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101.
The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5–7.0. β-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression. 相似文献
102.
Habibollah Faraji Mohammad Ramezani Baratali Mashkani Hamid R. Sadeghnia Hamid M. Benhangi Saeed Hosseini Teshnizi Fatemeh Soltani 《Biotechnology progress》2019,35(4):e2819
Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure. 相似文献
103.
Optimization of fed-batch feeding parameters was explored for a system with multiple mechanisms of product inactivation. In particular, two separate mechanisms of inactivation were identified for the recombinant tissue-type activator (r-tPA) protein. Dynamic inactivation models were written to describe particular r-tPA glycoform inactivation in the presence and absence of free-glucose. A glucose-independent inactivation mechanism was identified, and inactivation rate constants were found dependent upon the presence of glycosylation of r-tPA at N184. Inactivation rate constants of the glucose-dependent mechanism were not affected by glycosylation at N184. Fed-batch optimization was performed for r-tPA production by CHO cell culture in a stirred-tank reactor with glucose, glutamine and asparagine feed. Feeding profiles in which culture supernatant concentrations of free-glucose and amino acids (combined glutamine and asparagine) were used as control variables, were evaluated for a wide variety of set points. Simulation results for a controlled feeding strategy yielded an optimum at set points of 1.51 g L(-1) glucose and 1.18 g L(-1) of amino acids. Optimization was also performed in absence of metabolite control using fixed feed-flow rates initiate during the exponential growth phase. Fixed feed-flow results displayed a family of optimum solutions along a mass flow rate ratio of 3.15 of glucose to amino acids. Comparison of the two feeding strategies showed a slight advantage of rapid feeding at a fixed flow rate as opposed to metabolite control for a product with multiple mechanisms of inactivation. 相似文献
104.
105.
The prediction of the complex structure of a small ligand with a protein, the so-called protein–ligand docking problem, is
a central part of the rational drug design process. For this purpose, we introduce the docking algorithm PLANTS (Protein–Ligand
ANT System), which is based on ant colony optimization, one of the most successful swarm intelligence techniques. We study
the effectiveness of PLANTS for several parameter settings and present a direct comparison of PLANTS’s performance to a state-of-the-art
program called GOLD, which is based on a genetic algorithm and frequently used in the pharmaceutical industry for this task.
Last but not least, we also show that PLANTS can make effective use of protein flexibility giving example results on cross-docking and virtual screening experiments for protein kinase A.
This article is based on a paper that won the best paper award at ANTS 2006, the 5th International Workshop on Ant Colony
Optimization and Swarm Intelligence held in Brussels, Belgium, 2006. This article includes new types of experiments and also
the possibility of considering flexibility of protein side-chains. 相似文献
106.
α-Crystallin is known to act as a molecular chaperone by preventing the aggregation of partially unfolded substrate proteins.
It is also known to assist the refolding of a number of denatured enzymes, but the activity yield is often less than 20%.
In this paper, we have tried to tune the refolding ability of α-crystallin in vitro by optimizing various external parameters. We wanted to find out the best possible condition under which it can exhibit maximum refolding capacity. We found that under
suitable condition in vitro α-crystallin can refold denatured malate dehydrogenase, carbonic anhydrase and lactate dehydrogenase to recover more than
40% activity. We also measured the effect of several external factors such as nucleotides, osmolytes, electrolytes, temperature
etc. on the in vitro α-crystallin mediated reactivation of above stated enzymes. We found that nucleotides and electrolytes had little effect
on the refolding ability of α-crystallin. However, in presence of different osmolytes, we found that its ability to reactivate
denatured substrate proteins enhanced significantly. Refolding in presence of pre-incubated α-crystallin reveals that hydrophobicity
had stronger influence on the refolding capacity of α-crystallin than its oligomeric size. 相似文献
107.
de Duve C 《化学与生物多样性》2007,4(4):574-583
Life is the product of chemistry, which obeys deterministic laws, and of natural selection, which operates on variants offered to it by chance, but may, in a number of cases, have been provided with a sufficiently extensive array of variants to be optimizing. Thus, the origin and evolution of life have been largely shaped by the contingency of environmental conditions. The possibility remaining open for consideration is that certain critical conditions are sufficiently reproducible for life to arise and even to evolve into conscious, intelligent beings elsewhere in the universe. 相似文献
108.
109.
为了探讨虎耳草总酚(TP)与抑制五步蛇毒中磷脂酶A2(PLA2)之间的关系,本研究通过单因素试验及Box-Behnken试验对虎耳草TP的提取条件与抑制五步蛇毒中PLA2活性进行优化,考察各试验因素对超声辅助提取虎耳草TP得率及其对五步蛇毒PLA2活性抑制率(PIR)的作用,并对虎耳草TP含量与PIR的相关性进行分析。结果表明,响应面法提取虎耳草TP和PIR的最佳工艺条件为:粒径为60目,料液比为1∶50,超声功率为250 W,超声时间为1.8 h。此优化工艺条件下虎耳草TP得率为(8.69±0.46) mg·g-1,PIR为(40.91±0.21)%;相关性分析结果显示虎耳草TP含量与PIR具有极显著正相关(P<0.01),说明TP可能为虎耳草抑制五步蛇毒中PLA2活性的物质基础。 相似文献
110.
Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine. 相似文献