理性设计提高蛋白质热稳定性的研究进展

通过理性设计提高蛋白质的热稳定性一直是当今计算生物学及蛋白质工程领域中的一个研究热点。与传统的定向进化的方法相比,该方法具有目的性强、效率高的优点,对扩大蛋白质的应用范围与探究蛋白质结构和功能的关系均具有重要意义。本文详细介绍了影响蛋白质热稳定性的因素,以及一些常用的通过理性设计来提高蛋白质的热稳定性的策略。由于影响蛋白质热稳定性的因素众多,并且众多因素之间还具有千丝万缕的联系,到目前为止研究人员还没有提出一个公认的适合于所有蛋白质的理性设计的策略,这也是现代计算生物学家及蛋白质工程学家们努力研究的一个重要方向。     

理性设计二硫键提高华根霉脂肪酶热稳定性

【背景】华根霉脂肪酶的工业应用前景广泛,但是受到酶热稳定性较差的限制。【目的】对华根霉Rhizopus chinensis CCTCC M201021脂肪酶r27RCL分子结构进行理性设计,以提高该酶热稳定性。【方法】以Disulfide by design软件筛选r27RCL分子表面能够形成二硫键的突变位点,共得到7对二硫键突变。利用全质粒PCR进行定点突变,并在毕赤酵母中表达获得突变酶。【结果】最佳突变酶m9/10 (S85C-Q145C)与野生型酶r27RCL相比,60°C下的半衰期分别提高了4.5倍,T_m值提高了4.2°C,而催化活性保持不变。蛋白质晶体结构模拟显示,位于β2折叠上的85C和位于α4螺旋上的145C可形成二硫键,从而提高酶的热稳定性。【结论】酶分子中引入新增二硫键可以显著提升酶的热稳定性。     

半理性设计进化土曲霉来源的ω-转氨酶AtTA热稳定性

转氨酶(ω-transaminase,ω-TA)作为一种天然的生物催化剂,在手性胺类化合物的合成中具有较好的应用前景。但ω-TA在催化非天然底物的反应过程中存在稳定性差、活性低的缺陷,大大限制了ω-TA的应用。为改善此缺陷,针对来源于土曲霉(Aspergillus terreus)的(R)-ω-TA(At TA),采用基于分子动力学模拟的计算机辅助设计与随机突变、组合突变相结合的策略进行酶的热稳定性改造,获得了热稳定性与活性同步提高的最佳突变酶At TA-E104D/A246V/R266Q (M3)。与At TA野生酶(wild-type, WT)相比,M3的半衰期t_1/2 (35℃)由17.8 min提升至102.7 min,提升了4.8倍,半失活温度T₅₀¹⁰比WT (38.1℃)提高2.2℃。最佳突变酶M3对丙酮酸和1-(R)-苯乙胺的催化效率分别是野生酶的1.59倍和1.56倍。分子动力学模拟与分子对接结果表明,分子内氢键与疏水相互作用的增加所导致α-螺旋的加固稳定是酶热稳定性提升的主要原因;底物分子与结合口袋氨...     

牛肠激酶催化亚基工程菌培养条件的探索与鉴定

目的：探索牛肠激酶催化亚基工程菌高效表达的培养条件。方法：牛肠激酶催化亚基基因的初步表达,筛选构建工程菌,从以下方面入手优化培养条件：培养基的组成、摇瓶发酵培养条件、培养基的PH、种龄、接种量、培养时间等,并且通过Western Blotting和SDS-PAGE等鉴定不同条件下的实验结果,从而确定最佳培养条件。结果：通过鉴定实验结果,从不同种平行培养条件中选择产量最高的培养条件,作为最优培养条件。结论：成功地探索并鉴定了工程菌的适宜培养条件。     

牛肠激酶催化亚基工程菌培养条件的探索与鉴定

目的:探索牛肠激酶催化亚基工程菌高效表达的培养条件。方法:牛肠激酶催化亚基基因的初步表达,筛选构建工程菌,从以下方面入手优化培养条件:培养基的组成、摇瓶发酵培养条件、培养基的PH、种龄、接种量、培养时间等,并且通过Western Blotting和SDS-PAGE等鉴定不同条件下的实验结果,从而确定最佳培养条件。结果:通过鉴定实验结果,从不同种平行培养条件中选择产量最高的培养条件,作为最优培养条件。结论:成功地探索并鉴定了工程菌的适宜培养条件。     

米根霉α-淀粉酶热稳定性的理性设计

真菌α-淀粉酶被广泛应用于麦芽糖浆生产工业,但其热稳定性普遍较差,在制糖工艺中增加了由于酶活力损失而引起的追加生产成本。在充分研究了热稳定性对于真菌α-淀粉酶应用于工业生产的重要性的基础上,为提高米根霉α-淀粉酶(ROAmy)的热稳定性,基于酶蛋白B-factor分析和分子动力学模拟,利用重叠PCR技术分别对ROAmy中的3个氨基酸残基G128、K269和G393进行了单点突变及组合突变。结果表明,所获得的7个突变体均比原酶具有更好的热稳定性,其中效果最好的为组合突变体G128L/K269L/G393P,其在55℃下的热失活半衰期(t_(1/2))约为原酶的5.63倍。同时,该突变体的最适温度由50℃提高到了65℃,最大反应速率(V_(max))和催化效率(k_(cat)/K_m)分别提高了65.38%和99.86%。通过蛋白结构功能比较分析,发现氢键数目的增多或脯氨酸在特殊位置中的引入可能是突变体热稳定性得到提高的主要因素。     

定向进化和半理性设计改造顺式环氧琥珀酸水解酶

【背景】D(-)-酒石酸是非天然有机酸,在保健品、食品和肿瘤药物合成等行业具有重大应用潜力,目前主要通过生物转化法生产,即顺式环氧琥珀酸水解酶[cis-epoxysuccinic acid hydrolase,CESH(D)]水解顺式环氧琥珀酸(cis-epoxysuccinic acid, ESH)生成D(-)-酒石酸。该法简单温和,但存在CESH(D)酶活转化效率低下的瓶颈问题。【目的】通过基因工程改造,提高CESH(D)的酶活力、温度和pH稳定性。【方法】利用定向进化和半理性设计体外改造CESH(D),高通量筛选出正向突变体;然后对其进行酶学性质研究,包括比酶活、温度和pH对酶催化效率的影响、酶的温度稳定性、pH稳定性及酶促动力学分析;最后通过分子对接等手段分析突变位点影响催化活性的初步机制。【结果】筛选获得4个正向突变体L231P/N226S、V77I、D183E和T223S。与野生型相比,4个突变体的比酶活分别提高2.2、1.6、1.5和1.4倍。其中,L231P/N226S的温度稳定性和pH稳定性较野生型均有显著提高,55℃时催化活性为野生型的1.6倍,pH 6.0时催化活...     

糖苷酶耐热性改造策略与应用

糖苷酶作为绿色温和的生物催化剂,能够水解包含糖苷、寡糖、多糖等在内的各种含糖化合物的糖苷键生成具有高生理和药理活性的衍生物,在食品、医药等工业领域应用广泛.而工业应用的糖苷酶经常需在高温条件下进行催化,以提高反应效率并减少污染,但大多数糖苷酶属于中温酶,在实际生产条件下的活性较低且损失较快,因此,提高糖苷酶在高温下的稳...     

理性设计提高酯合成催化反应脂肪酶的热稳定性

【背景】南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)具有优异的酯合成活性,是在非水相催化中应用极为广泛的工业用酶。【目的】在保留CALB优秀催化性能的基础上,提高CALB的热稳定性。【方法】采用预测软件PoPMuSiC和FoldX计算CALB潜在热稳定性突变位点,并根据氨基酸残基的空间位置进一步筛选。利用重叠延伸PCR技术在基因calb中引入10个单点突变,于毕赤酵母GS115中表达。【结果】点突变A146G、A151P、L278M均能有效提高CALB的热稳定性。在单点突变的基础上,组合突变体A146G-L278M和A146G-L278M-A151P的热稳定性得到进一步提高。与野生型相比,突变体A146G-L278M和A146G-L278M-A151P的最适反应温度均提高了5°C,T_m值分别提高了3.3°C和4.2°C。此外,合成己酸乙酯的酶促反应动力学分析表明,相比于野生型,突变体A146G-L278M和A146G-L278M-A151P对己酸和乙醇均具有更高的亲和力,且对己酸的催化效率k_(catA)/K_(m A)是野生型的4.1倍。通过分子动力学模拟,从分子水平阐明了突变体A146G-L278M和A146G-L278M-A151P热稳定性提高的机制。【结论】本研究采用的理性设计策略对提高CALB的热稳定性是行之有效的,该策略可作为其他工业用酶提高热稳定性的参考。     

Engineering proteins for thermostability through rigidifying flexible sites

Engineering proteins for thermostability is an exciting and challenging field since it is critical for broadening the industrial use of recombinant proteins. Thermostability of proteins arises from the simultaneous effect of several forces such as hydrophobic interactions, disulfide bonds, salt bridges and hydrogen bonds. All of these interactions lead to decreased flexibility of polypeptide chain. Structural studies of mesophilic and thermophilic proteins showed that the latter need more rigid structures to compensate for increased thermal fluctuations. Hence flexibility can be an indicator to pinpoint weak spots for enhancing thermostability of enzymes. A strategy has been proven effective in enhancing proteins' thermostability with two steps: predict flexible sites of proteins firstly and then rigidify these sites. We refer to this approach as rigidify flexible sites (RFS) and give an overview of such a method through summarizing the methods to predict flexibility of a protein, the methods to rigidify residues with high flexibility and successful cases regarding enhancing thermostability of proteins using RFS.     

疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性

目的:通过对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础.方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的...     

Thermostability Engineering of Fungal Phytases Using Low-Mr Additives and Chemical Crosslinking

With the ultimate goal to develop preparations of phytase (myo-inositol hexakisphosphate phosphohydrolase) with improved thermal resistance for inclusion in animal feed, several thermostabilization approaches were investigated with a set of fungal (Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger phytase) and consensus phytases. Screening of different low-M_r additives revealed that polyethylene glycols increase the thermostability of all phytases in a molecular weight-dependent fashion. The polyols ribitol, xylitol (C₅ sugars) and sorbitol (C₆ sugar) also improved their thermostability, whereas polyols containing more or less carbon atoms, such as glycerol, erythritol and mannoheptulose, showed only minor effects. The stabilizing effects of PEGs and polyols were concentration dependent. In a second series of experiments, crosslinking of the carbohydrate chains of A. fumigatus and consensus phytase using sodium periodate and adipic acid dihydrazide resulted in the formation of oligomeric forms, which may explain the observed thermostability enhancement of 10–15d`C.     

Rational and Combinatorial Engineering of the Glucan Synthesizing Enzyme Amylosucrase

Rational engineering of amylosucrase required detailed investigations of the molecular basis of catalysis. Biochemical characterization of the enzyme coupled to structural analyses enabled the polymerization mechanism to be elucidated. This provided key information for successfully changing amylosucrase to a hydrolase or an improved polymerase using site-directed mutagenesis. In parallel, a combinatorial approach was developed to further improve the catalyst. The method, based on random mutagenesis and recombination of parent sequences, has generated libraries of variants. These were searched for improved polymer formation using a first step of selection followed by a screening test including a double detection method. Several mutants with desired properties have been isolated, their sequences revealed that they could not have been designed following a rational approach.     

Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in T_m. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability.     

P197E与ep8叠加突变对扩展青霉脂肪酶热稳定性的影响

为提高脂肪酶的热稳定性,作者利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行了体外定点突变,构建了P197E(即将第197位的脯氨酸突变为谷氨酸)与随机突变体ep8叠加突变的重组质粒pPIC3.5K-ep8-P197E。将该质粒电转化至毕赤酵母Pichiapastoris GS115中,进行异源表达。与野生型酶和单点突变酶PEL-ep8的酶学性质比较,结果表明:叠加突变体PEL-ep8-P197E在40°C温育处理30min后,残余酶活分别比野生型PEL和随机突变体PEL-ep8提高了42.13%和37.3%。叠加突变体PEL-ep8-P197E的Tm值为41.51°C,比野生型酶PEL提高了2.81°C,比随机突变体脂肪酶PEL-ep8提高了2.25°C。通过对脂肪酶PEL的叠加突变,提高了该酶的热稳定性,并为结构与功能的进一步研究提供了材料。     

Thermostability improvement of maltogenic amylase MAUS149 by error prone PCR

The thermostability of maltogenic amylase from Bacillus sp. US149 (MAUS149) was improved by random mutagenesis using error prone PCR. The library constructed for the mutants obtained was subjected to screening, leading to the selection of a thermostable mutant enzyme named MA-A27. The latter was noted to contain four single mutations, namely D46V, P78L, V145A, and K548E. The half-life times recorded for MA-A27 at 50 °C and 55 °C were 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively. The results from molecular modeling attributed the increase in thermostability observed for MA-A27 to P78L and K548E substitutions that led to new hydrogen bond and salt bridge formations. Further site-directed mutagenesis studies showed that the P78L and K548E single mutations underwent an increase in thermostability, thus confirming the joint contribution of both substitutions to the increase in thermostability observed for MA-A27.