used in consolidated process designs has been exemplified and clues revealing how cellulose consumption can be further enhanced using commercial cellulolytic cocktails are provided.Hide Abstract
Directed evolution of the bacterial endo-β-1, 4-glucanase from Streptomyces sp. G12 towards improved catalysts for lignocellulose conversion.
Cecchini, D. A., Pepe, O., Pennacchio, A., Fagnano, M. & Faraco, V. (2018). AMB Express, 8(1), 74.
With the aim to develop biocatalysts for enhanced hydrolysis of (hemi)cellulose into monosaccharides, random diversity by directed evolution was introduced in the gene coding for the endo-β-1,4-glucanase from Streptomyces sp. G12 which had been recombinantly expressed in Escherichia coli and named rCelStrep. The main objectives were therefore to set up a complete strategy for creation and automated screening of rCelStrep evolved direct mutants and to apply it to generate and screen a library of 10,000 random mutants to select the most active variants. The diversity was introduced in the gene by error-prone polymerase chain reaction. A primary qualitative screening on solid plates containing carboxymethylcellulose as the substrate allowed selecting 2200 active clones that were then subjected to a secondary quantitative screening towards AZO-CMC for the selection of 76 improved variants that were cultured in flasks and characterized. Five rCelStrep mutants exhibiting the highest hydrolytic activities than the wild-type enzyme were further characterized and applied to the bioconversion of the pretreated Arundo donax lignocellulosic biomass. It is worth of noting that one of the five tested mutants exhibited a 30% improvement in bioconversion yields compared to the wild-type enzyme, despite the absence of the carbohydrate binding module domain in this variant. Homology models of the three-dimensional structures of the catalytic and binding modules of rCelStrep were obtained and localization of mutations on these models allowed us to speculate on the structure-function relationships of the mutants.Hide Abstract
Takano, M. & Hoshino, K. (2018). Bioresources and Bioprocessing, 5(1), 16.
Background: Rice straw is one of the abundant lignocellulosic biomass with potential as a feedstock for bioethanol production. To produce ethanol from the biomass biologically, enzymatic hydrolysis is necessary that can effectively degrade rice straw into fermentable sugars such as glucose and xylose. Many researches utilized many kinds of commercial cellulase reagents on the hydrolysis of cellulose. Since these have different enzyme activities, enzyme reagents suitable for each biomass must be selected. In this study, three appropriate cellulase reagents were selected by multivariate analysis technique and then optimized by design of experiments method for efficient hydrolysis of alkali-pretreated rice straw. Moreover, an ethanol production from the treated straw was performed by simultaneous saccharification and fermentation (SSF) with the optimized enzyme cocktail and xylose-fermenting fungus of Mucor circinelloides. Results: Pretreatment by alkali solution of rice straw resulted in the increase of fermentable sugar content from 56.3 to 80.0%. The desirable commercial enzyme reagents for saccharification of the straw were determined as a combination of “Cellulase Onozuka 3S”, “Cellulase T Amano 4”, and “Pectinase G Amano” by a multivariate analysis based on various cellulolytic activities of each reagent. The optimum mixing ratio was calculated by response surface method based on design of experiment method. The optimized cocktail successfully achieved 75.3 g/L in production of the total fermentable sugar by hydrolysis of alkali-treated rice straw that agreed with the hydrolysis efficiency of 94.1%. SSF of 100 g/L treated rice straw with the optimal cocktail and M. circinelloides under aerobic condition resulted in 30.5 g/L ethanol concentration for 36 h. Conclusion: The construction of cellulase cocktail by the proposed statistical method enabled efficient hydrolysis of alkali-treated rice straw. SSF process combining the optimized cocktail and a xylose-fermenting fungus could be expected as a promising system for ethanol production from various lignocellulosic biomasses.Hide Abstract
Wikee, S., Chumnunti, P., Al Kanghae, A., Chukeatirote, E., Lumyong, S. & Faulds, C. (2017). Journal of Bacteriology and Mycology, 4(2), np.
The Dothideomycetes represent the largest fungal class of Ascomycota. It is an ubiquitous class of fungi whose members span a wide spectrum of lifestyles and host interactions. The endophytic fungus Phyllosticta is one members of the Dothideomycetes, causing disease in economic crops. Phyllosticta was screened for the degradation of lignocellulosic biomass of commercial relevance, such as rice straw, rice husk, sorghum, wheat straw, miscanthus, lavender flower, and lavender straw. The highest degrading strains were identified from an initial screen and further analyzed for the secretion of lignocellulosic enzymes during growth on the different biomasses. With Phyllosticta capitalensis (MFLUCC14-0233), maximum activity of arabinase (944.18 U/ml culture), cellulase (27.10 U/ml), xylanase (10.85 U/ml), pectinase (465.47 U/ml), and laccase (35.68 U/ml) activities could be detected in the secretome during growth on lavender flowers and lavender straw. Phyllosticta capitalensis is thus an interesting new strain for the production of lignocellulosic enzymes during growth on cheap agro-industrial biomass.Hide Abstract
Allman, A. L., Williams, E. P. & Place, A. R. (2017). Journal of Shellfish Research, 36(1), 283-291.
The blue crab [Callinectes sapidus (Rathbun, 1896)] is a benthic decapod with a varied diet. The diet includes invertebrates and detrital material that can have relatively large amounts of chitin and cellulose, both of which can be difficult to digest for many organisms and often require the aid of specific bacteria in the gut microbiome. In this study, juvenile blue crabs were fed an optimized defined pelleted diet with a 20% replacement of wheat flour filler with either chitin, cellulose, or a 14%/6% mix of both, followed by a diet switch to the opposing ingredient. Crabs had increasing growth performance with increasing amounts of cellulose in the diet versus chitin and had an additional molt in most cases. This occurred during the initial phase and following the switch, indicating that performance can be recovered. Subsequently, cellulose and chitin digestion assays were used to show that the foregut, midgut, and hindgut were all able to significantly digest more cellulose than chitin with the majority of activity in the foregut and midgut. Implications for rearing and diet formulations as well as the role of cellulose and chitin digestion in the natural diet are discussed.Hide Abstract
Guo, Z. P., Duquesne, S., Bozonnet, S., Cioci, G., Nicaud, J. M., Marty, A. & O’Donohue, M. J. (2017). Biotechnology for Biofuels, 10(1), 132.
Background: Yarrowia lipolytica, one of the most widely studied “nonconventional” oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous β-glucosidases in Y. lipolytica, thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic Y. lipolytica for use in consolidated bioprocessing of cellulose. Results: Initially, different essential enzyme components of a cellulase cocktail (i.e,. cellobiohydrolases and endoglucanases) were individually expressed in Y. lipolytica in order to ascertain the viability of the strategy. Accordingly, the Trichoderma reesei endoglucanase I (TrEG I) and II (TrEG II) were secreted as active proteins in Y. lipolytica, with the secretion yield of EG II being twice that of EG I. Characterization of the purified His-tagged recombinant EG proteins (rhTrEGs) revealed that rhTrEG I displayed higher specific activity than rhTrEG II on both cellotriose and insoluble cellulosic substrates, such as Avicel, β-1, 3 glucan, β-1, 4 glucan, and PASC. Similarly, cellobiohydrolases, such as T. reesei CBH I and II (TrCBH I and II), and the CBH I from Neurospora crassa (NcCBH I) were successfully expressed in Y. lipolytica. However, the yield of the expressed TrCBH I was low, so work on this was not pursued. Contrastingly, rhNcCBH I was not only well expressed, but also highly active on PASC and more active on Avicel (0.11 U/mg) than wild-type TrCBH I (0.065 U/mg). Therefore, work was pursued using a combination of NcCBH I and TrCBH II. The quantification of enzyme levels in culture supernatants revealed that the use of a hybrid promoter instead of the primarily used TEF promoter procured four and eight times more NcCBH I and TrCBH II expressions, respectively. Finally, the coexpression of the previously described Y. lipolytica β-glucosidases, the CBH II, and EG I and II from T. reesei, and the N. crassa CBH I procured an engineered Y. lipolytica strain that was able to grow both on model cellulose substrates, such as highly crystalline Avicel, and on industrial cellulose pulp, such as that obtained using an organosolv process. Conclusions: A Y. lipolytica strain coexpressing six cellulolytic enzyme components has been successfully developed. In addition, the results presented show how the recombinant strain can be optimized, for example, using artificial promoters to tailor expression levels. Most significantly, this study has provided a demonstration of how the strain can grow on a sample of industrial cellulose as sole carbon source, thus revealing the feasibility of Yarrowia-based consolidated bioprocess for the production of fuel and chemical precursors. Further, enzyme and strain optimization, coupled to appropriate process design, will undoubtedly lead to much better performances in the future.Hide Abstract
Sarcina, R., Giosafatto, C. V. L., Faraco, V.,Lama, L.,Esposito, M. & Mariniello, L. (2017). International Journal of Environment, Agriculture and Biotechnology, 2(4), 1809-1813.
Cellulases are a important family of hydrolytic enzymes which catalyze the bond of cellulose and other related cello-oligosaccharide derivates. Industrial applications require enzymes highly stable and economically viable in terms of reusability. These costs can be reduced by immobilizing the cellulases, offering a potential solution through enzyme recycling and easy recovery. The covalent immobilization of enzymes is reported here: one is commercial cellulase from Aspergillus niger and other one is recombinant enzyme, named CelStrep it because was isolated from a new cellulolytic strain, Streptomyces sp. G12,. The optimal pH for binding is 4.6 for both cellulases and the optimal enzyme concentrations are 1 mg/mL and 5 mg/mL respectively. The support for immobilization is a poliacrylic matrix. Experiments carried out in this work show positive results of enzyme immobilization in terms of efficiency and stability and confirm the economic and biotechnical advantages of enzyme immobilization for a wide range of industrial applications.Hide Abstract
Deng, Y., Li, M., Chen, L. X., Chen, X. Q., Lu, J. H., Zhao, J. & Li, S. P. (2017). Carbohydrate Polymers, In Press.
The chain conformation, chemical characters and immunomodulatory activity of polysaccharide from Dendrobium devonianum (DDP) were investigated.Results showed that molecular weights, polydispersity index, radius of gyrations of DDP were 3.99 × 105 Da, 1.27, 74.1 nm, respectively. By applying the polymer solution theory, the exponent (v) values of <S2>z 1/2 = kMwv was calculated as 0.38, which revealed that DDP existed as a globular shape in aqueous solution, and further confirmed by AFM analysis. Furthermore, the main monosaccharide compositions were Man and Glc with the ratio of 29.61:1.00. Indeed, the main glycosidic linkages were β-1,4-Manp, and substituted with acetyl groups at O-2 and O-3 position. Notably, DDP could promote the immune functions of macrophages including NO release and phagocytosis. Thus, DDP could be explored as a natural immune-stimulating agent in the health and functional food area as well as pharmaceutical industries.Hide Abstract
Chang, J. J., Lin, Y. J., Lay, C. H., Thia, C., Wu, Y. C., Hou, Y. H., Huang,C. C. & Li, W. H. (2017). Biotechnology and Bioengineering, In Press.
Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.Hide Abstract
Marcolongo, L., Ionata, E., Cara, F. L., Amore, A., Giacobbe, S., Pepe, O. & Faraco, V. (2014). Fungal Genetics and Biology, 72, 162-167.
The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewer’s spent grains (BSG), after chemical or chemical–physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields.Hide Abstract