Cellobiohydrolase Research Articles

Effects of nitrogen fertilization and bioenergy crop species on central tendency and spatial heterogeneity of soil glycosidase activities

Extracellular glycosidases in soil, produced by microorganisms, act as major agents for decomposing labile soil organic carbon (e.g., cellulose). Soil extracellular glycosidases are significantly affected by nitrogen (N) fertilization but fertilization effects on spatial distributions of soil glycosidases have not been well addressed. Whether the effects of N fertilization vary with bioenergy crop species also remains unclear. Based on a 3-year fertilization experiment in Middle Tennessee, USA, a total of 288 soil samples in topsoil (0–15 cm) were collected from two 15 m2 plots under three fertilization treatments in switchgrass (SG: Panicum virgatum L.) and gamagrass (GG: Tripsacum dactyloides L.) using a spatially explicit design. Four glycosidases, α-glucosidase (AG), β-glucosidase (BG), β-xylosidase (BX), cellobiohydrolase (CBH), and their sum associated with C acquisition (Cacq) were quantified. The three fertilization treatments were no N input (NN), low N input (LN: 84 kg N ha−1 year−1 in urea) and high N input (HN: 168 kg N ha−1 year−1 in urea). The descriptive and geostatistical approaches were used to evaluate their central tendency and spatial heterogeneity. Results showed significant interactive effects of N fertilization and crop type on BX such that LN and HN significantly enhanced BX by 14% and 44% in SG, respectively. The significant effect of crop type was identified and glycosidase activities were 15–39% higher in GG than those in SG except AG. Within-plot variances of glycosidases appeared higher in SG than GG but little differed with N fertilization due to large plot-plot variation. Spatial patterns were generally more evident in LN or HN plots than NN plots for BG in SG and CBH in GG. This study suggested that N fertilization elevated central tendency and spatial heterogeneity of glycosidase activities in surficial soil horizons and these effects however varied with crop and enzyme types. Future studies need to focus on specific enzyme in certain bioenergy cropland soil when N fertilization effect is evaluated.

Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the Basidiomycete Phanerochaete chrysosporium.

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase Pc Cel6A from the fungus Phanerochaete chrysosporium , and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris . Comparison of the hydrolytic activities at 50 and 60 °C indicated that the thermal stability of Pc Cel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.

Insights into the cellulose degradation mechanism of the thermophilic fungus Chaetomium thermophilum based on integrated functional omics

BackgroundLignocellulose is the most abundant and renewable biomass resource on the planet. Lignocellulose can be converted into biofuels and high-value compounds; however, its recalcitrance makes its breakdown a challenge. Lytic polysaccharide monooxygenases (LPMOs) offer tremendous promise for the degradation of recalcitrant polysaccharides. Chaetomium thermophilum, having many LPMO-coding genes, is a dominant thermophilic fungus in cellulose-rich and self-heating habitats. This study explores the genome, secretomes and transcript levels of specific genes of C. thermophilum.ResultsThe genome of C. thermophilum encoded a comprehensive set of cellulose- and xylan-degrading enzymes, especially 18 AA9 LPMOs that belonged to different subfamilies. Extracellular secretomes showed that arabinose and microcrystalline cellulose (MCC) could specifically induce the secretion of carbohydrate-active enzymes (CAZymes), especially AA9 LPMOs, by C. thermophilum under different carbon sources. Temporal analyses of secretomes and transcripts revealed that arabinose induced the secretion of xylanases by C. thermophilum, which was obviously different from other common filamentous fungi. MCC could efficiently induce the specific secretion of LPMO2s, possibly because the insert in loop3 on the substrate-binding surface of LPMO2s strengthened its binding capacity to cellulose. LPMO2s, cellobio hydrolases (CBHs) and cellobiose dehydrogenases (CDHs) were cosecreted, forming an efficient cellulose degradation system of oxidases and hydrolases under thermophilic conditions.ConclusionsThe specific expression of LPMO2s and cosecretion of hydrolases and oxidases by the thermophilic fungus C. thermophilum play an important role in cellulose degradation. This insight increases our understanding of the cellulose degradation under thermophilic conditions and may inspire the design of the optimal enzyme cocktails for more efficient exploration of biomass resources in industrial applications.

Mini review: Advances in understanding regulation of cellulase enzyme in white-rot basidiomycetes

White-rot basidiomycetic fungi have gained a lot of scientific attention in recent years owing to their ability to produce cellulase enzymes that are of great importance in numerous industrial applications. This has seen a rise in number of studies seeking to comprehend both physical and molecular mechanisms that regulate the production of cellulase enzymes in these fungi. Cellulase has several applications in production of food and beverages, biofuel, biological detergents, pharmaceuticals, and deinking in paper and pulp industry. Enhanced understanding of genetic mechanisms that regulate cellulase production would have utility for optimal cellulase production in white-rot basidiomycetes using biotechnology approaches. Carbon catabolite repression and various transcriptional factors such as XlnR, Cre, Clr, Ace, and gna1 control expression of genes encoding cellobiohydrolase (CBH), endoglucanase (EGL) and β-glucosidase (BGL). In this review, we have consolidated and summarised some of recent findings on genetic regulation of cellulase with an aim of highlighting the general regulatory mechanisms that underlie cellulase expressions in white-rot fungi. This review further outlines some of important transcription factors that regulate cellulase genes, and key research gaps that may need to be addressed by future research.

The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials.

The ability of white-rot fungi to degrade polysaccharides in lignified plant cell walls makes them a suitable reservoir for CAZyme prospects. However, to date, CAZymes from these species are barely studied, which limits their use in the set of choices for biomass conversion in modern biorefineries. The current work joined secretome studies of two representative white-rot fungi, Phanerochaete chrysosporium and Trametes versicolor, with expression analysis of cellobiohydrolase (CBH) genes, and use of the secretomes to evaluate enzymatic conversion of simple and complex sugarcane-derived substrates. Avicel was used to induce secretion of high levels of CBHs in the extracellular medium. A total of 56 and 58 proteins were identified in cultures of P. chrysosporium and T. versicolor, respectively, with 78–86% of these proteins corresponding to plant cell wall degrading enzymes (cellulolytic, hemicellulolytic, pectinolytic, esterase, and auxiliary activity). CBHI predominated among the plant cell wall degrading enzymes, corresponding to 47 and 34% of the detected proteins in P. chrysosporium and T. versicolor, respectively, which confirms that Avicel is an efficient CBH inducer in white-rot fungi. The induction by Avicel of genes encoding CBHs (cel) was supported by high expression levels of cel7D and cel7C in P. chrysosporium and T. versicolor, respectively. Both white-rot fungi secretomes enabled hydrolysis experiments at 10 FPU/g substrate, despite the varied proportions of CBHs and other enzymes present in each case. When low recalcitrance sugarcane pith was used as a substrate, P. chrysosporium and T. versicolor secretomes performed similarly to Cellic® CTec2. However, the white-rot fungi secretomes were less efficient than Cellic® CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic® CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes highlighted T. versicolor enzymes for providing high glucan conversions, even at lower proportion of CBHs, probably because the other enzymes present in this secretome and CBHs lacking carbohydrate-binding modules compensate for problems associated with unproductive binding to lignin.

Activity of fungal \u03b2-glucosidases on cellulose

BackgroundFungal beta-glucosidases (BGs) from glucoside hydrolase family 3 (GH3) are industrially important enzymes, which convert cellooligosaccharides into glucose; the end product of the cellulolytic process. They are highly active against the β-1,4 glycosidic bond in soluble substrates but typically reported to be inactive against insoluble cellulose.ResultsWe studied the activity of four fungal GH3 BGs on cellulose and found significant activity. At low temperatures (10 ℃), we derived the approximate kinetic parameters kcat = 0.3 ± 0.1 s−1 and KM = 80 ± 30 g/l for a BG from Aspergillus fumigatus (AfBG) acting on Avicel. Interestingly, this maximal turnover is higher than reported values for typical cellobiohydrolases (CBH) at this temperature and comparable to those of endoglucanases (EG). The specificity constant of AfGB on Avicel was only moderately lowered compared to values for EGs and CBHs.ConclusionsOverall these observations suggest a significant promiscuous side activity of the investigated GH3 BGs on insoluble cellulose. This challenges the traditional definition of a BG and supports suggestions that functional classes of cellulolytic enzymes may represent a continuum of overlapping modes of action.

Rhizobioaugmentation of Casuarina glauca with N-fixing actinobacteria Frankia decreases enzymatic activities in wastewater irrigated soil: effects of Frankia on C. glauca growth.

The use of wastewater for irrigation in agroforestry is cost-effective for water management. It is well established that rhizospheric microorganisms such as N2-fixing bacteria are able to modulate rhizobioaugmention and to boost phyoremediation process. To date, no study has been conducted to evaluate biological effects of rhizobioaugmentation in Casuarina glauca trees induced by their symbiont N-fixing actinobacteria of the genus Frankia. The objective of the present study was to evaluate the main effects of rhizobioaugmentation on the biological activity in the C. glauca's rhizosphere and on C. glauca growth in soils irrigated with industrial wastewater. Two Frankia strains (BMG5.22 and BMG5.23) were used in a single or dual inoculations of C. glauca seedlings irrigated with industrial wastewater. Soil enzymes activity related to carbon, phosphorus, sulfur and nitrogen cycling were measured. Results revealed that the BMG5.22 Frankia strain increases significantly the size (dry weight) of C. glauca shoots and roots while dual inoculation increased significantly the root length. Surprisingly, β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), leucine aminopeptidase (LAP), and peroxidase (PER) activity in the rhizosphere decreased significantly in soils treated with the two strains of symbionts. This suggests no positive correlations between enzymatic activity and C. glauca growth.

Modification of a cellulase system by engineering Penicillium oxalicum to produce cellulose nanocrystal

In this study, three endoglucanases (EGs; Cel7B, Cel5B, and Cel12A), one cellobiohydrolase (CBH), and two auxiliary proteins (swollenin: SWO1 and SWO4) were used to hydrolyze microcrystalline cellulose (MCC) for cellulose nanocrystal (CNC) preparation. The mixture experiment of the three EGs showed that high CNC yield was obtained when the ratio of Cel7B and Cel5B is 1:1.11 (protein weight). Moreover, the addition of CBH (1 mg/g) and SWO1 from Trichoderma reesei effectively increased the yield of CNC. On the basis of the results, the cellulase-producing strain of Penicillium oxalicum M12 was engineered to improve its cellulase system. An engineered strain of cEES performed well in CNC preparation. CNC with a yield of 11.79 % and a crystallinity of 83.85 % were produced using the crude enzyme from cEES as a means to hydrolyze MCC, and the size and shape of CNC were uniform and fusiform.

Characterization of Cellobiohydrolases from Schizophyllum commune KMJ820.

A novel cellobiohydrolase (CBH)-generating fungi have been isolated and categorized as Schizophyllum commune KMJ820 based on morphology and rDNA gene sequence. Cellulose powder was used as carbon source, the total enzyme activity was 11.51U/ml is noted; which is among the highest amounts of CBH-generating microbes studied. CBH have been purified to homogenize, with pursual of serial chromatography using S. commune supernatants and two different CBHs were found; CBH 1 and 2. The filtered CBHs showed greater activity (V max = 51.4 and 20.8U/mg) in contrast to CBHs from earlier studies. The MW (molecular weights) of S. commune CBH 1 and 2 were verified to be approximately 50kDa and 150kDa, respectively, by size exclusion chromatography. Even though CBHs have been evaluated from other sources, but S. commune CBH is prominent in comparison to other CBHs by its high enzyme activity.

Influence of cellulase gene expression and cellulolytic activity on cellulose utilization in different Volvariella volvacea strains

Volvariella volvacea is delicious and nutritious while the biological efficiency is low, so the price keeps at a high level all the year round in China. V. volvacea uses crop straw enriched in cellulose as a substrate. Six cellulase genes (egI, egII, cbhI-I, cbhI-II, cbhII, bgl) encoding endoglucanase (EG) (E.C. 3.2.1.4), cellobiohydrolase (CBH) (E.C. 3.2.1.91), and β-glucosidase (BGL) (E.C. 3.2.1.21) proteins, all of which restrict the conversion of cellulose to glucose, were confirmed in genome. The investigate of enzymatic activity and biological efficiency showed better cellulose-degrading enzyme activity ensured higher biological efficiency. The cellulase genes cbhI-I, cbhI-II, and bgl clustered with those from Agaricus bisporus. Other genes, namely, egI, egII, and cbhII, clustered closely with those of wood decay fungi, which have weak cellulose-degrading activity. egI expression was significantly higher in strains 0229 and Vb1 (high biological efficiency) than in strain A8 (low biological efficiency). EG activity was positively correlated with biological efficiency in V. volvacea. EG、CBH and BGL are the three crucial endo and exo cellulytic enzymes involved in the conversion of cellulose to glucose. However, there was no relationship between CBH or BGL activity and biological efficiency. The egI gene is a key gene in the cellulase system. And there is a linear correlation between EG activity and egI expression in V. volvacea.

The dissociation mechanism of processive cellulases.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates

BackgroundCellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood.ResultsIn this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from Podospora anserina (PaLPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of PaLPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of PaLPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network.ConclusionsAbsence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and non-oxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels.

Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei

BackgroundFilamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production.ResultRecombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei.ConclusionBased on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.

Effects of forest conversion on carbon-degrading enzyme activities in subtropical China

The mineralization of soil organic carbon (SOC) is primarily mediated by carbon (C) degrading enzyme. In the current study, we determined how the activities of four soil C-degrading enzymes, the hydrolases β-glucosidase (BG) and cellobiohydrolase (CBH) and the oxidases polyphenol oxidase (PPO) and peroxidase (POD), responded to forest conversion of natural broadleaf forests (BF) to secondary forests (SF) and plantation forests (PF) in subtropical China. We also quantified SOC, dissolved organic C (DOC), permanganate oxidase organic C (PXC), recalcitrant C (RC), microbial biomass C (MBC), mineral-associated C (MOC), soil particle-sizes distribution, pH, and moisture content, and C: nitrogen (N) ratio. Results showed that, the activities of all four C-degrading enzymes (BG, CBH, PPO and POD) decreased by 23.1, 9.5, 6.9 and 1.8%, respectively by forest conversion of BF to SF and 30.5, 15.3, 28.1 and 27.8%, respectively by conversion of BF to PF and were higher in the topsoil than in the subsoil. Relative to SF and PF, BF had higher hydrolase activities, which were related to its higher concentrations of MBC, DOC, and PXC, and to its lower C:N ratio. The BF also had higher oxidase activities, which were related to its higher concentrations of MBC, RC, and MOC, and to its lower C:N ratio. PF had higher specific enzyme activities (i.e., enzyme activities per unit of SOC) than BF and SF, indicating faster C turnover rates in PF. In addition to being affected by the concentrations of SOC and SOC components, forest conversion-induced changes in soil enzyme activities were affected by clay content and soil moisture content. These results revealed the different underlying mechanisms between soil hydrolases and oxidases in their responses to forest conversion.

A lytic polysaccharide monooxygenase from Myceliophthora thermophila and its synergism with cellobiohydrolases in cellulose hydrolysis

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.

Phosphorus addition accelerates fine root decomposition by stimulating extracellular enzyme activity in a subtropical natural evergreen broad-leaved forest

Nitrogen (N) plays a vital role in litter decomposition by interacting with microbial activity in temperate and boreal ecosystems. However, in tropical and subtropical forests, where low soil phosphorus (P) availability is widespread, how P affects litter decomposition remains unclear. In the present study, litter-bag method was used to conduct fine root decomposition by P addition experiment in a subtropical natural evergreen broad-leaved forest. Fine roots were collected from two tree species: Cunninghamia lanceolata and Castanopsis carlesii, and their mixture in equal proportion as well. At the end of 2-year experiment, we found an enhancement of soil total P, available P, pH and microbial biomass carbon content, and increased abundance of all the examined microbial groups and total PLFA biomass after P addition. Annual decomposition constants of fine roots of both tree species and the mixture (0.615–1.049) were all elevated, but to different extents by P addition, with fine roots of lower initial P concentration corresponding to higher increase in decomposition rate. Average acid phosphatase activities during decomposition were significantly decreased, but there was a general increase for β-glucosidase (βG), cellobiohydrolase (CBH), phenol oxidase, peroxidase (PerOx) and N-acetyl glucosaminidase (NAG) activities. The response ratios of enzyme activities, which differed for the three experimental fine roots, corresponded to the individual increase in decomposition rates. Correlation analysis demonstrated that fine root decomposition rates were significantly related to initial P concentration, extractives and acid-insoluble fractions of fine roots, and activities of βG, CBH, NAG and PerOx as well. Taken together, our results demonstrate that fine root decomposition can be constrained by P availability through affecting microbial activity in this subtropical natural forest.

Using a linear pH-responsive zwitterionic copolymer to recover cellulases in enzymatic hydrolysate and to enhance the enzymatic hydrolysis of lignocellulose

A linear pH-responsive zwitterionic copolymer P(MDB) was synthesized from functional monomers methacrylic acid, 2-(dimethylamino) ethyl methacrylate (DMA) and benzyl methacrylate (BMA) through free radical polymerization to recover cellulases and enhance the enzymatic hydrolysis. The hydrophobicity and molecular weight of the copolymer was controlled by the feed ratio of monomers and the mass ratio of initiator to monomers, respectively. UV-turbidity showed P(MDB) exhibited sensitive pH-responsiveness with dissolution–precipitation ΔpH about 1 pH unit. The precipitation pHφ of P(MDB) was 5.0–5.5 and over 90% of P(MDB) could precipitate in the vicinity of its isoelectric point. Increasing the hydrophobicity or molecular weight of P(MDB) could improve its pH sensitivity and precipitation rate. P(MDB) could enhance the enzymatic hydrolysis of dilute acid pretreated Eucalyptus by 10–25%. P(MDB) could recover the free cellulases in enzymatic hydrolysate by co-precipitation. SDS-PAGE analysis showed that P(MDB) could recover 100% β-glucosidase (β-GL) and the recovery rates of cellobiohydrolase (CBH) and endoglucanase (EG) increased as the hydrophobicity and molecular weight of P(MDB) increased. The mechanism was proposed that hydrophobic affiliation was the main binding force of P(MDB) to cellulases. Increasing the hydrophobicity and molecular weight of copolymer could increase its binding force and the number of binding sites to cellulases thus the overall cellulase recovery performance was improved.

Effects of elevated atmospheric CO2 and nitrogen deposition on leaf litter and soil carbon degrading enzyme activities in a Cd-contaminated environment: A mesocosm study

Rising atmospheric CO2 concentration and nitrogen (N) deposition are changing terrestrial carbon (C) cycle; however, little has been known about such impacts in a heavy-metal-contaminated environment. This study conducted an open-top chamber experiment to explore the impacts of rising atmospheric CO2 concentration and N deposition on the leaf litter and soil C cycle in cadmium (Cd)-contaminated environment. The experiment include five treatments: control, Cd (30 g ha−1 yr−1) addition, Cd addition under elevated CO2 (700 ppm CO2), Cd and N(100 kg ha−1 yr−1) additions, and Cd and N additions under elevated CO2, with three replicates per treatment. Leaf litter and soil C cycle were indexed by microbial biomass C concentration and the activities of four key C-degrading enzyme (β-glucosidase (BG), cellobiohydrolase (CBH), polyphenol oxidase (PPO), and peroxidase (POD)) in litter and soil. Results showed that, after one year treatment, Cd addition negatively affected the activities of all four C-degrading enzyme in litter and soil; while elevated CO2 and N addition essentially alleviated these negative effects. Elevated CO2 and N addition increased C-degrading enzyme activities more of the non-legume (i.e., Cinnamomum camphora) litter than those of the legume (i.e., Acacia auriculiformis) litter. Elevated CO2, N addition, and Cd addition all affected C-degrading enzyme activities via their effects on the microbial biomass C concentration and C and N availability of the litter and soil samples. We suggest that rising atmospheric CO2 concentration and N deposition can offset the detrimental effect of Cd on the litter and soil C-degrading enzyme activities in forest ecosystems.

Effects of soil warming on soil microbial extracellular enzyme activities with different depths in a young Chinese fir (Cunninghamia lanceolata)plantation of subtropics.

Extracellular enzyme activitie (EEAs) are a sensitive indicator of microbial function and soil organic matter decomposition in response to climate warming. Up to now, most studies of climate warming and their effects on EEAs have been restricted on the relatively carbon rich topsoil (the upper 20 cm of the soil), whereas little is known about EEAs in subsoil (below 30 cm depth). This study focused on the responses of EEAs to soil warming in a subtropical forest at depths of 0-10 cm, 10-20 cm, 20-40 cm and 40-60 cm. The examined extracellular enzymes included β-glucosidase (BG), cellobiohydrolase (CBH), phenoloxidase (PHO) and peroxidase (PEO), all being involved in the C-cycle. The results showed that, 1) warming significantly increased all EEAs (18%-69%) at the depth of 0-10 cm and 10-20 cm. Below the depth of 20 cm, warming did no affect or suppressed EEAs (13%-31%), except increasing PHO (10%) at 20-40 cm. 2) Results from the redundancy analysis showed that the EEAs were mainly driven by ammonium nitrogen (NH4+-N) and soil moisture (M) in organic carbon rich topsoil. Warming enhanced nutrient competition between soil microorganisms and plants. Thus, it increased EEAs to meet NH4+-N demands of microorganisms. In subsoil with relatively low substrate availability, the EEAs were dominated by dissolved organic matter and microbial biomass (MBC). Warming increased dissolved organic matter and thus provided more substrates for microorganisms, which relieved the dependence of microbes on EEAs. Consequently, warming diminished EEAs in subsoils. Our results suggested that EEAs at the four depths showed different responses to warming. In addition, environmental factors accounting for the variances in EEAs under soil warming condition were different at topsoil and subsoil. Paying more attention to microbes at different soil depths has important implications to precisely predict ecosystem C cycling in response to global warming.

Acetone-butanol-ethanol solvents improved enzymatic hydrolysis of pretreated energy grass

Simultaneous saccharification and fermentation (SSF) is a promising process for conversion of lignocellulosic materials to biofuels. However, the fermentation end products might inhibit the saccharification of lignocellulosics by cellulose-degrading enzymes. This study investigated the effects of acetone-butanol-ethanol (ABE) fermentation products on enzymatic hydrolysis of energy grasses. After addition of 5 g/L butanol during the hydrolysis, the glucose yields of pretreated Hybrid Pennisetum and Avicel apparently increased from 62.8% and 33.4% to 77.5% and 63.8%, respectively. The acetone and ethanol also showed the promotional effect at higher concentrations (>10 g/L), but the acetic acid and butyric acid exhibited slight inhibition on the hydrolysis. The accumulative effect of these solvents was observed by addition of solvent mixtures. The ABE solvents improved the activity of cellobiohydrolase (CBH) I and endoglucanase (EG) II remaining in hydrolysis solution, particularly butanol increased the activity of EG II to 142.8%, which probably contributed to the enhanced hydrolysis. Thus, the inhibition of ABE end products on hydrolysis of lignocellulosic materials during SSF was negligible, but instead, the promotion on enzymatic hydrolysis provided an opportunity for effective utilization of enzymes.