Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.     

A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

High‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.     

PslG,a self-produced glycosyl hydrolase,triggers biofilm disassembly by disrupting exopolysaccharide matrix

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.     

The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction

Background

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.

Methods

Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.

Results

Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations < 10 nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.

General significance

Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1–10 nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.     

Potential applications of enzymes immobilized on/in nano materials: A review

Several new types of carriers and technologies have been implemented in the recent past to improve traditional enzyme immobilization which aimed to enhance enzyme loading, activity and stability to decrease the enzyme biocatalyst cost in industrial biotechnology. These include cross-linked enzyme aggregates, microwave-assisted immobilization, click chemistry technology, mesoporous supports and most recently nanoparticle-based immobilization of enzymes. The union of the specific physical, chemical, optical and electrical properties of nanoparticles with the specific recognition or catalytic properties of biomolecules has led to their appearance in myriad novel biotechnological applications. They have been applied time and again for immobilization of industrially important enzymes with improved characteristics. The high surface-to-volume ratio offered by nanoparticles resulted in the concentration of the immobilized entity being considerably higher than that afforded by experimental protocols based on immobilization on planar 2-D surfaces. Enzymes immobilized on nanoparticles showed a broader working pH and temperature range and higher thermal stability than the native enzymes. Compared with the conventional immobilization methods, nanoparticle based immobilization served three important features; (i) nano-enzyme particles are easy to synthesize in high solid content without using surfactants and toxic reagents, (ii) homogeneous and well defined core-shell nanoparticles with a thick enzyme shell can be obtained, and (iii) particle size can be conveniently tailored within utility limits. In addition, with the growing attention paid to cascade enzymatic reaction and in vitro synthetic biology, it is possible that co-immobilization of multi-enzymes could be achieved on these nanoparticles.     

Salivary secretions from the ectoparasitic wasp, Eulophus pennicornis contain hydrolases, and kill host hemocytes by apoptosis

The current work demonstrates that larvae of the ectoparasitic wasp, Eulophus pennicornis, produce salivary secretions (Ep S) that contain a variety of hydrolases. This includes medium to high levels of N-acetyl-β-glucosaminidase activity, relatively low levels of esterase (C 4) and lipase (C 14) activity, and trace levels of esterase lipase (C 8), acid phosphatise, β-galactosidase, β-glucuronidase, and α-glucosidase activity. In addition, in vitro monolayer assays indicate that both a 4 h and an 18 h incubation of hemocytes from host Lacanobia oleracea larvae in Ep S significantly increases (P < 0.001) the percentage of cells dying by apoptosis compared to the controls. Moreover, an 18 h incubation of hemocytes in Ep S (but not a 4 h incubation), also significantly increased the percentage of dead cells detected using a trypan-blue exclusion assay. The role of ectoparasitoid salivary secretions in conditioning the host by disabling hemocyte-mediated wound healing responses, and providing food for the developing wasp larvae is discussed.     

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.     

转基因表达OPH提高番茄果实降解有机磷农药能力的研究

拟通过基因工程提高番茄果实降解有机磷农药残留的能力。构建了E8启动子基因驱动有机磷降解基因(OPD)的植物表达载体pSE8OP,经农杆菌介导遗传转化番茄子叶后,进行GUS染色、PCR和Southern blotting分析。证明OPD基因已整合进转基因植株基因组中,为1个拷贝。HPLC比较分析发现,转基因番茄果实能显著提高降解毒死蜱和对硫磷的能力,大大减少了番茄中的农药残留。     

Distinctive structural motifs co‐ordinate the catalytic nucleophile and the residues of the oxyanion hole in the alpha/beta‐hydrolase fold enzymes

The alpha/beta‐hydrolases (ABH) are among the largest structural families of proteins that are found in nature. Although they vary in their sequence and function, the ABH enzymes use a similar acid–base‐nucleophile catalytic mechanism to catalyze reactions on different substrates. Because ABH enzymes are biocatalysts with a wide range of potential applications, protein engineering has taken advantage of their catalytic versatility to develop enzymes with industrial applications. This study is a comprehensive analysis of 40 ABH enzyme families focusing on two identified substructures: the nucleophile zone and the oxyanion zone, which co‐ordinate the catalytic nucleophile and the residues of the oxyanion hole, and independently reported as critical for the enzymatic activity. We also frequently observed an aromatic cluster near the nucleophile and oxyanion zones, and opposite the ligand‐binding site. The nucleophile zone, the oxyanion zone and the residue cluster enriched in aromatic side chains comprise a three‐dimensional structural organization that shapes the active site of ABH enzymes and plays an important role in the enzymatic function by structurally stabilizing the catalytic nucleophile and the residues of the oxyanion hole. The structural data support the notion that the aromatic cluster can participate in co‐ordination of the catalytic histidine loop, and properly place the catalytic histidine next to the catalytic nucleophile.     

Effect ofMethanobrevibactersp MF1 Inoculation on Glycoside Hydrolase and Polysaccharide Depolymerase Activities, Wheat Straw Degradation and Volatile Fatty Acid Concentrations in the Rumen of Gnotobiotically-reared Lambs

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 ofRuminococcus flavefaciensand at the age of 4 months with a pure culture ofMethanobrevibactersp. MF1. Following the establishment of MF1, the population ofR. flavefaciensslightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.