Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20–50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20–40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.     

Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates

In the model plant Arabidopsis thaliana, four Dicer‐like proteins (DCL1–4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII‐like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4‐dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4‐dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold‐back double‐stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR‐dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.     

Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.     

Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

Background  

Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water.     

Biochemical analysis of BmNPV attachment toBombyx mori BmN-4 cells

Binding characteristics ofBombyx mori nuclear polyhedrosis virus (BmNPV) toB. mori BmN-4 cells was determined. The cells had a single class of BmNPV-binding sites and displayed a low binding capacity for BmNPV in the range of 70 sites per cell. The biochemical nature of BmNPV-binding sites on the cell surface was identified. Treatment of BmN-4 cells with protease, wheat germ agglutinin, metabolic inhibitors, or lysosomotropic agents reduced BmNPV binding to BmN-4 cells, whereas treatment with phospholipase C showed no effect on virus binding. These results indicate that the structure of the binding site moiety is a protein-oligosaccharide complex.     

Ca(2+)-induced folding of a family I.3 lipase with repetitive Ca(2+) binding motifs at the C-terminus.

In order to understand a role of the Ca(2+) ion on the structure and function of a Ca(2+)-dependent family I.3 lipase from Pseudomonas sp. MIS38, apo-PML, holo-PML, holo-PML*, and the N-terminal domain alone (N-fragment) were prepared and biochemically characterized. Apo-PML and holo-PML represent refolded proteins in the absence and presence of the Ca(2+) ion, respectively. Holo-PML* represents a holo-PML dialyzed against 20 mM Tris-HCl (pH 7.5). The results suggest that the C-terminal domain of PML is almost fully unfolded in the apo-form and its folding is induced by Ca(2+) binding. The folding of this C-terminal domain may be required to make a conformation of the N-terminal catalytic domain functional.