Production of a halotolerant endo-1,4-ß-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-ß-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC-MS analysis showed 40% identity with an endo-1,3-1,4-ß-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley ß-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-ß-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65 °C. It was fairly thermotolerant, retaining 76.9% of the activity at 70 °C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a Vmax of 625 U/min/mL and a high affinity with barley ß-glucan resulting a Km of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-ß-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.

Exosomes in the phloem and xylem of woody plants.

MAIN CONCLUSION: Exosomes in the secondary phloem and secondary xylem of angiosperms and gymnosperms have physiological roles in the storage and transport of endoglucanases. Knowledge of plant extracellular vesicles (EVs) is limited by their presence in the apoplastic fluid of seeds and leaves. The contents of plant EVs and their biological functions are unclear. The aim of the present study was to expand our knowledge of EVs in woody plants. Sample splits were prepared from branch and stem samples from angiosperms and gymnosperms after cryomechanical destruction with liquid nitrogen. The study methods included scanning electron (SEM), atomic force microscopy (AFM), endoglucanase activity measurement. EVs visualized on the internal layers of the cell walls proved to be exosomes according to their diameter (65-145 nm). SEM revealed cup-shaped structures characteristic of exosomes in a dry state. Plant exosomes in the form of globules in the native state were visualized for the first time by AFM. Exosomes were present both in the active and dormant cambium. Erosion zones were observed at the sites of exosome localization. The activity of endo-1,4-ß-glucanase was detected in Picea xylem, while the RNA level was very low, suggesting that endo-1,4-ß-glucanases were preserved in the exosomes. There are grounds to assert that endo-1,4-ß-glucanases delivered by exosomes participated in pit cavity formation in the S1 layer of xylary fibres. A possible mechanism of endo-1,4-ß-glucanase action in the biosynthesis of the secondary wall is proposed. These results demonstrate that the physiological role of the exosomes in the phloem and xylem is the storage and transport of endo-1,4-ß-glucanases participating in cell wall remodeling in woody plants. Present study expands our knowledge about plant exosomes.

An auxin-induced ß-type endo-1,4-ß-glucanase in poplar is involved in cell expansion and lateral root formation.

MAIN CONCLUSION: PtrGH9A7, a poplar ß-type endo-1,4-ß-glucanase gene induced by auxin, promotes both plant growth and lateral root development by enhancing cell expansion. Endo-1,4-ß-glucanase (EGase) family genes function in multiple aspects of plant growth and development. Our previous study found that PtrCel9A6, a poplar EGase gene of the ß subfamily, is specifically expressed in xylem tissue and is involved in the cellulose biosynthesis required for secondary cell wall formation (Yu et al. in Mol Plant 6:1904-1917, 2013). To further explore the functions and regulatory mechanism of ß-subfamily EGases, we cloned and characterized another poplar ß-type EGase gene PtrGH9A7, a close homolog of PtrCel9A6. In contrast to PtrCel9A6, PtrGH9A7 is predominantly expressed in parenchyma tissues of the above-ground part; in roots, PtrGH9A7 expression is specifically restricted to lateral root primordia at all stages from initiation to emergence and is strongly induced by auxin application. Heterologous overexpression of PtrGH9A7 promotes plant growth by enhancing cell expansion, suggesting a conserved role for ß-type EGases in 1,4-ß-glucan chains remodeling, which is required for cell wall loosening. Moreover, the overexpression of PtrGH9A7 significantly increases lateral root number, which might result from improved lateral root primordium development due to enhanced cell expansion. Taken together, these results demonstrate that this ß-type EGase induced by auxin signaling has a novel role in promoting lateral root formation as well as in enhancing plant growth.

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.

A novel automatable enzyme-coupled colorimetric assay for endo-1,4-ß-glucanase (cellulase).

endo-1,4-ß-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-ß-D-cellopentaoside. Graphical Abstract Principle of the CELLG5 assay.

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-ß-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two ß-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-ß-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-ß-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Mutation in the Endo-ß-1,4-glucanase (KORRIGAN) Is Responsible for Thick Leaf Phenotype in Sorghum.

Sorghum [Sorghum bicolor (L.) Moench] is an important crop for food, feed, and fuel production. Particularly, sorghum is targeted for cellulosic ethanol production. Extraction of cellulose from cell walls is a key process in cellulosic ethanol production, and understanding the components involved in cellulose synthesis is important for both fundamental and applied research. Despite the significance in the biofuel industry, the genes involved in sorghum cell wall biosynthesis, modification, and degradation have not been characterized. In this study, we have identified and characterized three allelic thick leaf mutants (thl1, thl2, and thl3). Bulked Segregant Analysis sequencing (BSAseq) showed that the causal mutation for the thl phenotype is in endo-1,4-ß-glucanase gene (SbKOR1). Consistent with the causal gene function, the thl mutants showed decreased crystalline cellulose content in the stem tissues. The SbKOR1 function was characterized using Arabidopsis endo-1,4-ß-glucanase gene mutant (rsw2-1). Complementation of Arabidopsis with SbKOR1 (native Arabidopsis promoter and overexpression by 35S promoter) restored the radial swelling phenotype of rsw2-1 mutant, proving that SbKOR1 functions as endo-1,4-ß-glucanase. Overall, the present study has identified and characterized sorghum endo-1,4-ß-glucanase gene function, laying the foundation for future research on cell wall biosynthesis and engineering of sorghum for biofuel production.

Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-ß-Glucanase (CenC) from Ruminiclostridium thermocellum in a Mesophilic Host Through Various Cultivation and Induction Strategies.

Commonly, unintentional induction and inadvertently preparing medium for engineered Escherichia coli BL21 CodonPlus (DE3)-RIPL, give poor or variable yields of heterologous proteins. Therefore, to enhance the activity and production of an industrially relevant recombinant processive endo-1,4-ß-glucanase (CenC) propagated in Escherichia coli BL21 CodonPlus(DE3)-RIPL through various cultivation and induction strategies. Investigation of various growth media and induction parameters revealed that high-cell-density and optimal CenC expression were obtained in ZYBM9 medium induced either with 0.5 mM IPTG/150 mM lactose, after 6 h induction at 37 °C; and before induction, bacterial cells were given heat shock (42 °C) for 1 h when culture density (OD600nm) reached at 0.6. Intracellular enzyme activity was enhanced by 6.67 and 3.20-fold in ZYBM9 and 3×ZYBM9 medium, respectively, under optimal conditions. Using YNG auto-induction medium, activity was 2.5-fold increased after 10 h incubation at 37 °C. Approximately similar results were obtained by transferring the optimized process at the bioreactor level. Results showed that the effective process strategy is essential to enhance recombinant bacterial cell mass and enzyme production from small to large-scale. To the best of our knowledge, this is the first ever report on enhanced production of thermostable processive endo-1,4-ß-glucanase cloned from Ruminiclostridium thermocellum, which is a suitable candidate for industrial applications. Graphical Abstract Flow Chart Summary of Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-ß-glucanase from Ruminiclostridium thermocellum.

Purification, Characterization, and Gene Cloning of a Cold-Adapted Endo-1,4-ß-glucanase from Bellamya chinensis laeta.

An endo-1,4-ß-glucanase from Bellamya chinensis laeta was purified to electrophoretically homogeneous state. The molecular weight of the purified enzyme was estimated 70,000 by SDS-PAGE. The enzyme was most active at pH 5.5 and 50 °C, and stable at around pH 10 and 50 °C. The enzyme exhibited the significant activity at 20 °C (30 % of the activity at optimal 50 °C). The enzyme was hydrolyzed cellohexaose into cellobiose, cellotriose, and cellotetraose as main products. Three cDNAs (BC-EG70a, BC-EG70b, and BC-EG70c) encoding the endo-1,4-ß-glucanase were cloned by PCR-based method. Three endo-1,4-ß-glucanases consisted of 1758 bp encoding 586 amino acids. The three genes were almost the same nucleotide sequences. The deduced proteins were consisted of a signal sequence, cellulose binding domain, linker, and catalytic domain. The amino acid sequence of BC-EG70a shares sequence identity degree with the endo-1,4-ß-glucanases of Haliotis discus hannai (61 %), Ampullaria crossean (52 %), and Mizuhopecten yessoensis (51 %) which all belong to glycoside hydrolase family 9.

Expression of Gene Encoding Endo-1,4-ß-glucanase in Suspension-cultured Poplar (Poplus alba L.) Cells†.

The level of mRNA for endo-1,4-ß-glucanase was increased before the exponential phase of growth and decreased rapidly during the exponential phase in suspension-cultured poplar cells. The level of mRNA was increased for a short period after the addition of either 2,4-D or sucrose to the culture medium at the stationary phase. The level was increased to the maximal level when both 2,4-D and sucrose were provided together, but one did not increase the effect of the other. These findings suggest that expression of gene encoding poplar endo-1,4-ß-glucanase is controlled during cell growth by independent systems activated by auxin and sucrose.

Quantitative fluorometric assay for the measurement of endo-1,4-ß-glucanase.

There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-ß-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-ß-cellotrioside (BzMUG3) in the presence of an ancillary ß-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics.

Novel substrates for the measurement of endo-1,4-ß-glucanase (endo-cellulase).

A specific and sensitive substrate for the assay of endo-1,4-ß-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-ß-cellotrioside (BzCNPG3) in the presence of thermostable ß-glucosidase. Hydrolysis by exo-acting enzymes such as ß-glucosidase and exo-ß-glucanase is prevented by the presence of the benzylidene group on the non-reducing end d-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-ß-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free d-glucose by the ß-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-ß-glucanase in general.

Cloning and expression of the cold-adapted endo-1,4-ß-glucanase gene from Eisenia fetida.

Biofuel production from plant-derived lignocellulosic material using fungal cellulases is facing cost-effective challenges related to high temperature requirements. The present study identified a cold-adapted cellulase named endo-1,4-ß-glucanase (EF-EG2) from the earthworm Eisenia fetida. The gene was cloned in the cold-shock expression vector (pCold I) and functionally expressed in Escherichia coli ArcticExpress RT (DE3). The gene consists of 1,368 bp encoding 456 amino acid residues. The amino acid sequence shares sequence homology with the endo-1,4-ß-glucanases of Eisenia andrei (98%), Pheretima hilgendorfi (79%), Perineresis brevicirris (63%), and Strongylocentrotus nudus (58%), which all belong to glycoside hydrolase family 9. Purified recombinant EF-EG2 hydrolyzed soluble cellulose (carboxymethyl cellulose), but not insoluble (powdered cellulose) or crystalline (Avicel) cellulose substrates. Thin-layer chromatography analysis of the reaction products from 1,4-ß-linked oligosaccharides of various lengths revealed a cleavage mechanism consistent with endoglucanases (not exoglucanases). The enzyme exhibited significant activity at 10°C (38% of the activity at optimal 40°C) and was stable at pH 5.0-9.0, with an optimum pH of 5.5. This new cold-adapted cellulase could potentially improve the cost effectiveness of biofuel production.

PtrCel9A6, an endo-1,4-ß-glucanase, is required for cell wall formation during xylem differentiation in populus.

Endo-1,4-ß-glucanases (EGases) are involved in many aspects of plant growth. Our previous study found that an EGase, PtrCel9A6, is specifically expressed in differentiating xylem cells during Populus secondary growth. In this study, the xylem-specific PtrCel9A6 was characterized for its role in xylem differentiation. The EGase is localized on the plasma membrane with catalytic domain toward the outside cell wall, hydrolyzing amorphous cellulose. Suppression of PtrCel9A6 expression caused secondary cell wall defects in xylem cells and significant cellulose reduction in Populus. Heterologous expression of PtrCel9A6 in Arabidopsis enhanced plant growth as well as increased fiber cell length. In addition, introduction of PtrCel9A6 into Arabidopsis resulted in male sterility due to defects in anther dehiscence. Together, these results demonstrate that PtrCel9A6 plays a critical role in remodeling the 1,4-ß-glucan chains in the wall matrix and is required for cell wall thickening during Populus xylem differentiation.

Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis.

Endo-1,4-ß-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.

Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit.

To investigate the role of jasmonates (JAs) in the ripening of Fragaria chiloensis fruit, two concentrations of methyl jasmonate (MeJA, 10 and 100 µM) were evaluated at 2, 5 and 9 d using an in vitro ripening system. Fruit quality parameters; the contents of anthocyanin, lignin and cell wall polymers; and the transcriptional profiles of several ripening-related genes were analyzed. MeJA accelerated fruit ripening by means of a transitory increase in the soluble solid content/titratable acidity ratio, anthocyanin accumulation and an increase in softening at day 5. The expression of several phenylpropanoid-related genes, primarily those associated with anthocyanin biosynthesis, was increased under MeJA treatment, which correlated with an increased accumulation of anthocyanin. MeJA also altered the expression profiles of some cell wall-modifying genes, namely, EG1 and XTH1, and these changes correlated with a transient reduction in the firmness of MeJA-treated fruits. MeJA-responsive elements were observed in the promoter region of the EG1 gene. MeJA also increased the expression of LOX, AOS and OPR3, genes involved in the biosynthesis of JAs, and these changes correlated with the transient activation of fruit ripening observed. Conversely, the expression of ethylene and lignin biosynthesis genes (ACS, ACO, CAD and POD27) increased in MeJA-treated fruits at day 9. The present findings suggest that JAs promote the ripening of non-climacteric fruits through their involvement in anthocyanin accumulation, cell wall modification and the biosynthesis of ethylene and JAs.