Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

Bacterioplankton growth, grazing mortality and quantitative relationship to primary production in a humic and a clearwater lake

Bacterial growth and grazing mortality were estimated from Mayto October in two south Swedish oligotrophic lakes, one beinga clearwater lake (water colour 5–10 mg Pt l^–1 DOC2.9–3.4 mg l^–1, Secchi disk depth 5.0–9.4m) and the other a humic, brownwater lake (water colour 105–165mg Pt l^–1, DOC 13.7–22.7mg l^–1, Secchi diskdepth 1.3–2.1 m). Specific rates of growth and grazingmortality were generally similar for both lakes. However, theabundance of bacteria was consistently 2–3 times higherin the water of the humic lake, suggesting that the total productionand consumption of bacterial cells were also higher than inthe dearwater lake. The ratio of bacterial secondary productionto primary production was higher in the humic lake than in theclearwater lake, indicating that the bacterioplankton of thehumic lake utilize allochthonous substrates, in addition tosubstrates originating from autochthonous primary production.Most of the bacterial loss in both lakes could be attributedto small protozoan grazers. This implies that allochthonousand autochthonous organic carbon fixed by bacterioplankton isless important in terms of carbon flow to higher trophic levelsthan would be expected if macrozooplankton were the dominantbacterivores, providing a more direct and efficient transferof carbon to larger organisms.     

Transient growth‐enhancing effects of elevated maternal thyroid hormones at no apparent oxidative cost during early postnatal period

Maternal thyroid hormones (THs) have been proven crucial for embryonic development in humans, but their influence within the natural variation on wild animals remains unknown. So far the only two studies that experimentally investigated the potential fitness consequences of maternal THs in birds found inconsistent results. More studies are thus required to assess the general effects of maternal THs and their influences on more behavioral and physiological parameters. In this study, we experimentally elevated yolk TH content in a wild migratory passerine species, the collared flycatcher Ficedula albicollis, to investigate the effects on hatching success, nestling growth and oxidative stress. We found that TH‐injected eggs had a higher hatching success, and the nestlings hatched from TH‐injected eggs were heavier and larger than control nestlings, but only during the early postnatal period. These differences vanished by fledging. Nestlings from TH‐injected eggs exhibited lower activity of the glutathione‐s‐transferase, a major antioxidant enzyme, than control nestlings at day 12, a few days before fledging, but they did not differ in oxidative damage and overall intracellular oxidative state. These results suggest that the early growth‐enhancing effects incurred no observable oxidative stress. We hypothesize that such a transient growth‐enhancing effect might be adaptive in advancing the development and maturation of the offspring so they are well‐prepared in time for the upcoming migration. Further studies investigating whether such advancing effects can influence long‐term fitness, will be more than valuable.     

Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine

A critical component of an in vitro production process for baculovirus biopesticides is a growth medium that is efficacious, robust, and inexpensive. An in‐house low‐cost serum‐free medium, VPM3, has been shown to be very promising in supporting Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) production in H. zea insect cell suspension cultures, for use as a biopesticide against the Heliothine pest complex. However, VPM3 is composed of a significant number of undefined components, including five different protein hydrolysates, which introduce a challenging lot‐to‐lot variability to the production process. In this study, an intensive statistical optimization routine was employed to reduce the number of protein hydrolysates in VPM3 medium. Nearly 300 runs (including replicates) were conducted with great efficiency by using 50 mL TubeSpin® bioreactors to propagate insect cell suspension cultures. Fractional factorial experiments were first used to determine the most important of the five default protein hydrolysates, and to screen for seven potential substitutes for the default meat peptone, Primatone RL. Validation studies informed by the screening tests showed that promising alternative media could be formulated based on just two protein hydrolysates, in particular the YST‐AMP (Yeast Extract and Amyl Meat Peptone) and YST‐POT (Yeast Extract and Lucratone Potato Peptone) combinations. The YST‐AMP (meat‐based) and YST‐POT (meat‐free) variants of VPM3 were optimized using response surface methodology, and were shown to be just as good as the default VPM3 and the commercial Sf‐900 II media in supporting baculovirus yields, hence providing a means toward a more reproducible and scalable production process for HaSNPV biopesticides. © 2012 American Institute of Chemical Engineers Biotechnol. Prog.,, 2012     

Correction to: Predictive landscapes hidden beneath biological cellular automata

Journal of Biological Physics -     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

An ancient look at UCP1

Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure-function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.     

Transpiration by an emergent macrophyte: source of water and implications for nutrient supply

The contribution of sediment interstitial water and the water column to the transpiration stream of Myriophyllum aquaticum (Vellozo) Verdcourt was determined to estimate the significance of mass flow in supply of sediment nutrients for plant growth. Sediment interstitial water accounted for about 2% of the water transpired over a 37 day period. Because of the small volume of water that originated in the sediment we concluded that mass flow did not significantly enhance nutrient supply to the roots of M. aquaticum. Relative growth rate (RGR) of adventitious, water roots was greater than whole plant RGR, and RGR of sediment roots was not significantly different from zero, indicating a shift in the biomass allocation after emergence of the apical meristem into the air. Water use, measured by the transpiration coefficient, averaged 260 ml H₂O mg DW^-1, which is similar to C-4 terrestrial plants. M. aquaticum has leaf characteristics commonly associated with xerophytic habitats. These characteristics may be necessary if a high transpiration rate and a mechanical requirement for high cell turgor pressure, required by a reliance upon hydrostatic pressure for support of the aerial stems, are mutually exclusive because of morphological constraints on hydraulic conductivity.