In the present study, the young leaves of castor were used as materials to obtain protoplast, and the effects of enzymatic hydrolysis system on the yield and quality of protoplast were analyzed ...detailedly. The results shown that the treatment of the young leaves of castor with 1.5% Cellulase RS and 0.75% Macerozyme R-10 for 80 min could gain the maximum amount of protoplast. In addition, the activity of protoplast was observed with 1% Evans blue staining, and most of the protoplast (76.03%) from castor leaves had physical vitality. It lays a foundation for further studies on plant breeding and functional genes in castor.
Protein glycosylation is the most complex posttranslational modification process. Most cellulases from filamentous fungi contain N-glycosylation and O-glycosylation. Here, we discuss the potential ...roles of glycosylation on the characteristics and function of cellulases. The use of certain cultivation, inducer, and alteration of engineering glycosylation pathway can enable the rational control of cellulase glycosylation. Glycosylation does not occur arbitrarily and may tend to modify the 3D structure of cellulases by using specially distributed glycans. Therefore, glycoengineering should be considered comprehensively along with the spatial structure of cellulases. Cellulase glycosylation may be an evolution phenomenon, which has been considered as an economical way for providing different functions from identical proteins. In addition to gene and transcription regulations, glycosylation may be another regulation on the protein expression level. Enhanced understanding of the potential regulatory role of cellulase glycosylation will enable synthetic biology approaches for the development of commercial cellulase.
In this paper, we reported a simple and efficient protocol for preparation of Cu super(2+)-modified magnetic Fe sub(3)O sub(4)iO sub(2 ) core/shell microspheres for immobilization of cellulase. The ...uniform magnetic Fe sub(3)O sub(4)iO sub(2 ) core/shell microspheres with a thin shell of 20 nm were synthesized through a solvothermal method followed by a sol-gel process. An amino-terminated silane coupling agent of (3-aminopropyl)triethoxysilane (APTS) was then grafted on them for capturing Cu super(2+) ions. The reaction process is very simple, efficient, and economical. Noticeably, the content of Cu super(2+) ions on the magnetic core/shell microspheres can reach 4.6 Wt%, endowing them possess as high immobilization capacity as 225.5 mg/g for cellulase. And the immobilized cellulase can be retained over 90 % on the magnetic microspheres after six cycles. Meanwhile, the magnetic microspheres decorated with Cu super(2+) ions show a superparamagnetic character with a high magnetic saturation of 58.5 emu/g at room temperature, suggesting conveniently and rapidly recycle the enzyme from solution. This facile, recyclable, high immobilization capacity and activity strategy may find potential applications in enzyme catalytic reactions with low cost.
•Accessory enzymes (AE) deviate cellulase hydrolysis to the lignocellulose from FPA.•AE can boost the cellulase activity on different substrates to varying degrees.•The boost is realized apparently ...by the synergy/cohydrolysis of AE with cellulase.•New insights are provided into AE, which is helpful to customize cellulase cocktails.
The potential of cellulase enzymes in the developing and ongoing “biorefinery” industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates.
Microbial cellulases have been receiving worldwide attention, as they have enormous potential to process the most abundant cellulosic biomass on this planet and transform it into sustainable biofuels ...and other value added products. The synergistic action of endoglucanases, exoglucanases, and β-glucosidases is required for the depolymerization of cellulose to fermentable sugars for transformation in to useful products using suitable microorganisms. The lack of a better understanding of the mechanisms of individual cellulases and their synergistic actions is the major hurdles yet to be overcome for large-scale commercial applications of cellulases. We have reviewed various microbial cellulases with a focus on their classification with mechanistic aspects of cellulase hydrolytic action, insights into novel approaches for determining cellulase activity, and potential industrial applications of cellulases.
•The first overview of advances in improving the performance of cellulase in ILs.•Screening natural IL-tolerant cellulases from different microhabitats are described.•Physical and chemical methods ...for stabilizing cellulases in ILs are discussed.•Improving IL tolerance of cellulase through enzyme engineering are reviewed.•Inactivation mechanisms of cellulase in ILs are addressed.
Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.
The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes ...specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.
Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to ...cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular β-glucosidase enzymes and one intracellular β-glucosidase lacks β-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple β-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.
The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked ...glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.
•A strain of Bacillus subtilis with endoglucanase activity was isolated from the wastewater of a pulp and paper mill.•The endoglucanase gene was inserted into a modified plasmid (E. coli) and into pYD1 (S. cerevisiae).•The endoglucanase in pYD1 is expressed with good activity in E. coli, being the first report of this finding.•A truncated recombinant endoglucanase is obtained and is very stable at pH (2-10) and temperature (30–55 °C).
•The binding mechanisms of enzymes onto lignin thin films are enzyme specific.•Of the studied enzymes, the hydrolysis yields of TrCel6A and TrCel7B were most affected by lignin.•Soluble compounds ...from isolated lignin increased β-glucosidase activity up to 28%.
Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a β-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw. The enzymes exhibited decreasing affinity onto lignin model films in the following order: TrCel7B>TrCel6A>TrCel5A>AnCel3A>TrCel7A>TrXyn11. As analysed in Avicel hydrolysis, TrCel6A and TrCel7B were most inhibited by lignin isolated from pretreated spruce. This could be partially explained by adsorption of the enzyme onto the lignin surface. Enzyme properties, such as enzyme surface charge, thermal stability or surface hydrophobicity could not alone explain the adsorption behaviour.
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