Inactivation and reactivation of proteins (enzymes)

The molecular mechanisms of protein inactivation, i.e. aggregation, thiol-disulphide exchange, alteration of the primary structure, dissociation of cofactor molecules from the active centre, dissociation of the oligomeric proteins into subunits and conformational changes have been analysed. All these mechanisms are closely interrelated during inactivation of proteins. However, in many cases, the conformational changes accompany and trigger other inactivation processes. Reactivation of irreversibly inactivated proteins is·discussed. Reactivation can be successful when inactivation has been caused by aggregation, modification of SH-groups (or S-S bonds) or as a consequence of irreversible conformational changes.     

Effect of modulation of enzyme inactivation on temperature optimization for reactor operation with chitin-immobilized lactase

Temperature is a critical variable to be optimized in any enzymatic process, producing opposite effects on enzyme activity and inactivation rate. Temperature functions for all kinetic and inactivation parameters were validated for chitin-immobilized yeast lactase (CIL). Enzyme inactivation was described by a two-stage series mechanism. The effect of galactose and lactose on inactivation was determined in terms of modulation factors that were positive for galactose and negative for lactose. Modulation factors were mild functions of temperature in the first stage and strong functions in the second stage of enzyme inactivation, where galactose positive modulation factors increase while lactose negative modulation factors decrease with temperature. Temperature-explicit functions for kinetic and inactivation parameters were incorporated into a scheme to optimize temperature in the simulation of a continuous packed-bed reactor operation with chitin-immobilized lactase, based on an annual cost objective function. Optimum temperature was 20°C at enzyme replacement of 25% residual activity, and increased only slightly at higher replacement frequencies. The effect of modulation factors on reactor design and temperature optimization is presented and discussed. Software for temperature optimization that allows the introduction of variations in all parameters and operational criteria to perform sensitivity analysis was developed.     

Effects of Magnesium Ions on Thermal Inactivation of Alkaline Phosphatase

The effect of Mg²⁺ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures and Mg²⁺ concentrations. Increasing the Mg²⁺ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the increased free Mg²⁺ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg²⁺ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence emission spectra showed that the emission maximum for Mg²⁺-containing system was always higher than that of Mg²⁺-free system, and the higher temperature enhanced this difference. In addition, Mg²⁺also enhanced the unfolding rate of the enzyme at 47°C. The potential biological significance of these results are discussed.     

Enzyme Deactivation in Reactors

The influence of enzyme deactivation on substrate conversion in different reactor types is examined. The influence of inter- and intra-particle diffusion on deactivation and on effectiveness factor is analyzed. Optimum temperature operations criterion and policies are presented for reactors with deactivating catalysts. Appropriate examples are provided to highlight the different concepts presented.     

Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

Inactivation of enzymes by organic solvents: New technique with well-defined interfacial area

A liquid-liquid bubble column apparatus allows exposure of enzyme solutions to water-immiscible organic solvents with a known total interfacial area and welldefined time scales and flow. It allows clear distinction of the different classes of inactivation mechanism. With urease as a model enzyme, octan-2-one and butylbenzene act only through the effects of solvent molecules dissolved in the aqueous phase, giving first-order inactivation at 0.34 and 0.21 h(-1), respectively. Hexane and tridecane act only through exposure to the interface. The amount of urease inactivated is proportional to the total area of interface exposed, rather than to elapsed time, and may be characterized by a rate of about 0.5 mukat m(-2). This is consistent with the formation and (partial) inactivation of a complete adsorbed monolayer of protein. With butan-1-ol, both mechanisms contribute significantly to the observed inactivation. The presence of O(2) increases the rate of interfacial inactivation, but not that by dissolved solvent. (c) 1994 John Wiley & Sons, Inc.     

Anaerobic Reactor Design Concepts for the Treatment of Domestic Wastewater

Since the earlier anaerobic treatment systems, the design concepts were improved from classic reactors like septic tanks and anaerobic ponds, to modern high rate reactor configurations like anaerobic filters, UASB, EGSB, fixed film fluidized bed and expanded bed reactors, and others. In this paper, anaerobic reactors are evaluated considering the historical evolution and types of wastewaters. The emphasis is on the potential for application in domestic sewage treatment, particularly in regions with a hot climate. Proper design and operation can result in a high capacity and efficiency of organic matter removal using single anaerobic reactors. Performance comparison of anaerobic treatment systems is presented based mostly on a single but practical parameter, the hydraulic retention time. Combined anaerobic reactor systems as well as combined anaerobic and non-anaerobic systems are also presented.     

低温胁迫下不同光照条件对锦熟黄杨抗氧化酶活性的影响

研究了锦熟黄杨（Buxus sempervirens L.）在低温胁迫下不同光照条件（12 h光照/12 h黑暗、24 h全光、24 h全黑）对其抗氧化酶活性的影响,结果表明：低温不同光照条件下细胞膜透性和丙二醛含量均高于对照（20℃ 12 h光照/12 h黑暗）,细胞膜透性和丙二醛含量在5℃ 24 h光照条件下最大;低温胁迫下SOD活性高于对照,并在5℃ 24 h光照条件下达到最高值;CAT的活性仍维持较高水平,5℃ 12 h光照/12 h黑暗显著高于对照及其它处理;在低温有光照条件下,POD活性升高,黑暗条件下POD活性低于对照。低温胁迫下POD、SOD和CAT的活性均呈上升趋势,可能是植株具有较强抗性的原因。     

Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme

Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half-life of 15 +/- 3 min at 50 degrees C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30 degrees C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half-life of 5 +/- 2 min at 50 degrees C     

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.     

双水相技术在酶分离纯化中的运用

阐述了双水相技术在酶分离纯化中的应用 ,分析了影响双水相技术分离纯化酶的各种因素 ,并探讨了双水相技术在酶分离纯化应用中的发展方向     

Inactivation of Salmonella Senftenberg strain W 775 during composting of biowastes and garden wastes

AIMS: Determination of the minimum requirements (time-temperature relationship and moisture content) that are needed for a sufficient eradication of an indicator organism. METHODS AND RESULTS: To determine the hygienic safety of composting processes, the indicator organism Salmonella enterica ssp. enterica serotype Senftenberg strain W 775 (further abbreviated as W 775) was artificially inoculated on a meat carrier and monitored subsequently. Different types of composting processes, e.g. composting in enclosed facilities, in open-air and in-vessel composting, were investigated. The waste feedstocks used in this work were either biowastes (i.e. vegetable, fruit and garden wastes; also called source-separated household wastes) or pure garden wastes. Beside these large-scale trials, we also conducted some lab experiments in order to determine the impact of temperature, moisture content and the presence of an indigenous microflora on the eradication of W 775. We found the temperature to be the most important parameter to eradicate W 775 from compost. When the temperature of the compost heap is 60 degrees C and the moisture content varies between 60-65%, W 775 (10(8) CFU g(-1)) will be inactivated within 10 h of composting. The moisture content is, beside temperature, a second parameter that influences the survival of W 775. When the water content of the composting materials or meat carriers is reduced, a higher survival rate of W 775 was observed (survival rate increases 0.5 log(10) unit when there is a reduction of 5% in moisture content). In addition, other parameters (such as microbial antagonism, toxic compounds, etc.) have an influence on the survival of W 775 as well. CONCLUSIONS: Our study demonstrates that all types of composting processes tested in this work were sufficient to eradicate W 775 providing that they are well managed in terms of temperature and moisture content. SIGNIFICANCE AND IMPACT OF THE STUDY: To give a better view on the parameters of importance for the eradication of W 775 during composting.     

一株纤溶酶高产菌株的发酵特性研究

筛选了一株产纤溶酶能力较强的芽孢细菌,本文对其液体发酵条件进行了优化,结果表明菌株产酶最佳条件为:可溶性淀粉4%,蛋白胨2%,柠檬酸铁铵0.1%,磷酸钙0.4%,接种量2%,pH 7.5,发酵时间96 h,装瓶量75/500(mL/mL),摇床温度37℃,转速150 rpm,在该条件下产酶酶活可达581.81 IU/mL。     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

低温下钙对黄瓜幼苗抗氧化酶活性及POD同工酶谱的影响

以耐冷性存在明显差异的栽培黄瓜‘长春密刺’和‘北京截头’为材料,研究了冷胁迫下C a2 、钙离子螯合剂(EGTA)和钙调素拮抗剂(CPZ)对低温胁迫黄瓜幼苗叶片3种抗氧化酶活性及POD同工酶的影响.对保护酶活性的研究结果表明:C aC l2、EGTA和CPZ处理均能引起3种抗氧化酶活性变化,但变化幅度因材料而异.胁迫处理中C a2 能提高2种材料的叶片抗氧化酶活性,减少其在低温逆境中的下降幅度,而EGTA和CPZ处理降低了2种材料的抗氧化酶活性,加大了低温逆境中的下降幅度.POD同工酶研究发现,C aC l2延缓冷胁迫中同工酶条带的丢失,EGTA和CPZ处理则使同工酶条带丢失提前,但材料不同对酶条带丢失时间也有影响.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

根霉液态发酵产生纤溶酶的培养条件优化

目的:目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵,因此研制新型溶栓药物的需求迫切。方法:研究了根霉Rhizopus chinensisYY-15液体摇瓶发酵产生纤溶酶的工艺条件。采用单因素试验对液体发酵培养基的碳源、氮源、碳氮比、初始pH进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果:实验范围内菌株液体发酵产纤溶酶的适宜培养基组成为:麸皮水浓度3%(w/v),豆粕浓度5%(w/v),初始培养基pH5.0。适宜培养条件为接种量6%,培养时间72h。优化条件下的摇瓶液体发酵纤溶酶产量平均达98.31 U/ml。     

重组肝素酶I热失活动力学模型的建立与应用

对组氨酸标签肝素酶I（His．HepI,Ec4．2．2．7）的热失活机制进行了研究。针对短暂低温处理可以使热失活His．HepI酶活部分恢复及添加二硫苏糖醇（D1Tr）使其热稳定性提高的现象,利用荧光探针法研究了失活过程His．HepI构象变化,证明了该酶构象存在可逆转变行为。为进一步明晰His．HepI的热失活机制,假设该酶热失活的主要途径包括去折叠及形成聚集体,并以此为基础建立模型进行拟合,模型与实验值吻合良好,表明假设的合理性。根据模型计算的活化能为Er=100．217kJ／mol、Eir=7．857kJ／mol和Ed=77．062kJ／mol,此数据从一定程度上解释了冷处理为何能使His．HepI部分恢复活性。进一步研究表明,任何能够抑制这两种途径发生的措施对于提高His．HepI热稳定性都是有效的。