Enzymatic production of β-D-glucose-1-phosphate from trehalose

β-D -Glucose-1-phosphate (βGlc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of βGlc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, βGlc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline βGlc1P is now available from the cheap substrates trehalose and inorganic phosphate.     

Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

A constitutive expression system for high‐throughput screening

To reduce the amount of consumables and number of pipetting steps in high‐throughput screening, a constitutive expression system was developed that comprises four different promoters of varying strength. The system was validated by the expression of different sucrose phosphorylase enzymes from Leuconostoc mesenteroides, Lactobacillus acidophilus and Bifidobacterium adolescentis in 96‐deep‐ and low‐well plates at three temperatures. Drastically improved soluble expression in mini‐cultures was observed for the enzymes from L. mesenteroides strains by reducing the promoter strength from strong to intermediate and by expressing the proteins at lower temperatures. In contrast, the enzymes from B. adolescentis and L. acidophilus were expressed most efficiently with a strong promoter. The constitutive expression of sucrose phosphorylases in low‐well plates resulted in a level of activity that is equal or even better than what was achieved by inducible expression. Therefore, our plasmid set with varying constitutive promoters will be an indispensable tool to optimize enzyme expression for high‐throughput screening.     

Ectoine accumulation in Brevibacterium epidermis

As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 m NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 m NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while l-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; l-glutamate is a better carbon source for ectoine synthesis than l-aspartate.     

The Genus Gluconobacter Oxydans: Comprehensive Overview of Biochemistry and Biotechnological Applications

ABSTRACT

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

Trophic structure of cold-water coral communities revealed from the analysis of tissue isotopes and fatty acid composition

The trophic structure of cold-water coral reef communities at two contrasting locations, the 800-m deep Belgica Mounds (Irish margin) and the 300-m deep Træna reefs (Norwegian Shelf), was investigated using stable isotope (δ¹³C and δ¹⁵N) and fatty-acid composition analysis. A broad range of specimens, with emphasis on (commercial) fish specie's, and organic matter sources were sampled using a variety of tools. Irrespective of the environmental and geographical setting, the δ¹⁵N values indicated that the food web encompasses roughly 1.5 to 3 trophic levels. Mobile echinoderms, i.e. sea urchins and sea stars, had highest δ¹⁵N values, indicative of a high trophic position in the food web. The fraction of bacterial fatty acids in reef fauna was generally low (<5%), indicating that enhanced bacterial production in the water column through seafloor seepage of nutrients (‘hydraulic theory’) does not form a significant energy pathway into the food web. The high fraction of algal and essential fatty acids in reef fauna and fish at both locations indicates a close coupling with surface productivity, but the transport mechanism depends on the hydrographic setting. At Træna, Calanus copepods and euphausiids form an additional link between primary production and fish, which is largely absent at Belgica Mounds. At Belgica Mounds, the reef community is primarily supported by phytodetritus, as evidenced by the high contribution of algal fatty acids in faunal tissue and seasonal chlorophyll a deposition and marine snow at the reef. The environmental setting of cold-water coral reefs influences the structure of the associated food web.