胰蛋白酶水解β-乳球蛋白获得ACE抑制肽的条件优化

采用三因素二次通用旋转设计和体外检测法,对胰蛋白酶水解β-乳球蛋白获得ACE抑制肽的条件进行优化。结果表明,底物浓度(X1)、温度(X2)、酶与底物的质量比(X3)对ACE抑制率的影响回归方程为:Y=50.62-2.33X1-1.97X2+5.81 X3-3.36X2X3-6.56X22-1.96X32,胰蛋白酶水解β-乳球蛋白获得ACE抑制肽的最优水解条件为:底物质量浓度为60 g/L,水解温度30℃,酶与底物的质量比为5.5%,水解时间6 h,水解产物对ACE抑制活性最大抑制率为53.86%。     

自组装蛋白质芯片——功能蛋白质组学的新工具

传统的蛋白质芯片制备需要进行繁琐的蛋白质表达与纯化。同时,由于蛋白质活性不稳定,蛋白质芯片不宜长期保存。新一代自组装蛋白质芯片,利用无细胞表达体系和DNA固定技术,能够将蛋白质即时、原位表达并固定在芯片上,有效地解决了传统蛋白质芯片的制备和保存问题。目前自组装蛋白质芯片已初步用于大规模蛋白-蛋白质相互作用的筛选,以及鉴定免疫优势抗原等研究。该文介绍了近年自组装蛋白质芯片技术的进展和应用研究。     

3-氰基吡啶水合酶产生菌的筛选及其酶形成条件

应用富集培养和梯度底物浓度定向筛选技术,从长期被腈化物污染的土壤中筛选到一株产 3-氰基吡啶水合酶(3-cyanopyridine hydratase)活性较高的马红球菌(Rhodococcus e-qui)SHB-121.研究了该菌3-氰基吡啶水合酶的最适形成条件.在最适条件下,酶的比活力达5.3u/mg干细胞,比在初筛条件下的酶活力提高95倍,而在其细胞内共存的尼克酰胺(烟酰胺)水解酶活力很低.     

豆豉中高产乳酸乳酸菌的筛选及其产酸条件的优化研究

目的 从云南传统发酵豆豉中分离并筛选得到一株高产乳酸的乳酸菌菌株,并对其产酸培养基组成及条件进行优化探讨.方法 运用紫外分光光度法,对豆豉由来高产有机酸菌株YM-4-3的碳源进行优化.结果 分子生物学鉴定结果表明该乳酸菌菌株属于植物乳杆菌,并将之命名为Lactobacillus plantarum YM-4-3,碳源优化结果表明,乳糖是L.plantarum YM-4-3 菌株生物合成乳酸的最佳碳源,而果糖则是最利于各种主要有机酸合成的碳源,由于此时各常见有机酸的含量均高于其他碳源时的含量,且其总酸含量高达9696.32 mg/L.结论 云南传统发酵豆豉可以成为功能性乳酸菌筛选的菌种资源库,所分离得到的L plantarum YM-4-3产酸能力较强,它可被应用于有机酸发酵及豆豉发酵的酸化处理.     

一株蟾毒灵转化菌株的筛选鉴定与转化条件优化

【背景】微生物转化是对天然产物进行结构修饰的重要手段,具有催化反应快、选择性强、反应条件易控制和污染小等特点。许多微生物能够对蟾毒配基类化合物进行生物转化,在不同位置进行结构修饰,并且产生毒性减弱的活性衍生物。【目的】筛选蟾毒灵生物转化菌株,以期发现活性更好、毒性降低的化合物。【方法】利用蟾毒灵为底物筛选其生物转化菌株,通过HPLC (High performance liquid chromatography)/LC-MS (Liquid chromatograph-mass spectrometer)鉴定转化产物;对可生物转化蟾毒灵的菌株进行菌落形态观察和分子生物学鉴定,并优化发酵条件提高转化率,同时测试该菌株对其他甾体化合物的转化作用。【结果】筛选获得一株蟾毒灵转化菌,经形态学与ITS (Internal transcribed spacer)分析鉴定为Naganishia属菌,转化产物为3-去氢蟾毒灵。该菌株生物转化的培养基最适底物浓度为8 mg/L,最适初始pH值为6.5,最佳接种量为3%,最佳转化时间为96 h,最终转化率为48.3%。该菌株可将雌酮E1转化为雌二醇E2,也可将雌二醇E2逆向转化为E1。【结论】首次发现Naganishia属菌株对甾体类化合物具有转化活性,其产生的弱细胞毒性的转化产物3-去氢蟾毒灵有望成为高效、安全的强心药物。微生物转化法选择性高、反应条件温和、操作简单,为大量制备活性化合物3-去氢蟾毒灵提供了一条简便易行的道路,也为更多甾体化合物结构修饰提供了可能。     

微乳体系中11β-羟基甲羟孕酮的C1,2生物脱氢

为改善过程传质,提高甾类药物中间体11β-羟基甲羟孕酮C1,2生物脱氢转化率,采用简单节杆菌Arthrobacter simplex UR016菌株在Tween-80/乙醇/食油/水构成的微乳体系中进行生物脱氢,并考察了微乳体系组成、转化温度、投料浓度对脱氢反应的影响。结果表明:以菌体培养液作为水相,食油作为油相构建微乳体系,食油最适加量为10g/L,表面活性剂Tween-80加量为4g/L;底物经醇溶后水析投料,乙醇最适加量为发酵液体积的7%(V/V);最适转化温度为33oC;当底物浓度为4g/L时,在构建的微乳体系中转化46h,脱氢转化率达88.6%,与水相转化工艺相比提高了66.2%。在该体系中疏水性11β-羟基甲羟孕酮底物得到了有效的增溶和扩散,生物脱氢转化率明显提高。     

一株产低温β-半乳糖苷酶微杆菌的筛选鉴定、产酶条件及其酶学特性

【背景】低温β-半乳糖苷酶能在低温下仍保持较高的乳糖水解活性,筛选酶学特性适合在牛乳体系中高效水解乳糖的β-半乳糖苷酶生产菌株,是低乳糖牛乳加工产业关注的焦点。【目的】对天山中国一号冰川沉积物中分离的一株产低温β-半乳糖苷酶菌株的产酶条件和酶学特性进行研究。【方法】结合X-Gal平板法初筛和测定粗酶液酶活复筛,获得产低温β-半乳糖苷酶的菌株。通过形态学、生理生化试验及16S rRNA基因测序分析对筛选菌株进行鉴定,单因素摇瓶实验优化菌株的产酶条件,硫酸铵分级沉淀初步纯化β-半乳糖苷酶并对其酶学特性进行分析。【结果】通过形态学、生理生化特征和16S rRNA基因鉴定,确定菌株LW106为微杆菌属(Microbacterium)菌株;该菌株最适产酶温度为25°C,最佳产酶碳源为可溶性淀粉,培养基初始pH为7.0,接种量为3%;对初步纯化的低温β-半乳糖苷酶酶学性质的研究表明,LW106所产β-半乳糖苷酶的最适pH为6.0,最适反应温度为35°C,4°C时酶活为最大酶活的78%,4°C和pH 7.0时的稳定性最好,10 mmol/L的Na+对酶活性基本没有抑制作用,Ca~(2+)对酶活性具有一定的激活作用。【结论】菌株LW106所产低温β-半乳糖苷酶的酶学特性表明该酶在乳品低温加工领域具有进一步研究和应用的价值。     

慢性肾功能衰竭早期患者血清CysC、RBP、β2-MG水平的关系及其诊断价值分析

目的:分析慢性肾功能衰竭(CRF)早期患者血清胱抑素C(CysC)、视黄醇结合蛋白(RBP)、β_2-微球蛋白水平(β_2-MG)的关系,探讨其对CRF早期的诊断价值。方法:选取2016年8月至2018年10月新疆医科大学第二附属医院收治的CRF早期患者240例作为研究组,同期体检的健康者240例作为对照组,比较两组血肌酐(Scr)、血清CysC、RBP、β_2-MG水平,并分析CysC、RBP、β_2-MG的相关性以及其对CRF早期的诊断价值。结果:研究组血清SCr、CysC、RBP、β_2-MG水平及阳性率均显著高于对照组,差异有统计学意义(P0.05)。Pearson相关分析显示,CRF患者血清CysC与RBP、β_2-MG水平呈正相关(r=0.532,0.784,P=0.012,0.000),RBP与β_2-MG水平呈正相关(r=0.518,P=0.015)。CysC、RBP、β_2-MG联合诊断的特异度、敏感度和准确度分别为98.75%、88.33%和93.54%。结论:CRF早期患者血清CysC、RBP、β_2-MG水平升高,三指标之间具有一定的相关性,CysC、RBP、β_2-MG联合检测对CRF早期具有较高的诊断价值。     

不同剂量硼替佐米联合地塞米松对多发性骨髓瘤T细胞亚群及血清C反应蛋白、β2-微球蛋白的影响

目的:探讨不同剂量硼替佐米联合地塞米松对多发性骨髓瘤(MM)T细胞亚群及血清C反应蛋白(CRP)、β_2-微球蛋白(β_2-MG)的影响。方法:选取2010年1月～2019年8月期间皖南医学院附属马鞍山中心医院收治的52例MM患者以及南京中医药大学附属泰州医院收治的28例MM患者,共计纳入患者例数80例。根据随机数字表法分为A组(n=40,给予1.3 mg/m~2硼替佐米联合地塞米松治疗)和B组(n=40,给予1.6 mg/m2硼替佐米联合地塞米松治疗),比较两组患者的疗效、T细胞亚群、血清CRP、β_2-MG水平,记录两组治疗期间不良反应情况。结果:两组临床总有效率比较差异无统计学意义(P0.05)。两组治疗5个疗程后CD3~+CD4~+、CD3~+CD4~+/CD3~+CD8~+均升高,且B组高于A组(P0.05);CD3~+CD8~+下降,且B组低于A组(P0.05)。两组不良反应发生率比较无差异(P0.05)。两组治疗5个疗程后β_2-MG、CRP均下降,且B组低于A组(P0.05)。结论:MM患者采用1.6 mg/m2硼替佐米或者1.3 mg/m2硼替佐米联合地塞米松治疗,可获得相当的疗效及安全性,但1.6 mg/m2硼替佐米联合地塞米松治疗可更好地改善患者T细胞亚群及血清CRP、β_2-MG水平。     

Self-assembly of protein superstructures by physical interactions under cytoplasm-like conditions

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)—genetically encoded protein nanoparticles that form ordered intracellular clusters—as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.     

Collagen self-assembly in vitro: electron microscopy of initial aggregates formed during the lag phase

Initial aggregates formed in collagen self-assembly were visualized by electron microscopy, using formaldehyde to fix the state of aggregation at various points in the turbidimetric lag phase. Measurements of the length distributions of monomers and small oligomers show that the first-formed aggregates are dimeric, with the most prevalent dimer having a maximal (approximately equal to 4D; D = 67 nm) stagger between constituent molecules.     

Reaction intermediates formed by myofibrils during the ATPase reaction under relaxed conditions

The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, M*ATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or M*ATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.     

Self-assembly of the ionic peptide EAK16: the effect of charge distributions on self-assembly

Amphiphilic peptides suspended in aqueous solution display a rich set of aggregation behavior. Molecular-level studies of relatively simple amphiphilic molecules under controlled conditions are an essential step toward a better understanding of self-assembly phenomena of naturally occurring peptides/proteins. Here, we study the influence of molecular architecture and interactions on the self-assembly of model peptides (EAK16s), using both experimental and theoretical approaches. Three different types of EAK16 were studied: EAK16-I, -II, and -IV, which have the same amino acid composition but different amino acid sequences. Atomic force microscopy confirms that EAK16-I and -II form fibrillar assemblies, whereas EAK16-IV forms globular structures. The Fourier transform infrared spectrum of EAK16-IV indicates the possible formation of a beta-turn structure, which is not found in EAK16-I and -II. Our theoretical and numerical studies suggest the underlying mechanism behind these observations. We show that the hairpin structure is energetically stable for EAK16-IV, whereas the chain entropy of EAK16-I and -II favors relatively stretched conformations. Our combined experimental and theoretical approaches provide a clear picture of the interplay between single-chain properties, as determined by peptide sequences (or charge distributions), and the emerging structure at the nano (or more coarse-grained) level.     

Practical aspects of using the microbatch method in screening conditions for protein crystallization

The microbatch technique is a simple and efficient method for screening for protein crystallization conditions both by hand and using automated systems. Many of the problems associated with more commonly used methods such as vapour diffusion can be overcome using this method. Despite its promise microbatch has not been widely utilized as a viable screening procedure. This review aims to describe the method from a practical point of view with an emphasis on screening crystallization conditions, outlining current progress and discussion of the advantages compared to other methods.     

Computational design of protein self-assembly

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Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion.

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.     

Geochemical characteristics of the carbonate constructions formed during microbial oxidation of methane under anaerobic conditions

The aragonite constructions of the Black Sea are formed in a stable anaerobic zone and are a perfect object to study the natural mechanism of anaerobic methane oxidation. The most probable pathway of methane oxidation is its methanogen-mediated reaction with bicarbonates, dissolved in seawater, with the formation of water and acetate, which is then consumed by other components of the anaerobic community. Comparison of the delta 13C values of carbonate minerals and organic matter once more demonstrated that the formation of the organic matter of biomass is accompanied by intense fractionation of carbon isotopes, as a result of which the total organic matter of biomass acquires an extremely light isotopic composition, characterized by delta 13C values as low as -83.8@1000.