Strategy in manipulating transglycosylation activity of glycosyl hydrolase for oligosaccharide production

Background: The increasing market demand for oligosaccharides has intensified the need for efficient biocatalysts. Glycosyl hydrolases (GHs) are still gaining popularity as biocatalyst for oligosaccharides synthesis owing to its simple reaction and high selectivity.

Purpose: Over the years, research has advanced mainly directing to one goal; to reduce hydrolysis activity of GHs for increased transglycosylation activity in achieving high production of oligosaccharides.

Design and methods: This review concisely presents the strategies to increase transglycosylation activity of GHs for oligosaccharides synthesis, focusing on controlling the reaction equilibrium, and protein engineering. Various modifications of the subsites of GHs have been demonstrated to significantly modulate the hydrolysis and transglycosylation activity of the enzymes. The clear insight of the roles of each amino acid in these sites provides a platform for designing an enzyme that could synthesize a specific oligosaccharide product.

Conclusions: The key strategies presented here are important for future improvement of GHs as a biocatalyst for oligosaccharide synthesis.     

Assessing the ecological health status using macrobenthic communities of tropical coastal water

The relative contributions of natural and anthropogenic fluctuations are different in shaping habitat health status and natural benthic communities in tropical coastal water. Understanding responses of coastal benthic communities to these fluctuations are still equivocal and thus available data are inadequate. Here, multiple analytical approaches were used to address the significant risk factors and their impacts on coastal benthic habitat health through space and time. A total abundance of 1436 ± 612 individuals of 33 benthic species were counted and identified from 22 sampling stations across eight sampling periods over the two years of study. Bioassay results showed that the benthic community is moderately exposed to anthropogenic pollutants in Klang Strait coastal water. The results showed that there were no significant temporal changes of habitat health status and macrobenthic community structure; however, spatial changes were significant and synchronized with anthropogenic and natural fluctuations. This study demonstrates that Cd and Hg levels and sediment characteristics played the primary role in shaping the habitat health and macrobenthic assemblages, whereas the influence of other factors were insignificant.     

Monophyletic clades of Macaranga‐pollinating thrips show high specificity to taxonomic sections of host plants

Thrips (Thysanoptera) have been recorded as pollinators of various plant species, but they are mostly regarded to be of low ecological relevance. In Southeast Asia, thrips were recently discovered to pollinate flowers of several taxonomic sections of the pioneer tree genus Macaranga (Euphorbiaceae), which is particularly well known as an ant‐plant, and for its importance in early forest succession. The lack of taxonomic treatment and of knowledge about systematic relationships among extant thrips, however, has prevented firm conclusions on the specificity of this plant–pollinator interaction. Here, results from sequencing of the mitochondrial cytochrome oxidase subunit support our previous morphospecies concept of Macaranga flower thrips, and confirm the genetic identity of five recently described species. They were remarkably all assigned to the genus Dolichothrips (Phlaeothripidae), which typically consists of phytophagous species. In addition, the molecular data revealed one cryptic species. A first phylogenetic tree of the Dolichothrips associated with Macaranga provides insights into their systematic position. In particular, we identify monophyly of all important Macaranga pollinator species, all species being largely specific to particular taxonomic host plant sections. Our results suggest a closely matched diversification of pollinating thrips with Macaranga trees. This adds a novel type of association to thrips pollinator–plant interactions, which have been so far documented as single‐species interactions or generalist thrips species visiting multiple plant taxa.     

In silico identification and characterization of a putative phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gene in Eimeria tenella

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) play diverse roles in the cellular biology of many organisms, including signal transduction, secretion and vesicular trafficking, and regulation of cytoskeleton assembly. Discovery of the PIP5K gene in Eimeria tenella may shed light on its role in the biology of this avian protozoan, and afford further understanding of the cell-host interaction, particularly during the invasion process. In this study, we report the identification of the PIP5K coding region in the genome sequence of Eimeria tenella using in silico gene prediction approaches. Prediction of the PIP5K coding sequence was confirmed by mapping the full-length cDNA sequence, generated via the Rapid Amplification of cDNA Ends (RACE) method, to the genomic sequence. The putative PIP5K gene of Eimeria tenella is located on the complementary strand of the E1080B12.b1 contig, and comprises 12 exons. Further analysis showed that the coding region spans from exon 1 to exon 7, with all exons obeying the adopted 'gt...ag' splicing rule of intronic sequences. Consensus of the hexameric 5' donor-splice site was deduced as GTRDBB... and the consensus for the 3' acceptor-splice sites as ...BHDYAG. The gene encodes a 252-amino acid residue protein. Domain search and protein fold recognition analyses provide compelling evidences that the deduced protein is a PIP5K.     

Resource availability and roosting ecology shape reproductive phenology of rain forest insectivorous bats

Bats in temperate and subtropical regions typically synchronize birth of a single young with peaks in resource availability driven by local climate patterns. In tropical rain forest, insects are available throughout the year, potentially allowing departures from seasonal monoestry. However, reproductive energy budgets may be constrained by the cost of commuting to foraging grounds from distant roosts. To test these hypotheses, we simultaneously tracked female reproductive activity of 11 insectivorous bat species, insect biomass, and local weather variables for 20 months in a Malaysian rain forest. Five species roost in forest structures and hence have low commuting costs, whereas six species depend on caves, which are limited in the landscape, and are presumed to incur higher commuting costs to foraging sites. Monthly insect biomass was positively correlated with monthly rainfall, and there was a significant relationship between insect biomass and lactation in cave‐roosting but not forest‐roosting species. Cave‐roosting species were seasonally monoestrus, with parturition confined to a two‐month period, whereas in forest‐roosting species, pregnancy and lactation were recorded throughout the year. Our results suggest that the energetic costs of commuting from roosts to foraging grounds shape annual reproductive patterns in tropical rain forest insectivorous bats. Ongoing changes in forest landscapes are likely to increase these costs for cave‐roosting bats, further restricting reproductive opportunities. Climate change is projected to influence the timing of rainfall events in many tropical habitats, which may disrupt relationships between rainfall, insect biomass, and bat reproductive timing, further compromising reproductive success.     

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica

Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its K_m value was 256 μM and its catalytic efficiency was 81.7 s⁻¹. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.

     

Synthesis,Spectral Characterization and Crystals Structure of some Arsane Derivatives of Gold (I) Complexes: A Comparative Density Functional Theory Study

A series of complexes of the type LAuCl where L = tris(p-tolylarsane), tris(m-tolylarsane), bis(diphenylarsano)ethane, and tris(naphthyl)arsane have been synthesized. All of the new complexes, 1-4, have been fully characterized by means of ¹H NMR and ¹³C NMR spectroscopy and single crystal X-ray crystallography. The structures of complexes 1-4 have been determined from X-ray diffraction data. The linear molecules have an average bond distance between gold-arsenic and gold-chlorine of 2.3390Å and 2.2846Å, respectively. Aurophilic interaction was prominent in complex 1 and 3, whereas complex 2 and 4 do not show any such interaction. The intermolecular gold interaction bond length was affected by the electronegativity of the molecule. The computed values calculated at DFT level using B3LYP function are in good agreement with the experimental results.