α-Glucosidase Inhibitor from the Seeds of Balsam Pear (Momordica charantia) and the Fruit Bodies of Grifola frondosa

α-Glucosidase inhibitory activities were found in aqueous methanol extracts of the seeds of Momordica charantia and the fruit bodies of Grifola frondosa. An active principle against the enzyme prepared from rat small intestine acetone powders was isolated and characterized. The structure of the isolated compound was identified as D-(+)-trehalose by FDMS, ¹H-, ¹³C-NMR, and [α]_D measurements. The inhibitory activity of trehalose was compared with 1-deoxynojirimycin. Trehalose showed 45% inhibitory activity at the concentration of 2×10^?3 M, but 1-deoxynojirimycin had 52% inhibitory activity at 1×10^?7 M.     

Altered physiology in trehalose-producing transgenic tobacco plants: Enhanced tolerance to drought and salinity stresses

Transgenic tobaccoNicotiana tabacum L. var. SR1) plants that over-express theEscherichia coli trehalose-6-phosphate synthase (TPS) gene(otsA) synthesized small amounts of trehalose (<400 μg g^-1 leaf) while non-transformants produced no detectable trehalose. Some transgenic plants expressing a high level ofotsA exhibited stunted growth and morphologically altered leaves. We tested F₂2 homozygous plants devoid of phenotypic changes to determine their physiological responses to dehydration and salinity stresses. All transgenic plants maintained better leaf turgidity under a limited water supply or after treatment with polyethylene glycol (PEG). Furthermore, fresh weight was maintained at higher levels after either treatment. The initial leaf water potential was higher in transgenic plants than non-transformants, but, in both plant types, was decreased to a comparable degree following dehydration. When grown with 250 mM NaCl, transgenic plants exhibited a significant delay in leaf withering and chlorosis, as well as more efficient seed germination. Our results suggest that either trehalose or trehalose-6-phosphate can act as an osmoprotective molecule without maintaining water potential, in contrast to other osmolytes. Furthermore, both appear to protect young embryos under unfavorable water status to ensure subsequent germination.     

天蓝色链霉菌海藻糖合酶的异源表达、活性分析及重组菌全细胞转化合成海藻糖的条件优化

从天蓝色链霉菌Streptomyces coelicolor克隆得到海藻糖合酶基因 (ScTreS),在大肠杆菌Escherichia coli BL21(DE3) 中进行了异源表达,通过 Ni-NTA 亲和柱对表达产物进行分离纯化得到纯酶,经 SDS-PAGE 测定其分子量约为62.3 kDa。研究其酶学性质发现该酶最适温度35 ℃;最适pH 7.0,对酸性条件比较敏感。通过同源建模和序列比对分析,对该基因进行定点突变。突变酶K246A比酶活比野生酶提高了1.43倍,突变酶A165T相对提高了1.39倍,海藻糖转化率分别提高了14%和10%。利用突变体重组菌K246A进行全细胞转化优化海藻糖的合成条件并放大进行5 L罐发酵,结果表明：在麦芽糖浓度300 g/L、初始反应温度和pH分别为35 ℃和7.0的条件下,转化率最高达到71.3%,产量为213.93 g/L;当底物浓度增加到700 g/L时,海藻糖产量仍可达到465.98 g/L。     

Phialomyces macrosporus: Chemical Constituents,Antimicrobial Activity and Complete NMR Assignments for the 7,7′‐Biphyscion

Five known secondary metabolites, chrysophanol ( 1 ), 7,7′‐biphyscion ( 2 ), secalonic acid D ( 3 ), mannitol ( 4 ) and trehalose ( 5 ) were isolated for the first time from the extracts of the fungus Phialomyces macrosporus. Their structures were elucidated by NMR methods (1D and 2D NMR analysis), optical activity and ESI‐MS. Complete ¹H and ¹³C assignments were performed for compound 2 . The antimicrobial activity was evaluated by serial microdilution assay for compounds 2 and 3 and results showed that compound 3 exhibited a significant growth inhibition at concentrations of 15.6 mg/ml (S. aureus and S. choleraesius) and 0.97 mg/mL (B. subtilis), comparable to the positive control.     

Quantitative Proteomics Reveals Remodeling of Protein Repertoire Across Life Phases of Daphnia pulex

Although the microcrustacean Daphnia is becoming an organism of choice for proteomic studies, protein expression across its life cycle have not been fully characterized. Proteomes of adult females, juveniles, asexually produced embryos, and the ephippia‐resting stages containing sexually produced diapausing freezing‐ and desiccation‐resistant embryos are analyzed. Overall, proteins with known molecular functions are more likely to be detected than proteins with no detectable orthology. Similarly, proteins with stronger gene model support in two independent genome assemblies can be detected, than those without such support. This suggests that the proteomics pipeline can be applied to verify hypothesized proteins, even given questionable reference gene models. In particular, upregulation of vitellogenins and downregulation of actins and myosins in embryos of both types, relative to juveniles and adults, and overrepresentation of cell‐cycle related proteins in the developing embryos, relative to diapausing embryos and adults, are observed. Upregulation of small heat‐shock proteins and peroxidases, as well as overrepresentation of stress‐response proteins in the ephippium relative to the asexually produced non‐diapausing embryos, is found. The ephippium also shows upregulation of three trehalose‐synthesis proteins and downregulation of a trehalose hydrolase, consistent with the role of trehalose in protection against freezing and desiccation.     

杓兰果胶杆菌海藻糖酶的克隆表达及应用

海藻糖酶在工业发酵和食品医药等领域应用广泛,我国缺乏性能优良工业品种的海藻糖酶,同时在应用研究领域还不够深入。本研究从自然界筛选获得一株产酸性海藻糖酶的杓兰果胶杆菌(Pectobacterium cypripedii),克隆获得其海藻糖酶基因PCTre,在大肠杆菌(Escherichia coli)BL21(DE3)体系中实现了重组表达,在5L发酵罐上28h产酶达到4130U/mL。酶学性质研究显示,PCTre能特异性水解海藻糖,最适反应温度和pH分别是35℃和5.5,在pH4.0、4.5、5.0处理8h后残余酶活仍超过80%,表现出良好的耐酸性。同时该酶还显示出优良的有机溶剂耐受性,在20%(V/V)乙醇溶液中处理24h仍能保留60%的酶活。进一步在含有20%(V/V)乙醇和7.5%海藻糖的模拟发酵体系中,当添加500U/L PCTre,可在16h内完全水解海藻糖,具有良好的应用于工业乙醇发酵的潜力。     

海藻糖强化高盐胁迫下异养硝化-好氧反硝化菌群的代谢机制

异养硝化-好氧反硝化(heterotrophic nitrification-aerobic denitrification,HN-AD)菌是一类可在高盐环境脱氮的好氧微生物,但其工程应用效果不理想。海藻糖作为相容性溶质,通过参与调节细胞渗透压帮助微生物抵抗高盐胁迫,对提升高盐环境菌群的脱氮效率起重要作用。本研究通过启动膜曝气生物膜反应器(membrane aerobic biofilm reactor,MABR)富集HN-AD菌,设计添加150μmol/L海藻糖的C₁₅₀实验组和未添加海藻糖的C₀对照组,开展了外源性海藻糖对高盐胁迫下HN-AD菌群代谢的强化机制研究。反应器运行性能及群落结构分析结果显示,C₁₅₀组相较C₀组,NH₄⁺-N、总氮(total nitrogen,TN)和化学需氧量(chemical oxygen demand,COD)去除率分别提高29.7%、28.0%和29.1%;以不动杆菌属(Acinetobacter)和假黄褐藻属(Pseudofulvimonas)为优势菌属的耐盐型HN-AD菌群总相对丰度在C₁₅₀组达到66.8%、较C₀组提高了18.2%,添加海藻糖促进高盐环境中耐性型HN-AD菌群富集并强化系统脱氮性能。代谢组学深度分析表明,外源性海藻糖加强脯氨酸合成,提高微生物对高盐胁迫的抵抗能力;通过调节细胞增殖信号通路(cGMP-PKG、PI3K-Akt)、磷脂代谢通路及氨酰基-tRNA合成通路的活性,促使甘油磷脂代谢物磷酸乙醇胺及嘌呤和嘧啶丰度上调,提升细菌聚集能力和细胞增殖,助推微生物在高盐环境生长;同时,添加海藻糖还加快三羧酸循环(tricarboxylic acid cycle,TCA cycle),为HN-AD菌群的碳、氮代谢提供更多电子供体与能量,进而优化系统脱氮性能。本研究结果为HN-AD菌在高盐高氮废水处理中的运用提供理论指导。