Targeting deoxynivalenol for degradation by a chimeric manganese peroxidase/glutathione system.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

Identification of WxL and S-Layer Proteins from Lactobacillus brevis with the Ability to Bind Cellulose and Xylan.

Xylanase releases xylo-oligosaccharides from dietary xylan, which stimulate the growth of the gut bacteria lactobacilli. Many lactobacilli adhere to dietary fibers, which may facilitate the assimilation of xylo-oligosaccharides and help them gain competence in the gut, but the underlying mechanisms remain elusive. Herein we report, from the highly abundant transcripts of Lactobacillus brevis cultured in wheat arabinoxylan supplemented with a xylanase, the identification of genes encoding four putative cell-surface WxL proteins (Lb630, Lb631, Lb632, and Lb635) and one S-layer protein (Lb1325) with either cellulose- or xylan-binding ability. The repetitively occurring WxL proteins were encoded by a gene cluster, among which Lb630 was chosen for further mutational studies. The analysis revealed three aromatic residues (F30, W61, and W156) that might be involved in the interaction of the protein with cellulose. A homology search in the genome of Enterococcus faecium identified three WxL proteins with conserved counterparts of these three aromatic residues, and they were also found to be able to bind cellulose and xylan. The findings suggested a role of the cell-surface WxL and S-layer proteins in assisting the cellular adhesion of L. brevis to plant cell wall polysaccharides.

A GH51 α-L-arabinofuranosidase from Talaromyces leycettanus strain JCM12802 that selectively drives synergistic lignocellulose hydrolysis.

BACKGROUND: The development of sustainable technologies for plant cell wall degradation greatly depends on enzymes with hydrolytic activities against carbohydrates. The waste by-products of agricultural cereals are important biomass sources because they contain large amounts of saccharides. Achieving efficient debranching and depolymerization are two important objectives for increasing the utilization of such renewable bioresources. GH51 α-L-arabinofuranosidases are important in biomass pretreatment because they act synergistically with other enzymes during hemicellulose hydrolysis. RESULTS: A GH51 α-L-arabinofuranosidase from Talaromyces leycettanus JCM12802 was heterologously expressed in Pichia pastoris GS115 and characterized. The recombinant α-L-arabinofuranosidase, TlAbf51, showed an optimum temperature and pH of 55-60 °C and 3.5-4.0, respectively, and remained stable at 50 °C and pH 3.0-9.0. TlAbf51 showed a higher catalytic efficiency (5712 mM-1 s-1) than most fungal α-L-arabinofuranosidases towards the substrate 4-nitrophenyl-α-L-arabinofuranoside. Moreover, TlAbf51 preferentially removed 1,2- or 1,3-linked arabinofuranose residues from arabinoxylan and acted synergistically with the bifunctional xylanase/cellulase TcXyn10A at an activity ratio of 5:1. The highest yields of arabinose and xylooligosaccharides were obtained when TlAbf51 was added after TcXyn10A or when both enzymes were added simultaneously. High-performance anion-exchange chromatography analyses showed that (i) arabinose and xylooligosaccharides with low degrees of polymerization (DP1-DP5) and (ii) arabinose and xylooligosaccharides (DP1-DP3) were the major hydrolysates obtained during the hydrolysis of sodium hydroxide-pretreated cornstalk and corn bran, respectively. CONCLUSIONS: In contrast to other fungal GH51 α-L-arabinofuranosidases, recombinant TlAbf51 showed excellent stability over a broad pH range and high catalytic efficiency. Moreover, TlAbf51 acted synergistically with another hemicellulase to digest arabino-polysaccharides. These favorable enzymatic properties make TlAbf51 attractive for biomass pretreatment and biofuel production.

Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose.

Bifunctional glycoside hydrolases have potential for cost-savings in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme CbXyn10C/Cel48B from Caldicellulosiruptor bescii is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated. Herein, we discovered that the binding cleft of CbXyn10C would have at least six sugar-binding subsites by using isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo- and cello-oligosaccharides. This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides. The free form and complex structures of CbXyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. CbXyn10C was found to have a typical (ß/α)8-TIM barrel fold and "salad-bowl" shape of GH10 enzymes. In complex structures with xylo-oligosaccharides, seven sugar-binding subsites were found, and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that 6 and 10 amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on subsites -2 and -1 near the cleavage site, whereas residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues interacting with substrates in the distal regions directly or indirectly improved the activity of CbXyn10C on xylan and cellulose. Most of the key residues for cellulase activity are conserved across GH10 xylanases. Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity, indicating that the dual function may be a more common phenomenon than has been expected.

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency.

Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (kcat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

Revisiting overexpression of a heterologous ß-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

BACKGROUND: The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in ß-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous ß-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. RESULTS: The thermophilic ß-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed ß-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different ß-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a ß-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. CONCLUSIONS: We report herein the successful overexpression of a thermophilic N. fischeri ß-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved ß-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/ß-Glucanase That Can Degrade Crystalline Cellulose.

The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley ß-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/ß-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages.

Effectiveness of ruminal xylanase with an extra proline-rich C-terminus on lignocellulosic biomass degradation.

The efficient degradation of plant polysaccharides in agricultural waste requires xylanases with high catalytic activity. In this study, the C-terminal proline-rich GH10 xylanase XynA from sheep rumen was investigated using product analysis, structural characterization, truncated and site-directed mutagenesis, molecular dynamics simulation, and application evaluation, revealing that the proline-rich C-terminus contributes to the interaction at the substrate-binding pocket to reduce the binding free energy. Compared to the C-terminally truncated enzyme XynA-Tr, XynA has a more favorable conformation for proton transfer and affinity attack, facilitating the degradation of oligomeric and beechwood xylan without altering the hydrolysis pattern. Moreover, both the reduced sugar yield and weight loss of the pretreated wheat bran, corn cob, and corn stalk hydrolyzed by XynA for 12 h increased by more than 30 %. These findings are important to better understand the relationship between enzyme activities and their terminal regions and suggest candidate materials for lignocellulosic biomass utilization.

Boosting enzymatic degradation of cellulose using a fungal expansin: Structural insight into the pretreatment mechanism.

The recalcitrance of cellulosic biomass greatly hinders its enzymatic degradation. Expansins induce cell wall loosening and promote efficient cellulose utilization; however, the molecular mechanism underlying their action is not well understood. In this study, TlEXLX1, a fungal expansin from Talaromyces leycettanus JCM12802, was characterized in terms of phylogeny, synergy, structure, and mechanism of action. TlEXLX1 displayed varying degrees of synergism with commercial cellulase in the pretreatment of corn straw and filter paper. TlEXLX1 binds to cellulose via domain 2, mediated by CH-π interactions with residues Tyr291, Trp292, and Tyr327. Residues Asp237, Glu238, and Asp248 in domain 1 form hydrogen bonds with glucose units and break the inherent hydrogen bonding within the cellulose matrix. This study identified the expansin amino acid residues crucial for cellulose binding, and elucidated the structure and function of expansins in cell wall networks; this has potential applications in biomass utilization.

Deciphering the efficient cellulose degradation by the thermophilic fungus Myceliophthora thermophila focused on the synergistic action of glycoside hydrolases and lytic polysaccharide monooxygenases.

The thermophilic fungus Myceliophthora thermophila as an efficient decomposer secretes various glycoside hydrolases and auxiliary oxidation enzymes to deconstruct cellulose. However, the core enzymes critical for efficient cellulose degradation and their interactions with other cellulolytic enzymes remain unclear. Herein, the transcriptomic analysis of M. thermophila grown on Avicel exhibited that cellulases from GH5_5, GH6 and GH7, and lytic polysaccharide monooxygenases (LPMOs) from AA9 contributed to cellulose degradation. Moreover, the peptide mass fingerprinting analysis of major extracellular proteins and corresponding gene-knockout strains studies revealed that MtCel7A and MtCel5A were the core cellulolytic enzymes. Furthermore, synergistic experiments found that hydrolytic efficiencies of MtCel7A and MtCel5A were both improved by mixture C1/C4 oxidizing MtLPMO9H, but inhibited by C1 oxidizing MtLPMO9E and C4 oxidizing MtLPMO9J respectively. These results demonstrated the potential application of C1/C4 oxidizing LPMOs for future designing novel cellulolytic enzyme cocktails on the efficient conversion of cellulose into biofuels and biochemicals.

Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis.

Talaromyces leycettanus JCM12802 is a great producer of thermophilic glycoside hydrolases (GHs). In this study, two cellulases (TlCel5A and TlCel6A) belonging to GH5 and GH6 respectively were expressed in Pichia pastoris and functionally characterized. The enzymes had acidic and thermophilic properties, showing optimal activities at pH 3.5-4.5 and 75-80°C, and retained stable at temperatures up to 60°C and over a broad pH range of 2.0-8.0. TlCel5A and TlCel6A acted against several cellulose substrates with varied activities (3,101.1 vs. 92.9 U/mg to barley ß-glucan, 3,905.6 U/mg vs. 109.0 U/mg to lichenan, and 840.3 and 0.09 U/mg to CMC-Na). When using Avicel, phosphoric acid swollen cellulose (PASC) or steam-exploded corn straw (SECS) as the substrate, combination of TlCel5A and TlCel6A showed significant synergistic action, releasing more reduced sugars (1.08-2.87 mM) than the individual enzymes. These two cellulases may represent potential enzyme additives for the efficient biomass conversion and bioethanol production.

Efficient Coproduction of Mannanase and Cellulase by the Transformation of a Codon-Optimized Endomannanase Gene from Aspergillus niger into Trichoderma reesei.

Cellulase and mannanase are both important enzyme additives in animal feeds. Expressing the two enzymes simultaneously within one microbial host could potentially lead to cost reductions in the feeding of animals. For this purpose, we codon-optimized the Aspergillus niger Man5A gene to the codon-usage bias of Trichoderma reesei. By comparing the free energies and the local structures of the nucleotide sequences, one optimized sequence was finally selected and transformed into the T. reesei pyridine-auxotrophic strain TU-6. The codon-optimized gene was expressed to a higher level than the original one. Further expressing the codon-optimized gene in a mutated T. reesei strain through fed-batch cultivation resulted in coproduction of cellulase and mannanase up to 1376 U·mL-1 and 1204 U·mL-1, respectively.

Functional diversity of family 3 ß-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the ß-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 ß-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl ß-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl ß-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 ß-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

A thermophilic ß-mannanase from Neosartorya fischeri P1 with broad pH stability and significant hydrolysis ability of various mannan polymers.

A new ß-mannanase gene, man5P1, was cloned from the thermophilic fungus Neosartorya fischeri P1, and successfully expressed in Pichia pastoris. The predicted amino acid sequence of man5P1 consists of a putative 19-residue signal peptide at the N-terminus and a catalytic domain of glycoside hydrolase family 5. The purified recombinant Man5P1 (rMan5P1) was optimally active at pH 4.0 and 80 °C, and was acid and alkali tolerant, exhibiting >20% of the maximal activity at pH 2.0 and 9.0. rMan5P1 had better stability over a broad pH range of 2.0-12.0, and was highly thermostable at 60 °C and below. The enzyme was highly active towards galactomannan and glucomannan, and exhibited classic endo-activity producing a mixture of mannooligosaccharides (MOS). Moreover, it had strong resistance to SDS and Ag(+) and proteases. The superior properties make Man5P1 a potential candidate for use in various industrial applications.

A thermophilic cellulase complex from Phialophora sp. G5 showing high capacity in cellulose hydrolysis.

A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60-65 °C and pH 4.0-5.0, and had good long-term thermostability at 50 °C. Compared with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase for industrial applications, and the production ability could be improved by mutagenesis.