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AZCL-HE-Cellulose

AZCL-HE-Cellulose I-AZCEL
Product code: I-AZCEL
€200.00

3 g

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Content: 3 g
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 2 years under recommended storage conditions
Substrate For (Enzyme): endo-Cellulase
Assay Format: Spectrophotometer (Semi-quantitative), Petri-dish (Qualitative)
Detection Method: Absorbance
Wavelength (nm): 590

High purity dyed and crosslinked insoluble AZCL-HE-Cellulose for identification of enzyme activities in research, microbiological enzyme assays and in vitro diagnostic analysis.

Substrate for the assay of endo-cellulase.

We offer other insoluble substrates.

Documents
Certificate of Analysis
Safety Data Sheet
Application Note Assay Protocol
Publications
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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Publication

Expression and Characterization of a Small, Xylan/Cellulose-degrading GH43 Protein Derived from Biofertilizer Metagenome.

Oraintara, A. & Bhunaonin, P. (2022). The Open Microbiology Journal, 16(1).

Background: A putative glycosyl hydrolase gene biof1_09 was identified from a metagenomic fosmid library of local biofertilizers in previous report [1]. The gene is renamed as gh43kk in this study. Methods: The gene gh43kk, encoding a putative β-D-xylosidase was amplified by polymerase chain reaction (PCR) and successfully cloned and expressed in Escherichia coli. The expressed recombinant protein was purified by metal affinity chromatography. Its properties were initially verified by enzyme assay and thin layer chromatography (TLC). Results: The purified recombinant protein showed the highest catalytic activities at acidic pH 4 and 50°C toward beechwood xylan, followed by carboxymethylcellulose (CMC). TLC analysis indicated a release of xylose and glucose when xylan and CMC were treated with Gh43kk protein, respectively, whereas glucose and cellobiose were detected when avicel, cellulose and filter paper were used as substrates, suggesting its dual function as xylanase with cellulase activity. The enzyme indicated great stability in a temperature between 10 to 50°C and a wide range of pH from 4 to 8. Enzyme activity of Gh43kk was enhanced in the presence of magnesium and manganese ions, while calcium ions, Ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) inhibited the enzyme activity. Conclusion: These results suggest that Gh43kk could be a potential candidate for application in various bioconversion processes.

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Publication

A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei.

Subramanian, V., Farmer, S. J., Heiland, K. L., Moore, K. T., Vander Wall, T. A., Sun, W., Chaudhari, Y. B., Himmel, M. E. & Decker, S. R. (2022). Journal of Industrial Microbiology and Biotechnology, In Press.

Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold difference in protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

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Publication

Inhibition of LPMOs by Fermented Persimmon Juice.

Tokin, R., Ipsen, J. Ø., Poojary, M. M., Jensen, P. E., Olsson, L. & Johansen, K. S. (2021). Biomolecules, 11(12), 1890.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.

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Publication

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts.

Tokin, R., Frandsen, K. E., Ipsen, J. Ø., Lo Leggio, L., Poojary, M. M., Berrin, J. G., Grisel, S., Brander, S., Jensen, P. E. & Johansen, K. S. (2021). New Phytologist, 232(3), 1337-1349.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported. In this study, a diverse library of 100 plant extracts was screened for LPMO activity-modulating effects. By employing protein crystallography and LC-MS, we successfully identified a natural LPMO inhibitor. Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites: one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC-MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1. The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts.

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Publication

Biochemical and synergistic properties of a novel alpha‐amylase from Chinese nong‐flavor Daqu.

Chen, L., Yi, Z., Fang, Y., Jin, Y., He, K., Xiao, Y., Zhao, D., Luo, H., He, H., Sun, Q. & Zhao, H. (2021). Microbial Cell Factories, 20(1), 1-15.

Background: Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. Results: In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. Conclusions: This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

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Publication

A biochemical comparison of fungal GH6 cellobiohydrolases.

Christensen, S. J., Krogh, K. B. R. M., Spodsberg, N., Borch, K. & Westh, P. (2019). Biochemical Journal, 476(15), 2157-2172.

Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investigated by phylogenetic analyses to ensure that they were not closely related phylogenetically. The selected enzymes were all heterologously expressed in Aspergillus oryzae, purified and thoroughly characterized biochemically. This approach allowed direct comparisons of functional data, and the results revealed substantial variability. For example, the adsorption capacity on cellulose spanned two orders of magnitude and kinetic parameters, derived from two independent steady-state methods also varied significantly. While the different functional parameters covered wide ranges, they were not independent since they changed in parallel between two poles. One pole was characterized by strong substrate interactions, high adsorption capacity and low turnover number while the other showed weak substrate interactions, poor adsorption and high turnover. The investigated enzymes essentially defined a continuum between these two opposites, and this scaling of functional parameters raises interesting questions regarding functional plasticity and evolution of GH6 CBHs.

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Publication

Secretory overexpression of the endoglucanase by Saccharomyces cerevisiae via CRISPR-δ-integration and multiple promoter shuffling.

Sasaki, Y., Mitsui, R., Yamada, R. & Ogino, H. (2019). Enzyme and Microbial Technology, 121, 17-22.

Various recombinant proteins can be produced by the yeast Saccharomyces cerevisiae cell factories; therefore, efficient recombinant protein production techniques are desirable. In this study, to establish an efficient recombinant protein production technique in S. cerevisiae, the secretory production of recombinant protein endoglucanase II (TrEG) was tested. We developed 2 novel methods for TrEG production via clustered regularly interspaced short palindromic repeat (CRISPR) -δ-integration as well as multiple promoter shuffling, which involved the pre-breakdown of the δ-sequence by the CRISPR system and subsequent δ-integration as well as the conjugation of TrEG with various promoters and subsequent δ-integration, respectively. Moreover, simultaneous use of the CRISPR-δ-integration and multiple promoter shuffling methods was also examined. The CRISPR-δ-integration method was effective for improvement of the integrated TrEG copy number and its activity, and the multiple promoter shuffling method was also beneficial for enhancing the transcriptional level of TrEG and its activity. Furthermore, simultaneous use of CRISPR-δ-integration and multiple promoter shuffling methods was the most useful. The carboxymethyl cellulase activity of the TrEG expressing transformant YPH499/24CP constructed by the method reached 559 U/L, and it was 17.3-fold higher than that of the transformant constructed by the conventional YEp type vector. Overall, the simultaneous use of CRISPR-δ-integration and multiple promoter shuffling can be useful and easily applied for recombinant protein production.

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Publication

Strain improvement for industrial production of lignocellulolytic enzyme by Talaromyces cellulolyticus.

Fujii, T., Inoue, H., Yano, S. & Sawayama, S. (2018). Fungal Cellulolytic Enzymes, pp. 135-154. Springer, Singapore.

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is a commercial fungal source used for industrial enzyme production for silage preparation. In this chapter, the development of T. cellulolyticus strains to produce lignocellulolytic enzymes suitable for the hydrolysis of target biomass is reviewed. High-yield production and composition improvements of lignocellulolytic enzymes in T. cellulolyticus have been succeeded by mutagenesis, genetic engineering, and enzyme preparation up to the present date. Recent developments of T. cellulolyticus genetic tools including whole genome sequencing, homologous recombination, marker recycling, RNA interference, genome editing and transcriptional regulation, a concept of core and accessary enzymes, and their utilization for strain improvements will be discussed.

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Publication
Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass.

Dadheech, T., Shah, R., Pandit, R., Hinsu, A., Chauhan, P. S., Jakhesara, S., Kunjadiya, A., Rank, D. & Joshi, C. (2018). International Journal of Biological Macromolecules, 113, 73-81.

Cellulase hydrolyses the cellulose by cleaving the β-1,4-linkages to produce mono-, oligo- and shorter polysaccharide units. These enzymes have applications in various industries such as pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. In the present study we have cloned acid-cellulase gene (Cel-1) from the fosmid library of buffalo rumen metagenomic DNA and functionally expressed it in Escherichia coli. The ORF encoding cellulase consisted of 1176-bp, corresponding to protein of 391 amino acid and has catalytic domain belonging to glycosyl hydrolase family 5. The purified protein has a molecular weight of 43-kDa on SDS-PAGE and its expression was confirmed by western blotting. The tertiary structure of the cellulase (Cel-1) showed a classical (α/β) TIM-like barrel motif. Model surface charge of Cel-1 predicted that surface near active site was mostly negative which might be responsible for the stability of enzyme at lower pH. The pH and temperature for maximum enzyme activity were 4.5 and 45°C respectively. Various metal ions enhanced the enzyme activity and in presence of Mn+2 activity was significantly increased. Cel-1 hydrolyzed pre-treated wheat straw and released reducing sugars (62.60%). These desirable properties of Cel-1 make it attractive for the bioconversion of biomass.

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Safety Information
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Precautionary Statements : Not Applicable
Safety Data Sheet
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