Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity

The cellulase cocktail produced by marine Aspergillus niger exhibits a property of salt-tolerance,which is of great potential in cellulose degradation in high salt environment.In order to explain the mechanism on the salttolerance of the cellulase cocktail produced by marine A.niger,six cellulase components(AnCel6,AnCel7A,AnCel7B,AnEGL,AnBGL1 and AnBGL2)were obtained by directed expression.Studies on their enzymatic properties revealed that oneβ-glucosidase(AnBGL2)and one endoglucanase(AnEGL)exhibited an outstanding salttolerant property,and one cellobiohydrolase(AnCel7B)exhibited a certain salt-tolerant property.Subsequent study revealed that the salt-tolerant An EGL and AnCel7B endowed the cellulase cocktail with stronger salttolerant property,while the salt-tolerant An BGL2 had no positive effect.Moreover,after overexpression of AnCel6,AnCel7A,AnCel7B and AnEGL,the activity of cellulase cocktail increased by 80%,70%,63%and 68%,respectively.However,the activity of cellulase cocktail was not improved after overexpression of AnBGL1 and AnBGL2.After mixed-strain fermentation with cellobiohydrolase recombinants(cel6 a,cel7a and cel7b recombinants)and endoglucanase recombinant(egl recombinant),the the activity of cellulase cocktail increased by 114%,102%and91%,respectively.

A boronate-modified renewable nanointerface for ultrasensitive electrochemical assay of cellulase activity

The saccharification of cellulosic biomass to produce biofuels and chemicals is one of the most promising industries for gree n-power production and sustainable development.Cellulase is the core component in the saccharification process.Simple and efficient assay method to determine cellulase activity in saccharification is thus highly required.In this work,a boronate-affinity surface based renewable and ultrasensitive electrochemical sensor for cellulase activity determination has been fabricated.Through bo ronate-sugar interaction,celluloses are attached to the electrode surface,forming the cellulose na nonetwork at the sensing interface.Cellulase degradation can lead to the variation of electrochemical impedance.Thus,electrochemical impedance signal can reflect the cellulase activity.Importantly,via fully utilizing the boronate-affinity chemistry that enables reversible fabrication of cellulose nanonetwork,a renewable sensing surface has been firstly constructed for cellulase activity assay.Thanks to interfacial diffusion process of electrochemical sensor,the product inhibitory effect in the cellulase activity assays can be circumvented.The proposed electrochemical sensor is ultrasensitive for label-free cellulase activity detection with a very simple fabrication process,showing great potential for activity screen of new enzymes in saccharification conversion.

Screening and characterization of a novel ruminal cellulase gene(Umcel-1) from a metagenomic library of gayal(Bos frontalis)

Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase （GenBank accession no. YP_004310852.1 ） from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coil BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45~C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.

Two-step fermentation process and the corresponding study on its enzymology characteristics

Strain HIT-3,which has good performances in cellulose degradation,was isolated from the campus soil. Based on the identification of conservative sequences 16S rDNA and the analysis of physiological-biochemical characteristics,HIT-3 was identified as Achromobacter xylosoxidans. Denitrificans. A two-step fermentation process was conducted by adopting compound-bioflocculant-producing flora constructed by cellulose-degrading bacterium HIT-3 and flocculating bacterium F2. The cellulose degradation metabolites of HIT-3 was taken as substrates by flocculating bacterium F2,by which excellent compound bio-flocculant was obtained. In addition,the enzymology characteristics of HIT-3 were investigated when cultured in cellulose media,which utilized CMC-Na as its sole carbon. The results show that HIT-3 achieves a climax of 67.6 U/mL of enzyme production after incubation for 6 d,and the organic carbons produced are sufficient as the substrates required by the fermentation of flocculating bacterium F2(flocculating efficiency of 85.6%),which makes it feasible to reuse bioenergy.

Screening and Identification of Efficient Cellulose-degrading Strain and Its Preliminary Application in Sheep Dung Compost

The paper was to solve the problems of incomplete fermentation and more cellulose impurities in finished products caused by high cellulose content in sheep dung and accessory substances in composting production.[Method]Efficient cellulose-degrading strains were screened from different animal dung samples and three widely used commercial microbial composting strains(RW,BDM and JBB).The target strains were comprehensively screened by Congo red staining,degradation test of filter strips and cellulase activity test,and the strains screened were identified by physiological and biochemical tests and molecular tests.The strain was used to produce bioaugmentation strain,which was compared with composting fermentation of commercial strains.The temperature of composting process and the physical and chemical properties of finished products were tested to determine whether it met the national standards.[Result]A strain(M2)with strong cellulose-degrading ability from horse dung was screened,and its cellulase activity was(126.06±0.36)U/mL,higher than that of cellulose strain with the highest enzyme activity in commercial strains.Through morphological,physiological and biochemical identification and 16S rDNA sequence analysis,strain M2 was identified as Sphingomonas paucimobilis belonging to Sphingomonas sp.Compared with commercial strains,the bioaugmentation strain effectively improved the cellulose-degrading ability of strains after composting fermentation.The products were uniform in color,without obvious cellulose impurities,and the total nutrients were improved to a certain extent.[Conclusion]The strain M2 screened can be used for the development of specific strain for sheep dung composting.

Screening and Characterization of the High-Cellulase-Producing Strain Aspergillus glaucus XC9

Cellulose is a kind of renewable resource that is abundant in nature.It can be degraded by microorganisms such as mildew.A mildew strain with high cellulase activity was isolated from mildewy maize cob and classified as Aspergillus glaucus XC9 by morphological and 18S rRNA gene sequence analyses.We studied the effects of nitrogen source,initial pH,temperature,incubation time,medium composition,and surfactants on cellulase production.Maximal activities of carboxymethylcellulase(6,812 U/g dry koji)and filter paperase(172 U/g dry koji)were obtained in conditions as follows:initial pH,5.5–6.0;temperature,30℃;cultivation period,3–4 days;inoculum ratio,6%(vol/vol);sugarcane bagasse/wheat bran ratio,4:6.When bagasse was used as substrate and mixed with wet koji at a 1:1(wt/wt)ratio,the yield of reducing sugars was 36.4%.The corresponding conversion rate of cellulose to reducing sugars went as high as 81.9%.The results suggest that A.glaucus XC9 is a preferred candidate for cellulase production.

Reaction mechanism of dicofol removal by cellulase

It remains unclear whether dicofol should be defined as a persistent organic pollutant. Its environmental persistence has gained attention. This study focused on its degradation by cellulase. Cellulase was separated using a gel chromatogram, and its degradation activity towards dicofol involved its endoglucanase activity. By analyzing the kinetic parameters of cellulase reacting with mixed substrates, it was shown that cellulase reacted on dicofol and carboxyl methyl cellulose through two different active centers. Thus, the degradation of dicofol was shown to be an oxidative process by cellulase. Next, by comparing the impacts of tert-butyl alcohol（a typical OH free-radical inhibitor） on the removal efficiencies of dicofol under both cellulase and Fenton reagent systems, it was shown that the removal of dicofol was initiated by OH free radicals produced by cellulase. Finally, 4,4′-dichlorodibenzophenone and chloride were detected using gas chromatography mass spectrometry and ion chromatography analysis, which supported our hypothesis. The reaction mechanism was analyzed and involved an attack by OH free radicals at the orthocarbon of dicofol, resulting in the degradation product 4,4′-dichloro-dibenzophenone.