D-泛解酸内酯水解酶的定向进化

易错PCR结合DNA改组方法向D-泛解酸内酯水解酶基因中引入突变,并构建突变体库。利用酶的催化特点和产物特性建立了基于平板初筛和高效液相复筛的两步法D-泛解酸内酯水解酶活性筛选系统。用该筛选系统以酶活力和pH稳定性为指标对突变体库进行筛选,最终获得一株酶活力高且在低pH条件下稳定性好的突变体Mut E-861。该突变体的酶活力是野生型酶的5.5倍。对突变体和野生型酶在pH 6.0和pH 5.0条件下的残余酶活进行对比,在这两种pH条件下,突变体酶的酶活残留分别为75%和50%,而野生型酶只能保持原来的40%和20%。通过软件对突变体Mut E-861酶基因和野生型酶基因进行分析对比,发现突变体Mut E-861酶基因发生了三处点突变,其中突变使两处氨基酸取代,另一处为沉默突变,未引起氨基酸的变化。     

谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用

目的:建立谷氨酸依赖型氨基转移酶-谷氨酸脱氢酶偶联反应的96孔板高通量筛选方法,并用于大肠杆菌氨基转移酶Wec E突变库的筛选。方法:通过优化偶联指示酶-谷氨酸脱氢酶、信号分子NADH浓度及双酶偶联反应时间,建立了光学法测定氨基转移酶活性的氨基转移酶-谷氨酸脱氢酶偶联反应方法;通过定点饱和突变技术构建了大肠杆菌氨基转移酶WecE的突变库;采用96孔板高通量初筛、摇瓶复筛获得了高活性的转氨酶突变体,并对纯化的突变体进行催化活力分析。结果:建立了谷氨酸依赖型氨基转移酶目标反应与0.5 U/ml L-谷氨酸脱氢酶和0.4 mmol/L NADH信号指示反应相偶联的筛选方法;构建了氨基转移酶WecE Tyr 321饱和突变库,通过96孔板高通量筛选,获得了催化活性比野生型提高3.4倍的突变体Y321F。结论:所建立高通量筛选方法背景干扰小,准确性高,为谷氨酸依赖型氨基转移酶分子进化提供了可行性方案。     

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...     

高通量筛选工具的设计与应用

定向进化方法作为新兴的高效蛋白质工程手段,其内容包括蛋白质突变体文库的构建和有效突变体的快速筛选。高通量筛选方法是定向进化方法的重要组成部分,是成功获得有效突变体的关键。筛选的突变体数量越多,获得有效突变体的几率越大。以下介绍了目前已经成功应用于或有潜力应用于定向进化改造蛋白质的几种高通量筛选工具。高通量筛选工具的不断设计与开发将推动蛋白质工程领域的技术革新。     

基于体外分子进化技术提高弯曲芽孢杆菌CCTCC 2015368 β-淀粉酶的热稳定性

运用体外分子进化技术易错PCR方法,高通量筛选热稳定性提高的弯曲芽孢杆菌Bacillus flexus CCTCC2015368β-淀粉酶突变体。利用LB琼脂淀粉板显色、96-孔板DNS法测酶活和酶标仪检测等,最终筛选到了一株热稳定性显著提高的突变体D476N。野生型和突变体D476N分别纯化后,酶学性质测定表明:突变体D476N的最适pH为6.5,与野生型相比降低了0.5。突变体D476N和野生型的最适温度均为55℃,突变体D476N在55℃下的半衰期为35 min,比野生型提高了95%。突变体D476N的T_(50)值比野生型提高4℃。突变体D476N的K_m值为97.98μmol/L,是野生型(85.86μmol/L)1.14倍;突变体稳定性提高的同时,催化活力相对于野生型有略微下降。通过SWISS-MODEL同源模拟野生型和突变体D476N的三维结构,并通过PyMol软件分析,发现突变后的氨基酸残基Asn476位于蛋白质表面的loop环上,通过MOE软件计算,D476N的分子自由能(ΔG)为106.01kcal/mol,比野生酶降低10.3%,这一结果与蛋白质分子自由能和热稳定性呈负相关的理论相符。     

杰氏棒杆菌L-天冬氨酸α脱羧酶半理性改造及全细胞催化合成β-丙氨酸

β-丙氨酸是多个药物合成的重要砌块，可以通过天冬氨酸α脱羧酶（Pan D）催化L-天冬氨酸脱羧来合成，但普遍在用的Pan D酶活性不高是制约全细胞催化合成β-丙氨酸的瓶颈。因此，本研究通过酶的挖掘，选择将杰氏棒杆菌来源（Corynebacterium jeikeium）Pan D在Escherichia coli中异源表达。对杰氏棒杆菌来源Pan D进行Alaph Fold2建模和分子对接，采用Rosetta虚拟突变确定突变热点，结合薄层层析初筛和纯化后复筛，最终筛选到突变体L39A，其比酶活为13.45 U/mg，相比野生型酶的比酶活（9.6 U/mg）提升了1.4倍。酶学性质表征数据表明，野生型酶和L39A突变体最适p H均为6.5，且在p H 6.0-7.0之间酶活性稳定；两者最适温度为55℃，但L39A热稳定性较野生型提高；突变体酶的催化效率比野生型提升了1.4倍。对突变体进行结构解析发现，39位取代为侧链基团更小的丙氨酸，亲水性增强，增加了关键催化氨基酸58位酪氨酸与其他氨基酸的相互作用，使活性中心周围的区域稳定性提高，从而提高了催化活性。全细胞催化数据表明，在OD600=4...     

基于结构B因子分析指导的头孢菌素C酰化酶动力学稳定性改造

【目的】筛选Pseudomonas sp.SE83 acy Ⅱ定点饱和突变库,获得动力学稳定性提高的头孢菌素C(CPC)酰化酶突变体,并对突变酶进行初步的结构-功能关系分析。【方法】靶标酶Pseudomonas sp.SE83 acy Ⅱ与Pseudomonas diminuta N176具有较高的同源性,通过分析N176的结构B因子,构建CPC酰化酶SE83定点饱和突变库;基于pH指示剂显色法,采用Biomek FX~P自动工作站建立CPC酰化酶高通量筛选方法,获得优良突变酶,对其活性、稳定性等酶学性质进行表征;利用SWISS-MODEL对突变体进行同源建模,探讨突变体结构与功能的关系。【结果】通过B因子分析和同源结构比对,共找出9个靶标位点;经过3轮筛选,发现R218及K226位点突变显著提高酶的热稳定性,其中最显著的R218Q和K226V在40°C的半衰期分别为野生型的3.77和2.77倍,催化效率k_(cat)/K_m分别为野生型的1.8和3.1倍。同源建模分析表明氢键作用和疏水相互作用的增加可能是突变体稳定性提高的原因。【结论】B因子指导的酶分子改造是一种高效可靠的动力学稳定性改造策略,突变体R218Q和K226V均可提高CPC酰化酶的稳定性和催化效率,对进一步的CPC酰化酶分子改造具有一定的参考价值和指导意义。     

耐热植酸酶突变体的筛选及性质研究

目的:对大肠杆菌Escherichia coli植酸酶基因进行定向进化,获得热稳定性提高的植酸酶突变体。方法:利用易错PCR技术和96微孔板高通量筛选方法获得突变体基因,并对突变酶进行异源表达、纯化及性质研究。结果:通过筛选获得3株热稳定性明显提高的植酸酶突变体APPA1、APPA2、APPA3。酶学性质分析结果表明,3个突变体分子量均约为55kDa,最适pH均为4.5,与野生型无明显差别,热稳定性较野生型均有显著提高,其中突变体APPA3的最适温度为65℃,较野生酶提高5℃,在90℃处理10min后保留50%的酶活。酶的三维结构模拟显示,5个突变位点在植酸酶整体结构上均引入新氢键。结论:通过定向进化获得热稳定性提高的大肠杆菌Escherichia coli植酸酶突变体,对植酸酶的工业应用和研究植酸酶结构与功能关系具有重要意义。     

基于磷脂酶水解圈定向筛选高效聚对苯二甲酸乙二醇酯降解酶

【目的】目前自然环境中聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)废弃物的积累严重威胁生态健康,因此PET的降解问题已成为全球性的热点问题。生物酶法降解PET技术以其绿色环保而备受关注,但天然PET降解酶的催化活性普遍偏低,亟待进一步定向改造。现阶段定向进化为快速提高PET降解酶催化性能提供了可能,其中筛选方法是成功获得高性能突变体的关键所在。本研究旨在提出一种新型高效灵敏的筛选方法并应用于褐色喜热裂孢菌(Thermobifida fusca)来源角质酶Tfu-0883的定向改造,以期快速获得PET降解活性提高的突变体。【方法】基于易错PCR构建突变体文库,涂布于卵黄磷脂平板,以水解圈的大小作为筛选指标获得PET降解活性提高的突变体;对突变体进行酶学定性并筛选出潜在的分子改造位点,最终获得高性能突变体。【结果】从卵黄磷脂平板中挑取水解圈直径最大的单菌落,即突变体H10(N2D/D94H/A149E),其PET降解能力是野生型的1.5倍,最适温度与pH分别为60℃和8.0。突变体H10中第2位和第149位氨基酸残基远离底物结合凹槽,其突变会导致酶蛋白稳定性下降;第94位氨基酸残基则位于底物结合凹槽附近,由负电荷氨基酸Asp突变为正电荷氨基酸His,有利于吸附在带负电荷的PET表面,是突变体H10降解能力提升的关键因素;随后将野生型的第94位氨基酸残基Asp分别突变为His及同为正电荷且空间位阻更小的Lys和Arg,突变体D94H、D94K和D94R对PET降解能力均有提升,其中,突变体D94K降解PET能力是野生型的3.6倍。【结论】本研究基于磷脂酶水解圈构建了一种新的PET降解酶定向筛选方法,以此获得了降解活性提高的突变体,并证实角质酶Tfu-0883第94位氨基酸残基位点具有提升其PET降解活性的潜在能力。     

通过定点突变提高纳豆激酶纤溶活性和热稳定性

纳豆激酶（Nattokinase, NK）是一种纤溶酶，可溶解血栓，常用于治疗心血管疾病（CVDs）。然而，野生型NK往往表现出很少的纤溶能力和较低的热稳定性，针对如何提高NK的热稳定性和活性开展研究。使用重叠延伸PCR法将NK中位于194位的Ser替换为Pro，为验证突变后的热稳定性，将野生型NK和突变体S194P均在不同温度下孵育相同时间，使用纤维蛋白板法测定二者酶活，验证热稳定性。成功构建了pET-26b-NK_S194P，测量酶活结果显示，野生型NK和突变体S194P酶活分别达到101.30 IU/mL和123.23 IU/mL，结果表明突变体S194P的酶活比野生型NK高（18.10±2）%，将野生型NK和突变体S194P在60~65 ℃温度下孵育30 min后测量酶活，野生型NK在63 ℃时丧失酶活，突变体S194P在65 ℃时丧失酶活，耐热温度提高2 ℃。结果表明，突变体S194p的蛋白结构在突变后发生了改变，使NK提高了耐热温度。     

棉花枯萎病菌突变体库的构建及分析

[背景]棉花枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害,但关于棉花枯萎病菌的致病力、产孢量、生长速度及颜色变化等相关功能基因目前还不是十分明确.[目的]通过构建绿色荧光蛋白(green fluorescent protein,GFP)标记棉花枯萎病菌突变体库,筛选出由于T-DNA的随机插入而导致性状发生变异的突变体...     

裂殖壶菌高产油突变体的高通量筛选

二十二碳六烯酸(DHA)具有促进婴幼儿大脑和视网膜发育等多种生理功能,被广泛应用于食品、医药和养殖等行业。为了获得适合于工业化生产的高产油、高产DHA的裂殖壶菌工程株,文中建立了一套操作简单、快速准确的基于尼罗红染色的高通量筛选方案。首先利用紫外线(UVC)诱变的方式快速构建裂殖壶菌的随机突变体库。然后采用优化后的筛选条件如裂殖壶菌的最佳尼罗红染色条件(二甲基亚砜浓度为20%,尼罗红终浓度为2.0μg/mL,孵育时间为10 min,孵育温度为40℃)和更合理的筛选依据(多功能酶标仪实现高通量测量的单位细胞密度油脂量)等,对3 648株突变体进行筛选,得到了3株高产油突变体(D03432、D05106和D01521)。摇瓶发酵实验表明,这3株突变体在生物量、油脂含量和DHA产量上均高于野生型菌株,其中突变体D03432和D05106的油脂量分别达到了干重的64.74%和63.13%,远高于野生型菌株的43.19%。而且这两株突变体的DHA产量分别是野生型菌株的2.26倍和2.37倍。最后,对突变体D03432和D05106进行了5 L发酵罐发酵培养,相较于野生型菌株,这两株突变体不仅生物量和油脂含量有所增加,而且DHA产量更是分别增加了45.5 1%和66.46%,展现出较好的工业应用潜力。此外,本筛选方案对其他产油微生物高产油突变体的高通量筛选具有借鉴作用。     

Nisin高产菌株的高通量筛选

【目的】乳酸链球菌素(nisin)是一种天然生物活性抗菌肽,对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,而用作食品的防腐剂。本研究通过建立高通量筛选方法,实现高效快速省力的高产菌株筛选,为工业上筛选高产菌株提供研究方案。【方法】通过对Lactococcus lactis ATCC11454菌株进行紫外诱变,获得2511株突变株。利用Biomek FXP自动工作站建立96微孔板的高通量筛选方法,突变株经高通量挑选、菌种培养及菌液稀释后,加入到生长至对数中期的藤黄微球菌中,采用改进后的比浊法快速检测nisin生物活性。用此方法对突变株进行初筛、复筛后可得到nisin高产菌株,并通过摇瓶发酵评估高通量筛选方法。【结果】确定比浊法检测的条件为:nisin活性稀释在10–25 IU/m L范围内,与藤黄微球菌反应2 h后检测藤黄微球菌的菌体量(OD600)。2511株突变株经过2轮高通量筛选,最终获得约50株产量提升的菌株,对其中8株进行摇瓶精确测量,显示产量均有提高,并且其中一株产量提升了30%,成功建立了高通量筛选nisin高产菌株的方法。【结论】利用比浊检测法,在其基础上成功建立高通量筛选高产nisin菌的方法,经过初筛复筛,整个周期由1人耗时5 d即可完成2511株突变株的筛选工作。相较于传统的选育方法,高通量筛选具有快速、稳定、高效的特点,提高了筛选效率,缩短了选育周期,是工业上筛选高产nisin菌的有效手段。     

构建葡萄糖二酸指示系统筛选肌醇加氧酶突变株

葡萄糖二酸是一种高附加值天然有机酸,已经广泛应用于疾病防治、生产聚合物材料等领域。在葡萄糖二酸的合成途径中,肌醇加氧酶MIOX所催化的肌醇转换为葡萄糖醛酸的过程是整个途径的限速步骤。通过应用将葡萄糖二酸浓度与绿色荧光蛋白荧光强度相结合的筛选系统,从突变体文库中筛选出3株有潜力的肌醇加氧酶突变体(K59V/R60A、R171S和D276A),使MIOX活性得到提高。重组菌株Escherichia coli BL21(DE3)/MU-R171S的葡萄糖二酸产量相比于未突变菌株提高了36.5%。     

茎瘤固氮根瘤菌环二鸟苷酸代谢相关基因的功能鉴定

【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。     

Identifying functional residues in Arabidopsis thaliana zeta class glutathione S-transferase through screening inactive point mutants

The functional residues of z-class glutathione S-transferase were identified by screening inactive point mutants from a random mutagenesis library. First, a random mutant library was constructed using error-prone polymerase chain reaction, and then candidate inactive mutants were screened by a high-throughput colorimetric assay. Twenty-five mutants were obtained, and 12 that formed inclusion bodies were discarded. The remaining 13 mutants that expressed soluble protein were used for accurate quantification of enzymatic activity and sequencing. The mutants W15R, C19Y, R22H/K83E, P61S, S73P, S109P, and Q112R were found to have activity lower than 1% of the wild-type and were considered as “inactive mutants”, whereas the mutants K83E, Q102R, and L147F still have a large fraction of the activity and were thus considered as “partially inactivated mutants”. Molecular modeling experiments disclosed that mutations resulting in inactivation of the enzyme were found in or near the binding pocket, whereas mutations resulting in partial inactivation were distant from both substrates. The role of the residue Ser73 in the enzyme was verified by site-directed mutagenesis. The result suggested that screening inactive point mutants from a random mutagenesis library is an efficient way of identifying functional residues in enzymes.     

Modifying Thermostability of appA from Escherichia coli

In order to improve the thermostability of Escherichia coli AppA phytase, Error-prone PCR was used to randomize mutagenesis appA gene, and a gene mutation library was constructed. A mutant I408L was selected from the library by the method of high-throughput screening with 4-methyl-umbelliferylphosphate (4-MUP). The appA gene of the mutant was cloned and expressed in E. coli Origami (DE3). The recombinant protein was purified by Ni-affinity chromatography, and the enzymatic features were analyzed. The results indicated that AppA phytase activities of mutant I408L and wild-type (WT) strain remained at 51.3 and 28%, respectively, after treatment at 85°C for 5 min. It means that the thermostability enhancement of AppA phytase I408L was 23.3% more as compared with WT. The K _m of both phytase were 0.18 and 0.25 mM, respectively, which indicated that the catalyzing efficiency of I408L was improved. AppA phytase of mutant I408L showed a significant enhancement against trypsin, which was nearly three times compared with WT. In addition, AppA phytase of mutant could be activated by Mg²⁺ and Mn²⁺; in contrast, it could be inhibited by Ca²⁺, Co²⁺, Cu²⁺, and K⁺ in varying degrees, and the enzymatic activity was almost lost the presence of Fe³⁺ and Zn²⁺. It appears that screening thermotolerant phytase of E. coli by high throughput screening with a fluorescence substrate is a fast, simple, and effective method. The mutant I408L obtained in this study could be used for the large-scale commercial production of phytase.     

Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9^th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.     

Screening of engineered Pichia pastoris mutant with enhanced production of a functional rFIP-glu protein and investigating its potential bioactivities

An engineered Pichia pastoris GS115 with a FIP-glu gene was mutated using ultraviolet (UV) radiation, and a high-throughput screening method was established for screening of high-yield strains. Meanwhile, a preliminary study was conducted to determine the bioactivity of the rFIP-glu. Based on OD₆₀₀ value and the mortality of engineered P. pastoris GS115, the best UV irradiation time was determined. Bradford method and SDS-PAGE method were employed to analyze the concentration and yield of rFIP-glu. Melanoma B16 cells were employed to evaluate the biological activities of rFIP-glu in vitro. Results showed that the protein yield of the best mutant #4-336 screened from 3680 mutant strains increased from 242 to 469 μg ml⁻¹. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity and possessed the ability to affect melanin content and enhance tyrosinase activity in B16 cells. In conclusion, an effective high-throughput screening approach was established for screening mutant strains. The screened mutant possesses a good ability to enhance the production of rFIP-glu, and recombinant proteins display a better biological activity on melanoma B16 cells. The engineered P. pastoris mutant seems promising as a potential source for industrial production of rFIP-glu and should be a candidate industrial strain for further study.