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嗜热毛壳菌外切葡聚糖纤维二糖水解酶的纯化和部分性质研究 总被引:3,自引:0,他引:3
研究液体发酵嗜热毛壳菌(Chaetomium thermophilum)产生的一种外切葡聚糖纤维二糖水解酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Sephacryl S-100分子筛层析、Q Sepharose Fast Flow强阴离子层析等步骤后获得凝胶电泳均一的外切葡聚糖纤维二糖水解酶。经12.5%SDS-PAGE和凝胶过滤层析方法测得该酶的分子量大小约为66.3kDa和67.1kDa。该酶反应的最适温度和pH值分别为65℃和5.0。在60℃以下酶比较稳定,在70℃酶的半衰期为1h,在80℃下保温20min仍具有20%的活性,该酶的热稳定性较中温真菌的同类酶高,与国外报道的嗜热真菌的同类酶热稳定性接近。以pNPC为底物的Km值为0.956mmol/L。 相似文献
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《生物物理学报》2014,(2)
酶的热稳定性问题一直是生物科学领域的研究热点。构建氨基酸网络,从系统水平上研究酶热稳定性的影响因素是阐明嗜热酶耐热机制的重要途径。作者以超氧化物歧化酶(iron superoxide dismutase,Fe-SOD)的空间三维结构信息为基础,构建了不同类型的氨基酸相互作用网络。通过分析氨基酸网络的网络参数,发现热稳定性高的Fe-SOD氨基酸网络的平均度、平均连接强度及同配系数均高于常温的Fe-SOD氨基酸网络,而嗜热Fe-SOD氨基酸网络的特征路径长度小于常温的Fe-SOD氨基酸网络。此外,通过改变滑动窗口大小研究氨基酸网络中分子间相互作用区域范围,发现热稳定性高的Fe-SOD氨基酸网络中二级结构内部分子间连接紧密,二级结构之间及结构域之间的连接也较频繁。这些现象表明,嗜热Fe-SOD酶致密的内部结构缩短了氨基酸之间的距离,这更有利于稳定酶结构的作用力(如氢键和盐桥)的形成。实验结果进一步表明,通过研究氨基酸网络的网络参数可以阐述酶结构和功能之间的关系。 相似文献
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嗜热细菌的碱性磷酸酯酶的研究 总被引:13,自引:0,他引:13
从58株来自国内温泉的嗜热细菌中分离到一株菌,它所产生的耐热碱性磷酸酯酶在95℃中保温60min后仍保留原来活力的75%。测定了酶的一系列性质,包括酶作用的最适PH,最适离子强度,最适温度,以及酶的热稳定性,米氏常数,活化能等等,还研究了一些无机离子,氨基酸以及表面活性剂对酶活的影响。 相似文献
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嗜热脂肪芽孢杆菌过氧化氢酶Ⅰ及其取代突变酶88(25)的延伸突变是指在酶蛋白的C末端上连接一段随机肽链,从而改变了酶蛋白的结构.研究结果表明,这种延伸突变的方法非常有效地提高了酶的热稳定性,并且随机肽链的疏水性与其对应的延伸突变体酶的热稳定性呈现一定的负相关性,即随机肽链的疏水性越高,对应的突变体酶热稳定性越低 相似文献
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过氧化氢酶Ⅰ结构延伸突变改善酶热稳定性的初步研究 总被引:2,自引:0,他引:2
嗜热脂肪芽孢杆菌过氧化氢酶Ⅰ及其取代突变酶88(25)的延伸突变是指在酶蛋白在C末上连接一段随机肽链,从而改变了酶蛋白的结构,研究结果表明,这种延伸突变的方法非常有效地提高了酶的热稳定性,并且随机肽链的疏水性一与其对应的延伸突变体酶的热稳定性呈现一定的负相关性,即随机肽链的疏水性越高,对应的突变体酶热稳定性越低。 相似文献
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【目的】通过对一株地衣芽孢杆菌来源的角蛋白酶N端进行分子改造,研究其对角蛋白酶活力和热稳定性的影响,进而提高角蛋白酶的热稳定性。【方法】将角蛋白酶N端前5个氨基酸进行分段缺失,并通过序列比对将N端的前5个氨基酸替换为来源于Thermoactinomyces vulgaris的嗜热蛋白酶的N端,将野生型和突变体角蛋白酶基因在枯草芽孢杆菌WB600中进行表达,并对重组酶进行纯化与酶学性质研究。【结果】角蛋白酶N端不同长度的缺失大幅度地降低了角蛋白酶的活力,其中缺失前5个氨基酸完全丧失了酶活力。将角蛋白酶N端前5个氨基酸替换为嗜热蛋白酶N端前12个氨基酸,虽然降低了近70%的活力,但是却增加了角蛋白酶的热稳定性,60°C条件下的半衰期t1/2由原来的9 min提高到20 min。【结论】角蛋白酶的N端对其酶活力具有较大的影响,与嗜热蛋白酶来源的N端进行替换可以有效提高角蛋白酶的热稳定性。 相似文献
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嗜热与嗜常温微生物的蛋白质氨基酸组成比较 总被引:11,自引:0,他引:11
嗜热微生物的嗜热特性与其蛋白质的高度热稳定性紧密相关。为了探索嗜热蛋白质的热稳定机制,比较嗜热和嗜常温微生物的蛋白质在氨基酸组成上的差别,收集110对分别来自嗜热和嗜常温微生物的同源蛋白质序列,比较两组蛋白质各种氨基酸含量以及疏水性氨基酸组成、疏水性指数和荷电氨基酸组成的差别,结果两者在多种氨基酸含量上存在微小但统计学上显著的差别,嗜热蛋白质比嗜常温蛋白质具有较高的平均疏水性和荷电氨基酸组成。对两组蛋白质的“脂肪族氨基酸指数”进行分析,证明嗜热蛋白质之所以具有较高的脂肪族氨基酸指数是由于其亮氨酸含量较高,与影响该指数的其它几种氨基酸无关;从而认为该指数的意义值得怀疑。通过对大量同源嗜热蛋白质和嗜常温蛋白质氨基酸组成的比较,能够揭示一些有关蛋白质热稳定性的普遍规律。 相似文献
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Structural features of thermozymes 总被引:15,自引:0,他引:15
Enzymes synthesized by thermophiles and hyperthermophiles are known as thermozymes. These enzymes are typically thermostable, or resistant to irreversible inactivation at high temperatures, and thermophilic, i.e. optimally active at elevated temperatures between 60 and 125 degrees C. Enzyme thermostability encompasses thermodynamic stability and kinetic stability. Thermodynamic stability is defined by the enzyme's free energy of stabilization (deltaG(stab)) and by its melting temperature (Tm). An enzyme's kinetic stability is often expressed as its halflife (t1/2) at defined temperature. DeltaG(stab) of thermophilic proteins is 5-20 kcal/mol higher than that of mesophilic proteins. The thermostability mechanisms for thermozymes are varied and depend on the enzyme; nevertheless, some common features can be identified as contributing to stability. These features include more interactions (i.e. hydrogen bonds, electrostatic interactions, hydrophobic interactions, disulfide bonds, metal binding) than in less stable enzymes and superior conformational structure (i.e. more rigid, higher packing efficiency, reduced entropy of unfolding, conformational strain release and stability of alpha-helix). Understanding of the stabilizing features will greatly facilitate reengineering of some of the mesozymes to more stable thermozymes. 相似文献
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J. G. Zeikus Claire Vieille Alexei Savchenko 《Extremophiles : life under extreme conditions》1998,2(3):179-183
Recent findings on the biochemical and molecular features of the following thermozymes are presented, based on their biotechnological
use: α-amylase and amylopullulanase, used in starch processing; glucose isomerase, used in sweetener production; alcohol dehydrogenase,
used in chemical synthesis; and alkaline phosphatase, used in diagnostics. The corresponding genes and recombinant proteins
have been characterized in terms of sequence similarities, specific activities, thermophilicity, and unfolding kinetics. Site-directed
and nested deletion mutagenesis were used to understand structure–function relationships. All these thermozymes display higher
stability and activity than their counterparts currently used in the biotechnology industry.
Received: January 22, 1998 / Accepted: February 16, 1998 相似文献
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Lin YS 《Proteins》2008,73(1):53-62
Factors that are related to thermostability of proteins have been extensively studied in recent years, especially by comparing thermophiles and mesophiles. However, most of them are global characters. It is still not clear how to identify specific residues or fragments which may be more relevant to protein thermostability. Moreover, some of the differences among the thermophiles and mesophiles may be due to phylogenetic differences instead of thermal adaptation. To resolve these problems, I adopted a strategy to identify residue substitutions evolved convergently in thermophiles or mesophiles. These residues may therefore be responsible for thermal adaptation. Four classes of genomes were utilized in this study, including thermophilic archaea, mesophilic archaea, thermophilic bacteria, and mesophilic bacteria. For most clusters of orthologous groups (COGs) with sequences from all of these four classes of genomes, I can identify specific residues or fragments that may potentially be responsible for thermal adaptation. Functional or structural constraints (represented as sequence conservation) were suggested to have higher impact on thermal adaptation than secondary structure or solvent accessibility does. I further compared thermophilic archaea and mesophilic bacteria, and found that the most diverged fragments may not necessarily correspond to the thermostability-determining ones. The usual approach to compare thermophiles and mesophiles without considering phylogenetic relationships may roughly identify sequence features contributing to thermostability; however, to specifically identify residue substitutions responsible for thermal adaptation, one should take sequence evolution into consideration. 相似文献
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We probe the hypothesis of corresponding states, according to which homologues from mesophilic and thermophilic organisms are in corresponding states of similar rigidity and flexibility at their respective optimal temperatures. For this, the local distribution of flexible and rigid regions in 19 pairs of homologous proteins from meso- and thermophilic organisms is analyzed and related to activity characteristics of the enzymes by constraint network analysis (CNA). Two pairs of enzymes are considered in more detail: 3-isopropylmalate dehydrogenase and thermolysin-like protease. By comparing microscopic stability features of homologues with the help of stability maps, introduced for the first time, we show that adaptive mutations in enzymes from thermophilic organisms maintain the balance between overall rigidity, important for thermostability, and local flexibility, important for activity, at the appropriate working temperature. Thermophilic adaptation in general leads to an increase of structural rigidity but conserves the distribution of functionally important flexible regions between homologues. This finding provides direct evidence for the hypothesis of corresponding states. CNA thereby implicitly captures and unifies many different mechanisms that contribute to increased thermostability and to activity at high temperatures. This allows to qualitatively relate changes in the flexibility of active site regions, induced either by a temperature change or by the introduction of mutations, to experimentally observed losses of the enzyme function. As for applications, the results demonstrate that exploiting the principle of corresponding states not only allows for successful thermostability optimization but also for guiding experiments in order to improve enzyme activity in protein engineering. 相似文献
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Yunqi Li 《Biochemical and biophysical research communications》2010,396(3):736-741
Identification of the characteristic structural patterns responsible for protein thermostability is theoretically important and practically useful but largely remains an open problem. These patterns may be revealed through comparative study on thermophilic and mesophilic proteins that have distinct thermostability. In this study, we constructed several distance-dependant potentials from thermophilic and mesophilic proteins. These potentials were then used to evaluate the structural difference between thermophilic and mesophilic proteins. We found that using the subtraction or division of the potentials derived from thermophilic and mesophilic proteins can dramatically increase the discriminatory ability. This approach revealed that the ability to distinct the subtle structural features responsible for protein thermostability may be effectively enhanced through rationally designed comparative study. 相似文献
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We developed a technique to compute structural entropy directly from protein sequences. We explored the possibility of using structural entropy to identify residues involved in thermal stabilization of various protein families. Examples include methanococcal adenylate kinase, Ribonuclease HI and holocytochrome c(551). Our results show that the positions of the largest structural entropy differences between wild type and mutant usually coincide with the residues relevant to thermostability. We also observed a good linear relationship between the average structural entropy and the melting temperatures for adenylate kinase and its chimeric constructs. To validate this linear relationship, we compiled a large dataset comprised of 1153 sequences and found that most protein families still display similar linear relationships. Our results suggest that the multitude of interactions involved in thermal stabilization may be generalized into the tendency of proteins to maintain local structural conservation. The linear relationship between structural entropy and protein thermostability should be useful in the study of protein thermal stabilization. 相似文献
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Thermostabilizing mutations preferentially occur at structural weak spots with a high mutation ratio
We apply Constraint Network Analysis (CNA) to investigate the relationship between structural rigidity and thermostability of five citrate synthase (CS) structures over a temperature range from 37 °C to 100 °C. For the first time, we introduce an ensemble-based variant of CNA and model the temperature-dependence of hydrophobic interactions in the constraint network. A very good correlation between the predicted thermostabilities of CS and optimal growth temperatures of their source organisms (R2=0.88, p=0.017) is obtained, which validates that CNA is able to quantitatively discriminate between less and more thermostable proteins even within a series of orthologs. Structural weak spots on a less thermostable CS, predicted by CNA to be in the top 5% with respect to the frequency of occurrence over an ensemble, have a higher mutation ratio in a more thermostable CS than other sequence positions. Furthermore, highly ranked weak spots that are also highly conserved with respect to the amino acid type found at that sequence position are nevertheless found to be mutated in the more stable CS. As for mechanisms at an atomic level that lead to a reinforcement of weak spots in more stable CS, we observe that the thermophilic CS achieve a higher thermostability by better hydrogen bonding networks whereas hyperthermophilic CS incorporate more hydrophobic contacts to reach the same goal. Overall, these findings suggest that CNA can be applied as a pre-filter in data-driven protein engineering to focus on residues that are highly likely to improve thermostability upon mutation. 相似文献
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Felsovalyi F Patel T Mangiagalli P Kumar SK Banta S 《Protein science : a publication of the Protein Society》2012,21(8):1113-1125
Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. 相似文献
19.
Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability. 总被引:2,自引:1,他引:1
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D. H. Shin H. K. Song I. S. Seong C. S. Lee C. H. Chung S. W. Suh 《Protein science : a publication of the Protein Society》1996,5(11):2236-2247
Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure. 相似文献
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Haghani K Khajeh K Naderi-Manesh H Ranjbar B 《International journal of biological macromolecules》2012,50(4):1040-1047
It has been lately proposed that the interaction between like-charged residues stabilizes the native state of proteins. To explore this, we created a histidine-histidine pair in the Ca-III binding site of the Bacillus amyloliquefaciens α-amylase (BAA) and then examined the impact of this pairing on the BAA. For this purpose, we used site-directed mutagenesis (SDM) to substitute Pro407 with His, Ala, Gln, Arg, and Glu in the BAA. Subsequently, thermostability, kinetic parameters and structural properties of these variants were measured. Moreover, His-His pairing effect on the BAA thermostability was examined by simultaneous mutation of two residues (P407H/H406A and P407H/H406N). The data exhibited a significant improve in thermostability and structural features of enzyme by His replacement instead of Pro407. Other substitutions in this site did not have a significant effect on the enzyme properties, except for P407R, which yielded a partial improvement. The results also showed that the thermostabilities of double mutants significantly decreased compared with that of the P407H mutant. Moreover, the thermostability of P407H remarkably increased compared with that of other variants even in the absence of Ca(2+). Our data clearly demonstrated that His406-His407 pairing was the major cause for improved thermal stability. 相似文献