Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

MaEng1, an endo-1,3-glucanase, contributes to the conidiation pattern shift through changing the cell wall structure in Metarhizium acridum.

Microcycle conidiation has displayed the greater potential than normal conidiation in large-scale production of mycopesticides. Fungi require partial hydrolysis of the cell wall to achieve the necessary plasticity during their morphological changes. Therefore, various cell wall-associated hydrolases are crucial for fungal morphogenesis. Eng1, as an endo-ß-1,3-glucanase, is involved in the cell separation of fungi, but its role in morphological changes of entomopathogenic fungi is not yet clear. Here, the endo-ß-1,3-glucanase gene MaEng1 was characterized in the model entomopathogenic fungi M. acridum. MaEng1 possesses a typical carbohydrate hydrolase domain and belongs to the GH81 family. The functions of MaEng1 in fungal growth, stress tolerance, pathogenicity, and conidiation capacity were analyzed using targeted gene disruption. The results displayed that the absence of MaEng1 does not affect the fungal growth, stress tolerances, and pathogenicity in M. acridum. However, the knockout of MaEng1 led to the normal conidiation of M. acridum on the SYA medium, which can induce the microcycle conidiation. Moreover, the content of ß-1,3-glucan in the cell wall of the MaEng1-disruption strain were significantly reduced and the exposures of ß-1,3-glucan on the surface of the mature conidia and mycelia in ΔMaEng1 were declined, indicating that MaEng1 contributes to the conversion of conidiation mode in M. acridum by affecting the cell wall structure.

Multi-stage glucose/pachymaran co-feeding enhanced endo-ß-1,3-glucanase production by Trichoderma harzianum via simultaneous increases in cell concentration and inductive effect.

Endo-ß-1,3-glucanase is used to hydrolyze curdlan in a wide range of oligosaccharides production processes. Using pachymaran as the sole carbon source resulted in an endo-ß-1,3-glucanase activity of 86.1 U/mL and an Eendo/Etotal ratio of 0.43, which were 3.2 and 1.65 folds of the values from control (glucose as the sole carbon source), due to the inductive effect of pachymaran as a polysaccharide. However, the cell concentration decreased from 25 to 12 g/L during the late fermentation phase. Therefore, a novel multi-stage feeding strategy was developed wherein glucose was fed twice during the cell logarithmic growth phase (24 and 48 h) and pachymaran once during the early stage of the enzyme accumulation phase (72 h). Consequently, the cell concentration remained around 30 g/L during the late fermentation phase. Endo-ß-1,3-glucanase activity and Eendo/Etotal reached 160.0 U/mL and 0.76, respectively, which were 6.0 and 2.92 folds of the values from control. In addition, three typical polysaccharides with ß-1,3-linked glucose residues were successfully hydrolyzed by endo-ß-1,3-glucanase to produce multifunctional ß-1,3-oligoglucosides.

Pneumocystis encodes a functional endo-ß-1,3-glucanase that is expressed exclusively in cysts.

ß-1,3-glucan is a major cell wall component of Pneumocystis cysts. We have characterized endo-ß-1,3-glucanase (Eng) from 3 species of Pneumocystis. The gene eng is a single-copy gene that encodes a protein containing 786 amino acids in P. carinii and P. murina, and 788 amino acids in P. jirovecii, including a signal peptide for the former 2 but not the latter. Recombinant Eng expressed in Escherichia coli was able to solubilize the major surface glycoprotein of Pneumocystis, thus potentially facilitating switching of the expressed major surface glycoprotein (Msg) variant. Confocal immunofluorescence analysis of P. murina-infected mouse lung sections localized Eng exclusively to the cyst form of Pneumocystis. No Eng was detected after mice were treated with caspofungin, a ß-1,3-glucan synthase inhibitor that is known to reduce the number of cysts. Thus, Eng is a cyst-specific protein that may play a role in Msg variant expression in Pneumocystis.

Promoting substrates uptake and curdlan synthesis of Agrobacterium sp. by attenuating the exopolysaccharide encapsulation.

During curdlan production by Agrobacterium sp., the secreted exopolysaccharide (EPS) gradually encapsulated Agrobacterium sp., accompanied by cell aggregation, resulted in inhibited substrate uptake and curdlan synthesis. To relieve the EPS encapsulation effect, the shake-flask culture medium was quantitatively supplemented with 2 % to 10 % endo-ß-1,3-glucanase (BGN), while obtaining curdlan with a decreased weight-average molecular weight ranging from 18.99 × 104 Da to 3.20 × 104 Da. In a 7-L bioreactor, the 4 % BGN supplement substantially attenuated the EPS encapsulation, resulting in increased glucose consumption and curdlan yield to 66.41 g/L and 34.53 g/L after fermentation of 108 h, which improved 43 % and 67 %, respectively compared with the control. The disruption of EPS encapsulation with BGN treatment accelerated the regeneration of ATP and UTP, resulting in sufficient uridine diphosphate glucose for curdlan synthesis. The upregulation of related genes at the transcription level reveals that the respiratory metabolic intensity, the energy regeneration efficiency, and the curdlan synthetase activity were enhanced. This study presents a simple and novel strategy of relieving the effects of EPS encapsulation on the metabolism of Agrobacterium sp. for the high-yield and value-added production of curdlan, which could be potentially applied in producing other EPSs.

A First Expression, Purification and Characterization of Endo-ß-1,3-Glucanase from Penicillium expansum.

ß-1,3-glucanase plays an important role in the biodegradation, reconstruction, and development of ß-1,3-glucan. An endo-ß-1,3-glucanase which was encoded by PeBgl1 was expressed, purified and characterized from Penicillium expansum for the first time. The PeBgl1 gene was amplified and transformed into the competent cells of E. coli Rosetta strain with the help of the pET-30a cloning vector. The recombinant protein PeBgl1 was expressed successfully at the induction conditions of 0.8 mmol/L IPTG at 16 °C for 16 h and then was purified by nickel ion affinity chromatography. The optimum reaction temperature of PeBgl1 was 55 °C and it had maximal activity at pH 6.0 according to the enzymatic analysis. Na2HPO4-NaH2PO4 buffer (pH 6.0) and NaCl have inhibitory and enhancing effects on the enzyme activities, respectively. SDS, TritonX-100 and some metal ions (Mg2+, Ca2+, Ba2+, Cu2+, and Zn2+) have an inhibitory effect on the enzyme activity. The results showed that PeBgl1 protein has good enzyme activity at 50-60 °C and at pH 5.0-9.0, and it is not a metal dependent enzyme, which makes it robust for storage and transportation, ultimately holding great promise in green biotechnology and biorefining.

One-step production of functional branched oligoglucosides with coupled fermentation of Pichia pastoris GS115 and Sclerotium rolfsii WSH-G01.

Endo-ß-1,3-glucanase with high specific activity is a prerequisite for enzymatic preparation of valuable ß-oligoglucosides. Heterologous expression in Pichia pastoris GS115 with error-prone PCR technology was implemented, and the mutant strain 7 N12 was obtained. The mutant endo-ß-1,3-glucanase showed efficient specific activities for degrading curdlan (366 U mg-1) and scleroglucan (274.5 U mg-1). Thereafter, one-step production of functional branched oligoglucosides was established with coupled fermentation of Pichia pastoris and Sclerotium rolfsii. During the fermentation process, the endo-ß-1,3-glucanase secreted by Pichia pastoris GS115 can efficiently hydrolyse scleroglucan metabolized by Sclerotium rolfsii WSH-G01. The maximum yields of ß-oligoglucosides in the shake flasks and 7-L bioreactor reached 1.73 g L-1 and 12.71 g L-1, respectively, with polymerization degrees of 2-17. The successful implementation of heterologous expression with error-prone PCR and the coupled fermentation simplified the multi-step enzymatic ß-oligoglucoside preparation procedures, which makes it a potential strategy for industrial production of functional oligosaccharides.

Efficient endo-ß-1,3-glucanase expression in Pichia pastoris for co-culture with Agrobacterium sp. for direct curdlan oligosaccharide production.

The production of curdlan oligosaccharides, a multifunctional and valuable carbohydrate, by hydrolyzing polysaccharides is of great interest. The endo-ß-1,3-glucanase derived from Trichoderma harzianum was expressed in Pichia pastoris with three commonly used promoters (AOX1, GAP and FLD1). The purified recombinant endo-ß-1,3-glucanase expressed by Pichia pastoris with GAP promoter displayed high specific activity at pH 5.5 and 50 °C. Thereafter, a co-culture system of Pichia pastoris GS115 (GAP promoter) and Agrobacterium sp. was constructed in which Agrobacterium sp.-metabolized curdlan can be directly hydrolyzed by Pichia pastoris-secreted endo-ß-1,3-glucanase to produce functional curdlan oligosaccharides. The co-culture conditions were optimized and the process was carried out in a 7-L bioreactor. The maximum yield of curdlan oligosaccharides reached 18.77 g/L with 3-10 degrees of polymerization. This study presents a novel and easy curdlan oligosaccharide production strategy that can replace traditional sophisticated production procedures and could potentially be implemented for production of other oligosaccharides.

Novel Anti-Fungal d-Laminaripentaose-Releasing Endo-ß-1,3-glucanase with a RICIN-like Domain from Cellulosimicrobium funkei HY-13.

Endo-ß-1,3-glucanase plays an essential role in the deconstruction of ß-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-ß-1,3-glucanase (GluY) with a ricin-type ß-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei HY-13 was identified and biocatalytically characterized. The recombinant enzyme (rGluY: 57.5 kDa) displayed the highest degradation activity for laminarin at pH 4.5 and 40 °C, while the polysaccharide was maximally decomposed by its C-terminal truncated mutant enzyme (rGluYΔRICIN: 42.0 kDa) at pH 5.5 and 45 °C. The specific activity (26.0 U/mg) of rGluY for laminarin was 2.6-fold higher than that (9.8 U/mg) of rGluYΔRICIN for the same polysaccharide. Moreover, deleting the C-terminal RICIN domain in the intact enzyme caused a significant decrease (>60%) of its ability to degrade ß-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of ß-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against Candida albicans in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of C. albicans by rGluY (approximately 1.8 µM) was approximately 80% of the fungal growth. The superior anti-fungal activity of rGluY suggests that it can potentially be exploited as a supplementary agent in the food and pharmaceutical industries.

Specific hydrolysis of curdlan with a novel glycoside hydrolase family 128 ß-1,3-endoglucanase containing a carbohydrate-binding module.

The cbm6e gene from Saccharophagus degradans 2-40 T was cloned and expressed in Escherichia coli. CBM6E contains a glycoside hydrolase family 128 (GH128) catalytic module and a C-terminal carbohydrate-binding module (CBM) grouped into CBM family 6. The purified recombinant CBM6E displayed high substrate specificity toward curdlan as an endo-ß-1,3-glucanase and had maximal activity at pH 6.0 and 35 ℃. The hydrolytic products against curdlan were predominantly laminaritriose (L3) and laminaritetraose (L4) along with a lower amount of laminaripentaose (L5) and laminarihexaose (L6). The CBM6 module selectively enhanced the enzyme activity against curdlan and displayed strict binding specificity to curdlan, no matter in its powder or high-set gel forms. This study laid a foundation for enzymatic degradation of curdlan to produce high-value ß-1,3-glucooligosaccharides at moderate temperatures and provided a novel CBM tag for enzyme immobilization on curdlan.

Comparative study of ß-glucan-degrading enzymes from Coprinopsis cinerea for their capacities to induce stipe cell wall extension.

We previously reported endo-ß-1,3-glucanase ENG in combination with ß-glucosidase BGL2 at low concentration induced stipe cell wall extension. This study further explored ENG could be replaced by endo-ß-1,3(4)-glucanase ENG16A in combination with BGL2 to induce stipe cell wall extension; similarly, BGL2 could be replaced by ß-glucosidase BGL1 to cooperate with ENG to induce stipe cell wall extension. However, ENG could not be replaced by exo-ß-1,3-glucanase EXG in combination with BGL2 to induce stipe cell wall extension, although EXG alone released higher level of soluble sugars from the stipe cell walls during the reconstituted wall extension than that released from the stipe cell walls by a combination of ENG16A or ENG and BGL2 or BGL1, which was different from chitinase-mediated stipe cell wall extension. These results indicate endo-ß-1,3-glucanases loosen the stipe cell wall, whereas exo-ß-1,3-glucanases and ß-glucosidases play a synergistic role to maintain a low and efficient concentration of endo-ß-1,3-glucanases for stipe cell wall extension. Furthermore, ENG was expressed at a very high level in the matured pilei, in contrast, ENG16A was expressed at a very high level in the elongating apical stipe. Therefore, ENG16A might be involved in stipe elongation growth, while ENG might participate in autolysis of pilei.

Purification and characterization of a novel ß-1,3-glucanase from Arca inflata and its immune-enhancing effects.

A novel ß-1,3-glucanase from Arca inflata was purified using chromatography methods. It was determined as a glycoprotein comprising 23.65% carbohydrate content with O-linked glycan and showed specific activity of 90.01 ±â€¯1.2 U/mg against laminarin. The optimal pH and temperature for the activity of the glucanase were 6.0 and 40 °C, respectively. The affinity parameter of the glucanase using laminarin was determined as Kd = 13.09 µM. The activity of the glucanase was 27 ±â€¯2.6% enhanced by 2-mM Mn2+ ions and inhibited by 40-50% using 2-mM Zn2+, Cu2+, or Ba2+ ions. The glucanase showed an endo-type cleavage mode and hydrolyzed laminarin into glucoses, disaccharides, trioligosaccharides, and tetraoligosaccharides. Otherwise, the glucanase exhibited immune-enhancing effects via significantly increasing the phagocytic activity of macrophages and inducing the release of nitric oxide, tumor necrosis factor α, and interleukin-6 in RAW264.7 cells. It might be used as a bifunctional additive for the food industry.

Embedding inulin fructotransferase from Arthrobacter aurescens into novel curdlan-based mesoporous silica microspheres for efficient production of Difructose Anhydride III.

A novel strategy was used to produce inulin fructotransferase from Arthrobacter aurescens (Aa-IFTase) embedded in curdlan-based mesoporous silica microspheres (CMSiM-Aa-IFTase). The CMSiM-Aa-IFTase was constructed by co-entrapping cross-linked Aa-IFTase aggregates and curdlan into biomemitic silica, and the curdlan was subsequently removed by digestion with endo-ß-1,3-glucanase. During this process, the curdlan served as an agent to introduce pores in silica microspheres. The resulting CMSiM-Aa-IFTase showed higher stability and activity than free Aa-IFTase and mCLEAs-Aa-IFTase (modified cross-linked enzyme aggregates with Aa-IFTase). Furthermore, the CMSiM-Aa-IFTase displayed good reusability and excellent storage stability. The excellent catalytic performances were due to the combinational structure from the cross-linked enzyme aggregates and hard shell of mesoporous silica microspheres, which might decrease the negative interaction between support and enzyme, and improve the mechanical properties. The CMSiM-Aa-IFTase was applicable for efficient production of Difructose Anhydride III (DFA III), and this approach should be highly valuable for preparing various mesoporous composites for catalysis.

Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

Identification of a Vitis vinifera endo-ß-1,3-glucanase with antimicrobial activity against Plasmopara viticola.

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-ß-1,3-glucanases (EGases) belong to the PR-2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis.

Molecular cloning and functional characterization of an endo-ß-1,3-glucanase from Streptomyces matensis ATCC 23935.

A gene encoding an endo-ß-1,3-glucanase from Streptomyces matensis ATCC 23935 (SmßG) was characterised by cloning and expressing it in Escherichiacoli. The purified enzyme produced ß-1,3-glucooligosaccharides, mainly laminaripentaose, from insoluble curdlan. The optimum pH and temperature were 6.0 and 55 °C, respectively. SmßG inhibited the growth of Candida albicans, which indicates that this enzyme could be potentially used as an anti-fungal agent to control invasive Candida infections. The results suggest that SmßG may be a useful bioavailable ß-1,3-glucanase.

ß-1,3-glucan modifying enzymes in Aspergillus fumigatus.

In Aspergillus fumigatus like in other filamentous ascomycetes, ß-1,3-glucan constitutes a prominent cell wall component being responsible for rigidity of the cell wall structure. In filamentous fungi, softening of the cell wall is absolutely required during conidial germination and hyphal branching. Because of the central structure of ß-1,3-glucans, it is expected that ß-1,3-glucanases play a major role in cell wall softening. Based on in silico and experimental data, this review gives an overview of ß-1,3-glucan modifying enzymes in A. fumigatus genome and their putative role during morphogenesis.