产高温蛋白酶高温菌的筛选

利用平板透明圈法，从堆肥、土壤中筛选到88种产高温蛋白酶的高温菌，它们在酪素筛选平板上都产生明显的蛋白水解透明圈。这些产高温蛋白酶高温菌株的获得为进一步研究高温菌的耐热机制、高温酶的热稳定性机理等理论问题及高温蛋白酶的应用，奠定了基础。     

Hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (αs1-, β- and k-casein) after enzymatic hydrolysis with pepsin and enzymatic extract from Pleurotus eryngii

The aim of this study was to estimate the hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (α_s1-, β- and k-casein) after in vitro digestion with two different enzymatic systems: pepsin from porcine gastric mucosa (EP) and a crude enzymatic extract from the edible mushroom Pleurotus eryngii. The used enzymes showed a different mode of casein catalysis with a consequent production of peptides of different antioxidant activity. The CN haplotype significantly influenced peptides production; in fact, the amino acid substitutions caused by genetic polymorphisms at the α_s1-, β- and k-CN loci influenced the sites of enzymatic cleavage and therefore the produced peptides. The above is evidenced by the different antioxidant activity found in the hydrolysates depending on the used enzymatic system, the CN haplotype, and the CN haplotype × enzymatic treatment interaction. The findings of this study are a perspective for the production of specific foods that exert a biological effect in addition to the nutritional one.     

乳及乳制品中嗜冷菌的多样性及其腐败危害研究进展

乳是一种营养丰富的物质,同时也是微生物生长繁殖的理想培养基。生乳的质量是影响乳制品产业链的关键因素,随着低温储存和冷链运输技术的发展,生乳中大部分细菌的生长受到抑制,但嗜冷菌的生长并未受到抑制,并逐步成为生乳中的优势菌。生乳在冷藏运输或储存期间,嗜冷菌依旧可以生长繁殖,其分泌的蛋白酶和脂肪酶可耐高温,经过巴氏杀菌或超高温灭菌处理后依旧保持活性,因此,了解嗜冷菌的多样性及其产生的酶对提高乳及乳制品质量、减少腐败和浪费具有重要作用。本文通过介绍乳及乳制品中嗜冷菌污染现状及腐败危害研究,旨在为乳及乳制品行业的风险评估提供背景信息,从源头控制嗜冷菌对生乳的浪费,保证乳及乳制品的品质。     

Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells

Activity-based monitoring of cell-secreted proteases has gained significant interest due to the implication of these substances in diverse cellular functions. Here, we demonstrated a cell-based method of monitoring protease activity using fluorescent cell-permeable peptides. The activatable peptide consists of anionic (EEEE), cleavable, and cationic sequences (RRRR) that enable intracellular delivery by matrix metalloproteinase-2 (MMP2), which is secreted by living cancer cells. Compared to HT-29 cells (MMP2-negative), HT-1080 cells (MMP2-positive) showed a strong fluorescence response to the short fluorescent peptide via cell-secreted protease activation. Our approach is expected to find applications for the rapid visualization of protease activity in living cells.     

Druggability Assessment for Selected Serine Proteases in a Pharmaceutical Industry Setting

Target druggability assessment is an integral part of the early target characterization and selection process in pharmaceutical industry. Here, we investigate a set of five different serine proteases from the blood coagulation cascade. The aim of this study is twofold. Firstly, leveraging the wealth of available in-house high-throughput screening (HTS) data, we analyze HTS hit rates and discuss their predictive value for the development of small molecule (SMOL) candidates. Purely structure-activity relationship (SAR) based druggability ratings are compared with computational protein-structure based druggability assessments. Secondly, we evaluate the impact of using conformational ensembles from molecular dynamics (MD) simulations instead of single static crystal structures as basis for computational druggability assessments. Based on this study, we recommend incorporating molecular dynamics routinely into the early target characterization process, especially if only a single X-ray structure is available.     

酸性蛋白酶降解小麦面筋蛋白的研究

以DPPH自由基清除率为指标,采用单因素和正交实验研究了酸性蛋白酶木瓜蛋白酶(精)对小麦面筋蛋白水解最适条件.结果表明,最佳酶解条件为温度70℃,pH6.0,底物量7 g,水解时间5 h;在此条件下,DPPH自由基清除率达到92.65%,效果显著.     

丝胶蛋白酶解产物抗氧化活性的研究

本实验采用六种蛋白酶对丝胶蛋白进行酶解,并分析其酶解产物清除二苯代苦味酰基 (DPPH) 自由基能力.通过脂质过氧化体系、还原力、金属螯合能力评价其酶解4h酶解产物的抗氧化活性.结果表明:各种酶解产物都呈现一定的清除DPPH自由基的能力,其中以碱性蛋白酶最强 (p＜0.05);各种酶解产物都存在抑制脂质过氧化活性,与其他蛋白酶相比碱性蛋白酶的抑制活性显著的高于其他酶 (p＜0.05);各种蛋白酶解产物具有一定的还原力和金属螯合能力.这些结果表明:六种酶解产物都具有显著的抗氧化活性.     

Synthesis of Novel Phosphonic‐Type Activity‐Based Probes for Neutrophil Serine Proteases and Their Application in Spleen Lysates of Different Organisms

Neutrophils are a type of granulocyte important in the “first line of defense” of the innate immune system. Upon activation, they facilitate the destruction of invading microorganisms by the production of superoxide radicals, as well as the release of the enzymatic contents of their lysozymes. These enzymes include specific serine proteases: cathepsin G, neutrophil elastase, proteinase 3, as well as the recently discovered neutrophil serine protease 4 (NSP4). Under normal conditions, the proteolytic activity of neutrophil proteases is tightly regulated by endogenous serpins; however, this mechanism can be subverted during tissue stress, thereby resulting in the uncontrolled activity of serine proteases, which induce chronic inflammation and subsequent pathology. Herein, we describe the development of low‐molecular‐weight activity‐based probes that specifically target the active sites of neutrophil proteases.