Phosphorus alleviation of nitrogen‐suppressed methane sink in global grasslands

Grassland ecosystems account for more than 10% of the global CH₄ sink in soils. A 4‐year field experiment found that addition of P alone did not affect CH₄ uptake and experimental addition of N alone significantly suppressed CH₄ uptake, whereas concurrent N and P additions suppressed CH₄ uptake to a lesser degree. A meta‐analysis including 382 data points in global grasslands corroborated these findings. Global extrapolation with an empirical modelling approach estimated that contemporary N addition suppresses CH₄ sink in global grassland by 11.4% and concurrent N and P deposition alleviates this suppression to 5.8%. The P alleviation of N‐suppressed CH₄ sink is primarily attributed to substrate competition, defined as the competition between ammonium and CH₄ for the methane mono‐oxygenase enzyme. The N and P impacts on CH₄ uptake indicate that projected increases in N and P depositions might substantially affect CH₄ uptake and alter the global CH₄ cycle.     

Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the V_max value of the (R) form was 10 times larger than that of the (S) form.     

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

果胶甲酯酶的结构与功能研究进展

果胶甲酯酶(PME)是一种重要的果胶酶,其水解果胶中的甲酯基从而释放甲醇并降低果胶的甲酯化程度。目前在食品加工、茶饮料、造纸等生产工艺中有着广泛的应用前景。随着对PME的深入研究,已报道了几种不同来源的酶晶体结构,对这些已获得的晶体结构进行分析发现,PME属于右手平行β-螺旋结构,其催化残基为2个保守的天冬氨酸和1个谷氨酰胺残基,并且在催化过程中分别起到了一般酸碱、亲核试剂以及稳定中间体的作用。同时对其底物特异性进行分析,初步了解其底物与活性位点的识别机制。文中针对这几个相关方面进行了系统的综述。     

光生物反应器培养微藻研究进展

微藻具有固定CO2和净化有机废水的能力,在环保、食品饲（饵）料、医药和生物能源开发等领域备受关注,但规模化培养及其产业化仍是研究的难点,亟待解决。就常用于大规模培养微藻的光生物反应器的特点和结构进行了综述。其中,封闭式微藻光生物反应器能够较好地调控藻种的培养条件、不易遭受污染,藻种的纯度容易控制,但培养规模小,生产成本较高;而开放式微藻光生物反应器无法精确控制藻种生长环境,但生产规模大、产量高、生产成本低,因此应用广泛。最佳的方法是综合两者优点,即首先利用封闭式微藻光生物反应器进行中试放大,大量繁殖藻种,然后投入开放式微藻光生物反应器内进行大规模商业生产,此方法有望成为微藻光生物反应器的发展方向,以期为微藻大规模培养提供参考借鉴。     

Wireless bioreactor for anaerobic cultivation of bacteria

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

Bird diversity and endemism along a land‐use gradient in Madagascar: The conservation value of vanilla agroforests

Land‐use change is the most important driver of biodiversity loss worldwide and particularly so in the tropics, where natural habitats are transformed into large‐scale monocultures or heterogeneous landscape mosaics of largely unknown conservation value. Using birds as an indicator taxon, we evaluated the conservation value of a landscape mosaic in northeastern Madagascar, a biodiversity hotspot and the center of global vanilla production. We assessed bird species richness and composition by conducting point counts across seven prevalent land‐use types (forest‐ and fallow‐derived vanilla agroforests, woody and herbaceous fallow that are part of a shifting cultivation system, rice paddy, forest fragment and contiguous old‐growth forest). We find that old‐growth forest had the highest species richness, driven by a high share of endemics. Species richness and community composition in forest‐derived vanilla agroforest were similar to forest fragment, whereas fallow‐derived vanilla agroforest was most comparable to woody fallow. The open land‐use types herbaceous fallow and rice paddy had fewest species. Across forest fragments, vanilla agroforests, and woody fallows, endemic bird species richness was positively correlated to landscape‐scale forest cover. We conclude that both fallow‐ and forest‐derived vanilla agroforests play an important but contrasting role for bird conservation: Fallow‐derived agroforests are less valuable but take fallow land out of the shifting cultivation cycle, possibly preventing further degradation. Conversely, forest‐derived agroforests contribute to forest degradation but may avoid total loss of tree cover from forest fragments. Considering the land‐use history of agroforests may thus be a promising avenue for future research beyond the case of vanilla. Abstract in Malagasay is available with online material     

Spawning habits and embryonic development of the banded lampeye killifish Aplocheilichthys spilauchen (Duméril 1861) in ex situ fresh and brackish water environments

Aside from ornamental uses, there is growing interest in using killifishes for a multiplicity of purposes including baitfish and mosquito biocontrol. This experiment explored the spawning habits and embryonic development of the banded lampeye, Aplocheilichthys spilauchen in ex situ freshwater (0.04‰) and brackish water (5.01‰) to ascertain the captive breeding prospects for mosquito control in areas where they occur. Significantly higher number of eggs were laid in the brackish water than the freshwater (X² = 1613.0, P < 0.05), and black mop was the most preferred spawning substrate, followed by green, blue and white mops. Microscopic monitoring of embryos revealed that cleavage occurred within the first 30 min after fertilization, organogenesis commenced on average in the 25th hour and hatching in approximately 230 h. Although freshwater eggs were relatively bigger than brackish water eggs and certain embryonic developmental stages occurred faster in the freshwater than brackish water, these differences were overall not significant and had no effects on the development and hatching. The observed outcome that A. spilauchen can be optimally propagated with black mops in brackish water offers a significant step in its use for the mosquito biocontrol programme, as well as other potential uses not yet explored.