Biochemical Characterization of a Carboxylesterase from the Archaeon Pyrobaculum sp. 1860 and a Rational Explanation of Its Substrate Specificity and Thermostability

In this work, genome mining was used to identify esterase/lipase genes in the archaeon Pyrobaculum sp. 1860. A gene was cloned and functionally expressed in Escherichia coli as His-tagged protein. The recombinant enzyme (rP186_1588) was verified by western blotting and peptide mass fingerprinting. Biochemical characterization revealed that rP186_1588 exhibited optimum activity at pH 9.0 and 80 °C towards p-nitrophenyl acetate (K_m: 0.35 mM, k_cat: 11.65 s⁻¹). Interestingly, the purified rP186_1588 exhibited high thermostability retaining 70% relative activity after incubation at 90 °C for 6 h. Circular dichroism results indicated that rP186_1588 showed slight structure alteration from 60 to 90 °C. Structural modeling showed P186_1588 possessed a typical α/β hydrolase’s fold with the catalytic triad consisting of Ser₉₇, Asp₁₄₇ and His₁₇₂, and was further confirmed by site-directed mutagenesis. Comparative molecular simulations at different temperatures (300, 353, 373 and 473 K) revealed that its thermostability was associated with its conformational rigidity. The binding free energy analysis by MM-PBSA method revealed that the van der Waals interaction played a major role in p-NP ester binding for P186_1588. Our data provide insights into the molecular structures of this archaeal esterase, and may help to its further protein engineering for industrial applications.     

Influence of Methanol on Prion Reduction during High Temperature and High Pressure Oleochemical Processes

Prion‐reduction in standard biodiesel processes is caused by acidic and alkaline conditions. In the alternative RepCat biodiesel process, efficient prion‐reduction can be achieved by high‐temperature/pressure. However, a possible increase in thermostability of prions in the presence of methanol during these conditions has not previously been investigated, and is verified in this work. Samples are spiked with prions, treated with methanol, and incubated at 220 °C at 80 bar for 30 min. No traces of protease‐resistant prion protein (as proxy for prions) are detected in tallow or glycerine (as the final by‐product) after treatment. Serial dilutions of spiked prions show at least 6 log₁₀ prion reduction. More importantly, similar effects are detected using milder conditions of 200 °C at 70 bar for 15 min, representing the worst‐case conditions of the process. In conclusion, this study shows that methanol does not increase the thermostability of prions and the RepCat process can efficiently eliminate prions and is therefore safe for the usage of category 1 tallow. Practical Applications: The study further supports the applicability of RepCat process in reducing prion‐contamination in the presence of methanol. Furthermore, the conditions leads to a considerable reduction of prions in glycerine obtained as by‐product.     

COPNA树脂的热稳定性能及残炭率研究进展

COPNA树脂是一类新型热固性高分子材料,固化后的热稳定性非常高,其性能可与目前最耐热的聚酰亚胺树脂相媲美,并具有很高的残炭率,这为其合成耐高温耐摩擦材料奠定了基础.在树脂合成中,合成单体、交联剂、催化剂、反应时间温度、合适的分子结构键型、化学添加剂等因素都会影响其耐热性能及残炭率.本文主要从影响因素方面综述了近年来国内外有关COPNA树脂热稳定性及残炭率的研究进展状况.     

Residue Asn277 Affects the Stability and Substrate Specificity of the SMG1 Lipase from Malassezia globosa

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3₁₀4 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.     

Synthesis and air separation of soluble terpolymers from Aniline,Toluidine, and Xylidine

A series of terpolymers were synthesized by oxidative polymerization of aniline, o-toluidine, and 2,3-xylidine with three monomer ratios using ammonium persulfate as an oxidant in two acidic media. The yield and intrinsic viscosity of the terpolymers were studied by changing the polymerization temperature and medium. The resulting terpolymers were characterized by ¹H–NMR, wide-angle X-ray diffraction, differential scanning calorimetry, thermogravimetry, and constant pressure-variable volume methods. The results showed that the terpolymers are amorphous and exhibit enhanced solubility, high thermostability, and high char yield, greater than 35 wt % at 900°C in nitrogen. A blend membrane of the terpolymer with ethyl cellulose shows good air-separation ability. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 790–798, 2001     

基于随机森林和特征选择方法的蛋白质热稳定性影响因素预测

酶的耐热性对其在食品工业中实现应用至关重要。本文以随机森林算法通过蛋白质序列预测酶的热稳定性,并对影响热稳定性的重要特征进行了分析。计算了从Swiss-Prot数据库获得的1600个包含热稳定性信息的酶的430个特征。采用重复欠抽样法处理数据不平衡问题,采用向后递归特征消去法优选出30个最重要的特征。通过交叉验证和独立测试比较以各特征子集构建的模型,发现仅以氨基酸组成为特征集构建的模型获得了最佳预测效果,模型的总体预测准确率为85.83%、敏感性为89.16%、特异性为73.33%、精度为77.00%、F1度量为74.87%。结果表明氨基酸组成对酶热稳定性的影响最大,嗜热酶中含有更多的谷氨酸、异亮氨酸和赖氨酸,而常温酶中含有更多的谷氨酰胺、丝氨酸和苏氨酸。研究为蛋白质工程改造食品工业用酶的热稳定性提供了一定的理论和方法。     

玉竹挥发油超临界CO2萃取条件及抑菌活性研究

运用超临界流体CO2萃取技术提取玉竹挥发油。采用正交试验研究萃取温度、萃取压力、分离温度、分离压力因素对玉竹挥发油得率的影响,以得率为主要标准,确定最佳工艺条件。结果表明：萃取温度35℃、萃取压力30MPa、分离温度20℃、分离压力9MPa为最佳提取工艺。通过抑菌试验发现,玉竹挥发油对细菌、霉菌、酵母菌、放线菌均有一定的抑菌活性,并具有良好的热稳定性。     

Fe~(2+)和Fe~(3+)对菠萝蛋白酶活性和稳定性的影响

本文研究不同浓度的Fe~(2+)和Fe~(3+)对菠萝蛋白酶活性和60℃下酶稳定性的影响,以及通过圆二色谱检测Fe~(2+)和Fe~(3+)对酶构象的变化,并初步探究EDTA-2Na结合超滤膜法在菠萝蛋白酶制备工艺中清除铁离子的应用效果。结果表明:Fe~(2+)浓度在0~0.75 mmol/L范围内,对菠萝蛋白酶活性有促进作用,以0.5 mmol/L促进效果最佳,但浓度大于0.75 mmol/L时,抑制酶活,且随浓度增加,抑制程度增强。Fe~(3+)对菠萝蛋白酶只有抑制作用,抑制程度与Fe~(3+)浓度成正比,且Fe~(3+)对酶的抑制作用强于Fe~(2+)。60℃下,Fe~(2+)在浓度为0.5 mmol/L对菠萝蛋白酶的稳定性表现出促进作用,延长了酶的半衰期。Fe~(3+)对酶的稳定性呈现抑制作用。Fe~(2+)和Fe~(3+)浓度大于0.5mmol/L时,随离子浓度的升高,对酶稳定性的抑制作用增强。采用圆二色谱检测酶的二级结构表明:Fe~(2+)和Fe~(3+)对酶抑制作用表现为α-螺旋含量下降,β-折叠和β-转角含量稍下降,无规卷曲含量明显提高。EDTA-2Na结合超滤膜法除铁的效果很明显,铁含量(干重)从291.63 mg/kg降低到142.99 mg/kg,铁离子去除率达50.97%,其酶活也由597.27 U/mg上升到808.52 U/mg,酶活提高了26.13%。     

Pseudoalteromonas carrageenovora芳香基硫酸酯酶突变文库热稳定性提高突变体的筛选及鉴定

利用易错聚合酶链式反应技术引入随机诱变,构建一个Pseudoalteromonas carrageenovora芳香基硫酸酯酶突变体库。经过筛选,获得一个芳香基硫酸酯酶热稳定性提高的突变株4-153。序列分析表明,该突变体有2个氨基酸替换,包括D84A和H260L。以对硝基苯硫酸钾为底物,突变酶4-153(M4-153)的最适反应温度为55℃,在45、50、55、60℃处理30 min后,M4-153分别保留85%、83%、48%和13%的残留酶活力。野生型酶(WT)在45、50、55、60℃处理30 min后,分别保留79%、68%、21%和1%的残留酶活力。M4-153与WT相比具有更好的热稳定性。M4-153的最适反应pH值为8.0,在pH 5.0~9.0范围内保持稳定。EDTA对突变酶的抑制作用表明,金属离子在突变酶的催化过程中起重要作用。M4-153对一些洗涤剂,包括Triton X-100、Tween 20、Tween 80和Chaps,有好的耐受性。M4-153对龙须菜粗多糖硫酸基团的脱硫率为79.5%。