Influence of molecular parameters on the degradation of chitosan by a commercial enzyme

The degradative activities of neutral protease against chitosan samples with different molecular parameters were characterized. The effects of the degree of deacetylation (DD) and molecular weight (MW) of chitosan on its susceptibility to degradation were investigated. The DD and MW of the chitosans were determined using potentiometric titration and viscometry, respectively. The molecular weight distribution of initial and degraded commercial chitosan was investigated by gel permeation chromatography. Initial degradation rates (r) were determined from the plots of viscosity decrease against time of degradation. The time courses of degradation of chitosans with neutral protease were non-linear and the enzymatic hydrolysis was an endo-action. Classical Michaelis-Menten kinetic parameters were measured by analyzing the amount of reducing sugars and Eadie-Hofstee plots established that hydrolysis of chitosan by neutral protease obeyed Michaelis-Menten kinetics. Michaelis-Menten parameters and initial degradation rates were calculated and compared to determine the influences of DD and MW on hydrolysis. The results showed that higher DD and higher MW chitosans possessed a lower affinity for the enzyme and a slower degradation rate. Those samples with a lower DD and lower MW were more susceptible substrates.     

Computational Simulation of HIV Protease Inhibitors to the Main Protease (Mpro) of SARS-CoV-2: Implications for COVID-19 Drugs Design

SARS-CoV-2 is highly homologous to SARS-CoV. To date, the main protease (Mpro) of SARS-CoV-2 is regarded as an important drug target for the treatment of Coronavirus Disease 2019 (COVID-19). Some experiments confirmed that several HIV protease inhibitors present the inhibitory effects on the replication of SARS-CoV-2 by inhibiting Mpro. However, the mechanism of action has still not been studied very clearly. In this work, the interaction mechanism of four HIV protease inhibitors Darunavir (DRV), Lopinavir (LPV), Nelfinavir (NFV), and Ritonavire (RTV) targeting SARS-CoV-2 Mpro was explored by applying docking, molecular dynamics (MD) simulations, and MM–GBSA methods using the broad-spectrum antiviral drug Ribavirin (RBV) as the negative and nonspecific control. Our results revealed that LPV, RTV, and NFV have higher binding affinities with Mpro, and they all interact with catalytic residues His41 and the other two key amino acids Met49 and Met165. Pharmacophore model analysis further revealed that the aromatic ring, hydrogen bond donor, and hydrophobic group are the essential infrastructure of Mpro inhibitors. Overall, this study applied computational simulation methods to study the interaction mechanism of HIV-1 protease inhibitors with SARS-CoV-2 Mpro, and the findings provide useful insights for the development of novel anti-SARS-CoV-2 agents for the treatment of COVID-19.     

贝莱斯芽孢杆菌SW5菌株与外源蛋白酶协同发酵对鳀鱼鱼露品质的影响

为进一步研究接种贝莱斯芽孢杆菌(Bacillus velezensis) SW5菌株对发酵鳀鱼鱼露品质的影响,以低值鳀鱼为原料,通过接种贝莱斯芽孢杆菌SW5菌株,并添加多种蛋白酶协同发酵鳀鱼鱼露.测定氨基酸态氮(AA-N)质量浓度、pH值和总酸质量浓度的变化,采用电子舌和色差仪测定鱼露发酵完成后发酵液的鲜味成分含量和色泽,与商品鱼露和酶解液进行对比.结果表明在发酵过程中,采用贝莱斯芽孢杆菌SW5菌株与中性蛋白酶协同发酵,发酵第7天AA-N质量浓度达到最高,为13.93 g·L^-1,符合市售一级鱼露标准.3个处理组的色泽和鲜味成分含量与商品鱼露相近.综上所述,通过接种贝莱斯芽孢杆菌SW5菌株和蛋白酶对鳀鱼鱼露进行协同发酵可以缩短发酵周期,提高鱼露的产量以及AA-N和鲜味成分的含量.因此,该菌株可用作海洋蛋白源物质的发酵深加工.     

嗜麦芽寡养单胞菌胞外蛋白酶的化学修饰

分别采用苯甲基磺酰氟(PMSF)、对-氯汞苯甲酸(PCMB)、N-乙咪唑(N-AI)、氯胺-T(Ch-T)、N-溴化琥珀亚胺(NBS)、2-巯基乙醇(2-ME) 6种化学修饰剂处理嗜麦芽寡养单胞菌胞外蛋白酶, 研究酶分子中氨基酸侧链基团与酶活性的关系. 结果表明Ch-T, NBS和2-ME能显著抑制酶活性, 而N-AI, PMSF和PCMB对酶活性的影响不大, 说明蛋氨酸残基、色氨酸残基和二硫键是酶活性的必需基团, 而酪氨酸残基、丝氨酸残基和巯基与酶活性无直接关系. 同时检测了EDTA和金属离子对酶活性的影响. 实验结果证实, EDTA, Mg^2＋, Ca^2＋, Hg^2＋和Cu^2＋能显著影响酶活性, 说明该酶为一种金属蛋白酶.     

多酶复合水解微波加热制备小分子大豆肽

以水解度和AN为指标,确定了微波加热条件下,三种单酶(碱性蛋白酶、中性蛋白酶、酸性蛋白酶)水解大豆蛋白的最佳工艺条件及三种酶复合水解的加酶顺序,将大豆蛋白最大限度的分解为小分子大豆多肽和氨基酸。毛细管电泳实验表明:多酶复合水解优于单酶。     

宇佐美曲霉 L336酸性蛋白酶 发酵过程动力学研究

宇佐美曲霉栗色变种 L336是一株高产酸性蛋白酶的生产菌 ,采用转置培养法 ,对其发酵过程的 一些动力学特点进行了研究 ,结果表明 ,酸性蛋白酶是细胞利用内源营养从头合成的 ,属于非生长关联 型; 腺嘌呤是菌体生长的生长因子 ,代谢类似物对酶合成产生较大的抑制 ;而蛋白胨是酶合成的诱导因 子 ,发酵液溶氧水平较大地影响酶合成能力 . 本研究为其工业发酵工艺的构建提供了理论依据 .     

虚拟活性化合物的自动生成

虚拟活性化合物的自动生成是从药效团和预先设定的结构碎片出发,通过碎片选择、碎片组装和柔性构象搜索来获得虚拟活性结构。经过对HIV-1蛋白酶抑制剂药效团进行虚拟活性化合物生成,得到了16个虚拟活性化合物,通过构象分析发现生成的化合物满足药效团的限制条件。说明这一方法能够有效的生成虚拟活性结构,与药效团检索结果对比发现生成的虚拟活性结构新颖易于合成。     

Fast and selective synthesis of novel cyclic sulfamide HIV-1 protease inhibitors under controlled microwave heating

A novel and highly selective silver-promoted monobenzylation method was developed to promote synthesis of nonsymmetrical sulfamide-based HIV-1 inhibitors. Microwave-accelerated palladium-catalyzed N-amide arylation- and aminocarbonylation reactions were employed for rapid and reliable compound generation. With this class of inhibitory agents, six active inhibitors were identified, the most potent inhibitor possessing a K_i-value of 20 nM.