Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K_cat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5‐fold increased specific activity (15°C) and over 100 times prolonged half‐life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity‐altering amino acid substitutions were from non‐charged to non‐charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc.     

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on B_A and H_A via the appearance of the B_A and H_A anion bands. We observed an unexpectedly early rise of the H_A⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between B_A and H_A, and the effect of protein conformation on the electron transfer rate.     

定量蛋白质组学分析链霉素耐药和敏感结核分枝杆菌临床分离株

[目的]发现结核分枝杆菌(Mycobacterium tuberculosis)链霉素耐药相关的潜在菌体蛋白.[方法]以结核分枝杆菌临床分离链霉素敏感株01105和结核分枝杆菌H37Rv为对照,采用iTRAQ技术和生物信息学鉴定并相对定量结核分枝杆菌临床分离链霉素耐药株01108菌体蛋白,并通过WEGO功能注释聚类分析01108菌株差异表达蛋白的细胞组分、分子功能和生物进程.[结果]01108菌株分别与01105菌株和H37Rv菌株比较差异表达蛋白为194个和146个,01108菌株与01105菌株和H37Rv比较均差异表达蛋白121个(共同差异表达蛋白).差异表达蛋白理论相对分子量和等电点分布广泛,其生物进程主要参与中间代谢、呼吸作用和脂质代谢,分子功能主要为催化活性功能和结合功能.共同差异表达蛋白:7个核糖体蛋白(Rv2785c,Rv0056,Rv0641,Rv0652,Rv0701,Rv1630和Rv2442c)在01108菌株中表达下调;7个蛋白在01108菌株中显著差异表达(上调大于1.20倍或下调小于0.55倍),分别为巯基过氧化物酶(Rv1932)、酰基载体蛋白脱氢酶(Rv0824c)、30S核糖体蛋白S15 (Rv2785c)、丙酮酸脱氢酶E2部分(Rv2215)、双组份转录调控蛋白(Rv3133c)以及假定未知蛋白(Rv2466c和Rv2626c).[结论]iTRAQ发现了链霉素耐药结核分枝杆菌相对于链霉素敏感结核分枝杆菌和H37Rv共同差异表达蛋白,为进一步探讨结核分枝杆菌链霉素耐药机制奠定了基础.     

球毛壳菌(Chaetomium globosum)NK102 纤维素酶基因及其表达条件

[目的]生物质的利用是当前生物技术研究的一个热点.本小组分离到一株高效降解纤维素球毛壳菌(Chaetomium globosum)NK102,本文拟探索研究此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素.[方法]通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用数字基因表达谱升级版(RNA-Seq)的方法得到纤维素酶基因的表达差异,然后观察了营养、物理条件下纤维素酶基因表达和酶活性变化的情况.[结果]发现随着培养时间的延长,纤维素酶基因整体上表达量升高.在所选基因中,外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbh1,cdh和egl1)的表达量最高.糖代谢的负调控因子ACE I和CreA的随时间表达量均降低,而Hap2/3/5复合体的表达量反而升高.之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强阻遏因子,纤维二糖为其诱导物,而山梨醇没有影响.特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制酶基因的表达.[结论]转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响.本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考.     

应用基因敲除快速构建大肠杆菌突变体改造脂肪酸代谢途径

【目的】基因敲除技术是研究基因功能的重要手段。我们试图建立一种快速、高效的大肠杆菌基因敲除方法。【方法】利用大肠杆菌(Escherichia coli)BW25113单基因缺失体Keio文库,将经典的Red同源重组技术与P1噬菌体转导技术相结合,对E.coli MG1655脂肪酸代谢基因进行快速敲除。【结果】获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE;脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH-△fabF。敲除fadD和fadE对生长情况没有影响;敲除fabH后,生长速度明显减慢;敲除fabF对生长几乎没有影响。FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L,略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L,明显低于野生型。【结论】在单基因突变体文库基础上,利用P1噬菌体转导、Red同源重组和抗性基因消除进行基因敲除,简化了构建大肠杆菌单基因和多重突变体的方法。     

Halomonas nanhaiensis sp. nov., a halophilic bacterium isolated from a sediment sample from the South China Sea

A novel Gram-negative, aerobic, slightly halophilic, yellow-pigmented, oxidase-negative, Voges–Proskauer positive, non-spore-forming bacterium, designated YIM M 13059^T, was isolated from a sediment sample collected from the South China Sea at a depth of 310 m. Optimal growth was found to occur at 28–30 °C, pH 7.0 and in the presence of 3–4 % (w/v) NaCl. Cells were observed to be rod-shaped and motile by peritrichous flagella. The polar lipids of strain YIM M 13059^T were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a ninhydrin-positive phospholipid, one glycolipid and two unknown phospholipids. The predominant respiratory quinone was determined to be Q-9. The major fatty acids were identified as C_18:1 ω7c, C_16:1 ω6c/C_16:1 ω7c, C_16:0 and C_12:0 3-OH. The genomic DNA G+C content was determined to be 54.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belongs to the genus Halomonas in the family Halomonadaceae. The 16S rRNA gene sequence similarities between strain YIM M 13059 ^T and the type strains of members of the genus Halomonas were in the range 93.3–98.3 %. However, the levels of DNA–DNA relatedness values between YIM M 13059 and the type strains of the most closely related species, Halomonas zhangjiangensis, Halomonas variabilis, Halomonas neptunia, Halomonas boliviensis and Halomonas sulfadieris were 50.2 ± 0.68 %, 46.8 ± 1.9 %, 28.5 ± 0.74 %, 42.9 ± 0.55 % and 37.1 ± 0.68 %, respectively. Based on phylogenetic, chemotaxonomic and phenotypic data, the strain YIM M 13059^T is proposed to represent a novel member of the genus Halomonas, with the name Halomonas nanhaiensis sp. nov. The type strain is YIM M 13059^T (=JCM 18142^T =CCTCC AB 2012911^T).     

Coxsackievirus A16 infection triggers apoptosis in RD cells by inducing ER stress

Coxsackievirus A16 (CA16) infection, which is responsible for hand, foot and mouth disease (HFMD), has become a common health problem in Asia due to the prevalence of the virus. Thus, it is important to understand the pathogenesis of CA16 infection. Viruses that induce endoplasmic reticulum (ER) stress are confronted with the unfolded protein response (UPR), which may lead to apoptotic cell death and influence viral replication. In this study, we found that CA16 infection could induce apoptosis and ER stress in RD cells. Interestingly, apoptosis via the activation of caspase-3, -8 and -9 in the extrinsic or intrinsic apoptotic pathways in RD cells was inhibited by 4-phenyl butyric acid (4PBA), a chemical chaperone that reduces ER stress. These results suggest that CA16 infection leads to ER stress, which in turn results in prolonged ER stress-induced apoptosis. This study provides a new basis for understanding CA16 infection and host responses.