Azo-CM-Cellulose (Liquid)

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00:02   Principle of the Assay Procedure
00:34    Substrate & Kit Description
01:02    Dissolution of Azo-CM-Cellulose
03:10    Precipitant Solution
04:59    Preparation of Buffer Solution
05:10    Assay Procedure
08:49    Calculation

Reference code: S-ACMCL

100 mL (2% w/v)

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Content: 100 mL (2% w/v)
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Physical Form: Liquid
Stability: > 6 months under recommended storage conditions
Substrate For (Enzyme): endo-Cellulase
Assay Format: Spectrophotometer, Petri-dish (Qualitative)
Detection Method: Absorbance
Wavelength (nm): 590
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 (Liquid) is equivalent to this.

Display our full list of chromogenic substrates.

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Megazyme publication
New chromogenic substrates for the assay of alpha-amylase and (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.

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Constructing a cellulosic yeast host with an efficient cellulase cocktail.

Chang, J. J., Lin, Y. J., Lay, C. H., Thia, C., Wu, Y. C., Hou, Y. H., Huang, C. C. & Li, W. H. (2018). Biotechnology and bioengineering, 115(3), 751-761.

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.

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RGS4 impacts carbohydrate and siderophore metabolism in Trichoderma reesei.

Schalamun, M., Molin, E. M. & Schmoll, M. (2023). BMC genomics, 24(1), 372.

Background: Adaptation to complex, rapidly changing environments is crucial for evolutionary success of fungi. The heterotrimeric G-protein pathway belongs to the most important signaling cascades applied for this task. In Trichoderma reesei, enzyme production, growth and secondary metabolism are among the physiological traits influenced by the G-protein pathway in a light dependent manner. Results: Here, we investigated the function of the SNX/H-type regulator of G-protein signaling (RGS) protein RGS4 of T. reesei. We show that RGS4 is involved in regulation of cellulase production, growth, asexual development and oxidative stress response in darkness as well as in osmotic stress response in the presence of sodium chloride, particularly in light. Transcriptome analysis revealed regulation of several ribosomal genes, six genes mutated in RutC30 as well as several genes encoding transcription factors and transporters. Importantly, RGS4 positively regulates the siderophore cluster responsible for fusarinine C biosynthesis in light. The respective deletion mutant shows altered growth on nutrient sources related to siderophore production such as ornithine or proline in a BIOLOG phenotype microarray assay. Additionally, growth on storage carbohydrates as well as several intermediates of the D-galactose and D-arabinose catabolic pathway is decreased, predominantly in light. Conclusions: We conclude that RGS4 mainly operates in light and targets plant cell wall degradation, siderophore production and storage compound metabolism in T. reesei.

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Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae). 

Moye, J., Schenk, T. & Hess, S. (2022). BMC Biology, 20(1), 1-16.

Background: Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents. These phagotrophic “protoplast feeders” represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. Although their fascinating feeding behaviour has been observed for the last 150 years, it is still unknown how protoplast feeders produce the well-defined and species-specific perforations in biochemically diverse cell walls. Differential expression analyses of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria) suggested the involvement of a highly expressed putative glycoside hydrolase of family GH5_5. To assess the importance of this carbohydrate-active enzyme in the feeding act of Orciraptor, we recombinantly produced its catalytic domain and studied the enzymatic activity, cellular localisation and function. Results: The GH5_5 catalytic domain from Orciraptor showed pronounced activity on soluble cellulose derivatives and mixed-linkage glucans, with reaction optima comparable to known GH5_5 representatives. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. Immunocytochemistry with a polyclonal antibody raised against the GH5_5 domain revealed that the native endocellulase localises to the contact zone of Orciraptor and the algal cell wall (= perforation zone) and to intracellular granules, which were enriched during attack. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. Conclusions: Our experimental data from enzymatic assays, immunocytochemistry and inhibition experiments strongly suggest a key role of the GH5_5 endocellulase in cell wall dissolution by Orciraptor agilis. With that, we provide evidence that the well-defined perforations produced by protoplast feeders are caused by extracellular carbohydrate-active enzymes and made a first step towards establishing the molecular basis of a fascinating, yet poorly understood microbial feeding strategy.

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MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila.

Li, N., Liu, Y., Liu, D., Liu, D., Zhang, C., Lin, L., Zhu, Z., Li, H., Dai, Y., wang, X. & Tian, C. (2022). Applied and Environmental Microbiology, 88(19), e01263-22.

The thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila, the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila. We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5′-GNG/C-3′). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production.

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Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.

Zhang, C., Li, N., Rao, L., Li, J., Liu, Q. & Tian, C. (2022). Microbiology Spectrum, e02321-21.

Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi.

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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.

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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.

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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.

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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, manganese peroxidase EC and laccase EC, as well endo-cellulase EC and endo-xylanase EC 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%.

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