工业环境下酶蛋白的催化行为与适应性改造研究进展

酶在工业上有着广泛应用和巨大潜力,但工业生产中高温、强酸/碱、高盐、有机溶剂和高底物浓度等条件仍然制约着酶的大规模应用。为使酶能更好地在工业环境下发挥催化作用,目前的主要策略是对酶进行适应性改造(如理性或半理性设计、定向进化、固定化等)。文中简要阐述了酶在工业环境下的催化行为以及近年对其适应性改造的研究进展,以期为酶的适应性改造提供参考。     

复合微生态制剂对幼刺参营养成分和 酶活力的影响

目的研究复合微生态制剂对幼刺参体壁营养成分、几种消化酶和免疫酶活力的影响。方法在封闭式循环系统中投喂不同的微生态制剂进行30 d刺参养殖实验。结果投喂液态复合微生态制剂组和粉状复合微生态制剂组的刺参体壁的粗脂肪、总糖、粗蛋白含量最高,两组与对照组相比差异有统计学意义(Ps0.05)。添加微生态制剂的3个实验组刺参肠道蛋白酶、淀粉酶活力均比对照组高,且差异有统计学意义(Ps0.05)。实验组刺参的肠道、体壁、体液过氧化氢酶活力均比对照组高,其中投喂液态复合微生态制剂实验组活力最高。实验组刺参组织的酸性磷酸酶和碱性磷酸酶活力也明显高于对照组(与对照组相比差异有统计学意义,Ps0.05)。结论微生态制剂可以有效改善幼刺参体壁营养成分,促进机体消化活力,提高刺参免疫力。     

自噬抑制剂3-MA上调H2O2损伤的PC12细胞氧化应激水平

为探究自噬抑制剂6-氨基-3-甲基腺嘌呤（3-methyladenine,3-MA）对损伤细胞氧化应激水平的影响,将3-MA作用于H2O2诱导的PC12细胞损伤模型,以自噬增强剂雷帕霉素（rapamycin,Rap）作为对照,探讨自噬与氧化应激的关系。测定线粒体的膜电位和细胞内的活性氧（reactive oxygen species, ROS）与丙二醛(malondialdehyde, MDA)含量,以及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性,评价损伤细胞的氧化应激状态。单丹(磺)酰戊二胺（monodansylcadaverine,MDC）染色,观察损伤细胞的自噬情况。蛋白质印迹分析损伤细胞中的自噬相关蛋白质LC3-II/LC3-I比值变化。实验结果显示：与正常组相比,H2O2损伤细胞的ROS水平上升到正常组的141%,MDA含量增加(P<0.001);CAT与SOD酶活力显著降低(P<0.001),差异均有统计学意义,证明损伤细胞氧化应激水平增加;MDC染色结果表明,H2O2组自噬明显增加。Western印迹结果表明,LC3-II/LC3-I值显著升高（P<0.05）;与损伤组相比,3-MA组MDC染色结果表明,自噬水平降低。Western印迹结果表明,LC3-II/LC3-I值下降;细胞内ROS水平升高,增加到正常组的208%。MDA含量增加(P<0.001),CAT、SOD酶活力降低(P<0.001)。综上结果表明,自噬抑制剂可增加H2O2诱导的PC12细胞损伤模型的氧化应激水平,增加细胞凋亡。     

Antioxidant and anti‐inflammatory effects of the monocarbonyl curcumin analogs B2BRBC and C66 in monocrotaline‐induced right ventricular hypertrophy

For 22 days after monocrotaline injection two groups of rats received either of the monocarbonyl curcumin analogs (2E,6E)‐2,6‐bis(2‐bromobenzylidene)cycloxehanone (B2BrBC) and (2E,6E)‐2,6‐bis([2‐tri?uoromethyl]benzylidene)cyclohexanone (C66), and their right ventricle parameters were compared to those from the control and the monocrotaline injected animals. B2BrBC and C66 treatments did not prevent the monocrotaline‐induced right ventricular hypertrophy but attenuated the changes in antioxidant enzyme activities and reduced inflammation. The level of thiol‐based nonenzymatic antioxidants did not change in the function of monocrotaline or curcumin analogs treatment. However, due to its stronger antioxidant properties, only B2BrBC treatment was effective in the reduction of monocrotaline‐associated lipid peroxidation. The obtained results suggest that increasing the levels of antioxidant enzymes may not be sufficient to reduce oxidative stress and chronic inflammation optimally and our current study supports the potential of compounds with more than one beneficial biological activity as a promising treatment against the progression of cardiac hypertrophy.     

Ready-to-use thermally stable mastermix pills for molecular biology applications

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.     

Exogenous Glycine Betaine Reduces Drought Damage by Mediating Osmotic Adjustment and Enhancing Antioxidant Defense in <i>Phoebe hunanensis</i>

Drought stress negatively impacts growth and physiological processes in plants. The foliar application of glycine betaine (GB) is an effective and low-cost approach to improve the drought tolerance of trees. This study examined the effect of exogenously applied GB on the cell membrane permeability, osmotic adjustment, and antioxidant enzyme activities of Phoebe hunanensis Hand.-Mazz under drought stress. Two levels (0 and 800 mL) of water irrigation were tested under different applied GB concentrations (0, 50, 100, and 200 mM). Drought stress decreased the relative water content by 58.5% while increased the electric conductivity, malondialdehyde, proline, soluble proteins, soluble sugars, and antioxidant enzyme activities (superoxide dismutase, catalase, peroxidase) by up to 62.9%, 42.4%, 87.0%, 19.1%, 60.5%, 68.3%, 71.7%, and 83.8%, respectively, on the 25^th day. The foliar application of GB, especially at 100 mM, increased the relative water content of P. hunanensis leaves under drought stress. The concentration of GB from 50 to 100 mM effectively alleviated the improvement of cell membrane permeability and inhibited the accumulation of membrane lipid peroxidation products. Under drought stress, the concentrations of proline, soluble proteins, and soluble sugars in the leaves of P. hunanensis increased as the applied GB concentration was increased and the water stress time was prolonged. Exogenously applied GB decreased oxidative stress and improved antioxidant enzyme activities as compared with treatments without GB application. Furthermore, the physiological and biochemical indexes of P. hunanensis showed a certain dose effect on exogenous GB concentration. These results suggest that GB helps maintain the drought tolerance of P. hunanensis.