酶定向进化的研究进展

定向进化在改造酶的性质方面已得到广泛应用,各种建立突变库的方法不断涌现。对新近发展的几种突变技术(如寡核苷酸设计型装配重组技术ADO、非序列同源蛋白重组SHIPREC等)进行了简要地介绍与分类。与突变技术相对应的筛选方法也在逐渐改变和完善,这里仅介绍高通量筛选方面的一些最新进展。     

蛋白质定向进化技术的研究进展

蛋白质定向进化技术是蛋白质分子改造的一个重要策略.重点介绍了易错PCR、DNA改组等对编码蛋白质的基因进行随机突变和重组的技术,以及构建突变体库和高通量筛选的方法,并探讨了定向进化技术在蛋白质工程中的应用及前景.     

适用于高通量筛选的脂肪酶表达系统

突变库容量和高通量筛选方法是影响酶分子定向进化的两个决定因素,虽然巴斯德毕赤酵母pPIC9K表达系统已被广泛使用[1],但由于外源基因可通过单插入整合入基因组,产生多拷贝突变基因,从而干扰后续重组子的筛选;另一方面,pPIC9K表达系统需要甲醇诱导,需要每日补加甲醇来诱     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

体内连续定向进化研究进展

定向进化为合成生物学的发展提供了一种简单高效的工具,尤其在化学品合成和医药开发方面发挥着重要的作用.但是传统的定向进化技术存在操作繁琐、耗时和效率低的问题,不能满足大量突变文库的构建和筛选.近几年,一项将突变、翻译(进化非基因)、筛选和复制过程进行无缝连接的体内连续定向进化技术开始出现,该技术在噬菌体、细菌和真核细胞中...     

脂肪酶的表面展示及其应用

脂肪酶是一种广泛应用的水解酶类。脂肪酶的表面展示技术不仅是脂肪酶蛋白质工程中一种有效的高通量筛选方法,而且展示的脂肪酶与自由酶相比具备更高的温度稳定性、有机溶剂稳定性等优点,其作为全细胞催化剂与传统的固定化脂肪酶相比也具备诸多优点。脂肪酶表面展示的宿主包括噬菌体、细菌以及酵母等,本文将分别介绍这三种宿主中脂肪酶表面展示的概况以及其作为高通量筛选和全细胞等方面的应用。     

产酯酶微生物菌种的筛选研究

研究了产酯酶微生物的筛选,包括筛选模型、酶活力检测方法及菌株的分布。对30多份土样以及实验室保存的菌种进行了大量的筛选,以添加三醋酸甘油酯、乳酸乙酯酯类物质对土样等样品富集,采用添加显色剂溴甲酚紫的快速简便平板显色法,观察水解变色圈直径和菌落直径的大小进行初筛。获得两者直径之比相对大的菌株174株,采用平板打孔检测法和摇瓶发酵比色法测酶活力相结合进行复筛,最终得到酯酶活力较高的24株菌株。就初筛和复筛方法及结果加以比较分析,复筛菌株做不同底物的酶活力检测,建立了一个有效、简便及快速的微生物酯酶的筛选模型。并对酯酶产生菌的立体选择专一性进行了初步考察。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

基于易错PCR技术的黏质沙雷氏菌脂肪酶基因LipA的定向进化

利用易错PCR技术对黏质沙雷氏菌脂肪酶基因LipA进行定向进化,经过筛选最终获得一个比活力比野生酶提高了425 U/mg的突变体ep1,测序分析ep1有5个氨基酸发生了突变,与野生酶相比ep1的最适pH值由原来的8.5降低为7.5,Tm值提高3℃,Km值由原来的40 mg/mL降低为12.5 mg/mL.对其三维结构进行分析,推测酶学性质的改变可能与处在活性中心右前方双螺旋发卡结构上的158A、和处在下部β卷曲折叠拐角处的S375G的突变有关.     

酶的定向进化及其应用

综述了生物催化剂（酶）分子定向进化的产生、原理、方法及应用,深入阐述酶分子定向进化过程中的相关问题,重点介绍了易错PCR（Error-prone PCR）和DNA改组（DNA shuffling）等几种典型的酶定向进化方法与成功实例,展望了生物定向进化研究的发展前景。     

Enhancement of the organic solvent‐stability of the LST‐03 lipase by directed evolution

LST‐03 lipase from an organic solvent‐tolerant Pseudomonas aeruginosa LST‐03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent‐stability of LST‐03 lipase was attempted by directed evolution. The structural gene of the LST‐03 lipase was amplified by the error prone‐PCR method. Organic solvent‐stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri‐n‐butyrin and which overlaid a plate medium. And the organic solvent‐stability was also confirmed by measuring the half‐life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent‐stability in the presence of DMSO. The organic solvent‐stabilities of mutated LST‐03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half‐lives of the LST‐03‐R65 lipase in the presence of cyclohexane and n‐decane were about 9 to 11‐fold longer than those of the wild‐type lipase, respectively. Some substituted amino acid residues of mutated LST‐03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

定向进化-易错PCR方法提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力

运用定向进化-易错PCR的方法,提高了华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力。经过两轮易错PCR和pNPP顶层琼脂法筛选,从第一轮和第二轮突变库中分别筛选获得最佳突变株1-11和2-28,脂肪酶酶活与野生菌株相比分别提高2倍和4倍。基因比对结果表明,突变脂肪酶2-28有4个氨基酸发生了突变:A129S、K161R、A230T、K322R。蛋白质分子空间结构模拟显示,突变A129S、K161R、A230T位于脂肪酶分子表面。突变A230T增强了α-螺旋盖结构的稳定性。突变K322R处在loop上,靠近脂肪酶底物结合区域,与邻近的Asp(带负电)形成盐桥。静电引力将该loop向底物进入酶活性中心的通道口反方向牵引,使底物分子更易进入酶活性中心。酶学性质研究表明,突变株2-28脂肪酶的Km值比出发菌株下降了10%,Kcat值提高为原来的2.75倍。     

Methods to increase enantioselectivity of lipases and esterases

Lipases and esterases are frequently used in the synthesis of optically pure compounds; however, natural enzymes do not always show sufficiently high enantioselectivity. Variation of the structure of the substrates, modification of the reaction system or protein engineering (e.g. the expression of pure enzymes, rational design or directed evolution) are strategies that can be employed to improve the distinction between two enantiomers or enantiotopic groups.     

Ultrahigh-throughput screening system for directed polymer binding peptide evolution

Accumulation of plastics in the environment became a geological indicator of the Anthropocene era. An effective reduction of long-lasting plastics requires a treatment with micro-organisms that release polymer-degrading enzymes. Polymer binding peptides function as adhesion promoters and enable a targeted binding of whole cells to polymer surfaces. An esterase A-based Escherichia coli cell surface display screening system was developed, that enabled directed evolution of polymer binding peptides for improved binding strength to polymers. The E. coli cell surface screening system facilitates an enrichment of improved binding peptides from a culture broth through immobilization of whole cells on polymer beads. The polypropylene (PP)-binding peptide liquid chromatography peak I (LCI) was simultaneously saturated at five positions (Y29, D31, G35, E42, and D45; 3.2 million variants) and screened for improved PP-binding in the presence of the anionic surfactant sodium dodecylbenzenesulfonate (LAS; 0.25 mM). The cell surface system enabled efficient screening of the generated LCI diversity (in total ~10 million clones were screened). Characterization of identified LCI binders revealed an up to 12-fold improvement (eGFP-LCI-CSD-3: E42V/D45H) in PP-binding strength in the presence of the surfactant LAS (0.125 mM). The latter represents a first whole cell display screening system to improve adhesion peptides which can be used to direct and to immobilize organisms specifically to polymer surfaces (e.g., PP) and novel applications (e.g., in targeted plastic degradation).     

A comparison of directed evolution approaches using the beta-glucuronidase model system

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.     

Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulfolobus tokodaii by site-saturation mutagenesis

An archaeon GGG(A)X-type esterase (ST0071) can catalyze the hydrolysis of various acetates of secondary alcohols, but shows low enantioselectivity. Using structure-guided site-saturation mutagenesis, we successfully identified a G274W variant that has excellent selectivity compared with that of wild-type ST0071.     

定向进化技术的最新进展

筛选是制约酶定向进化改造的瓶颈。为解决这一难题,近年来一系列基于组合活性中心饱和突变(Combinatorial active-site saturation test,CAST)及迭代饱和突变(Iterative saturation mutagenesis,ISM)的半理性设计新方法被开发出来,包括单密码子饱和突变(Single code saturation mutagenesis,SCSM)、双密码子饱和突变(Double code saturation mutagenesis,DCSM)和三密码子饱和突变(Triple code saturation mutagenesis,TCSM)。通过构建"小而精"的高质量突变体文库,对特定靶点进行组合突变,并成功应用于多种生物催化剂的立体/区域选择性及催化活力等多参数的改造。文中综述了近年来定向进化技术的最新进展及其在生物催化剂定向改造中的应用。     

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I₂ assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.