酶立体选择性的定向进化及其高通量筛选方法

定向进化技术已成为开发新型生物催化剂的有力工具,特别是在对酶结构或催化机理信息缺乏的情况下。酶的立体选择性是个比较难处理的参数,其在定向进化过程中的技术瓶颈是建立快速有效的高通量筛选方法。本文概述了在酶立体选择性的定向进化方面所取得的进展,着重论述了酶立体选择性的高通量筛选方法。     

离子淌度质谱结合荧光光谱法研究5-氟尿苷与铜锌超氧化物歧化酶的相互作用

分别利用离子淌度质谱和荧光光谱方法研究了5-氟尿苷（5-Furd）与铜锌超氧化物歧化酶（SOD1）的相互作用.离子淌度质谱研究结果表明,5-Furd能够稳定SOD1构型,减缓其碰撞诱导去折叠;外源荧光光谱方法进一步证明5-Furd可以抑制SOD1的聚集.利用荧光光谱研究了不同温度下5-Furd与SOD1的相互作用,阐明了二者的作用机制、结合常数和结合位点数,并根据热力学函数推断二者的作用力类型为典型的疏水作用.     

Directed Evolution of L-Aspartase by Mobility of Domains

To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19. 7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their kcat, and KM changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg^2 concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.     

电感耦合等离子体原子发射光谱(ICP-AES)法测定人发中铜、锌、钙、镁、铁

样品经硝酸-高氯酸消化溶解,高氯酸冒烟,盐酸溶解盐类后,在盐酸(5%)介质中,在选定的测定条件下,用电感耦合等离子体原子发射光谱(ICP-AES)法测定人发中微量元素铜、锌、铁、镁、钙。选择Cu 327.3、Zn 206.2、Fe 238.2、Mg 279.5、Ca 315.8nm分别作为铜、锌、铁、镁、钙的分析线与混合标准溶液同时测定;方法加标回收率为98.6%～101%,铜、锌、铁、镁、钙的精密度(RSD,n=8)为0.37%～2%,准确度(RE)为-3.4%～1.15%,检出限分别为0.002 3、0.001 6、0.004 6、0.003 0、0.001 4μg/mL。方法克服了分光光度法和原子吸收光谱法操作繁琐、周期长、成本高、灵敏度低等缺点。用于测定人发样品中的铜、锌、铁、镁、钙元素,测定结果与原子吸收光谱法测定值基本一致。经GBWO7061标准物质和自制标样分析验证,测定值与标准值吻合,结果准确可靠。     

Directed Evolution of an Artificial Imine Reductase

Artificial metalloenzymes, resulting from incorporation of a metal cofactor within a host protein, have received increasing attention in the last decade. The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin‐streptavidin technology using a straightforward procedure allowing screening in cell‐free extracts. Two streptavidin isoforms were yielded with improved catalytic activity and selectivity for the reduction of cyclic imines. The evolved ATHases were stable under biphasic catalytic conditions. The X‐ray structure analysis reveals that introducing bulky residues within the active site results in flexibility changes of the cofactor, thus increasing exposure of the metal to the protein surface and leading to a reversal of enantioselectivity. This hypothesis was confirmed by a multiscale approach based mostly on molecular dynamics and protein–ligand dockings.     

A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform “FlOxi” for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe²⁺ to Fe³⁺ (Fenton reaction). Fe³⁺ mediates the immobilization of a His₆-tagged eGFP (eGFP^His) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases—a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)—yielding a GalOx variant (T521A) with a 4.4-fold lower K_m value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher k_cat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.     

Complete Depolymerization of PET Wastes by an Evolved PET Hydrolase from Directed Evolution

PETase displays great potential in PET depolymerization. Directed evolution has been limited to engineer PETase due to the lack of high-throughput screening assay. In this study, a novel fluorescence-based high-throughput screening assay employing a newly designed substrate, bis (2-hydroxyethyl) 2-hydroxyterephthalate (termed BHET-OH), was developed for PET hydrolases. The best variant DepoPETase produced 1407-fold more products towards amorphous PET film at 50 °C and showed a 23.3 °C higher T_m value than the PETase WT. DepoPETase enabled complete depolymerization of seven untreated PET wastes and 19.1 g PET waste (0.4 % W_enzyme/W_PET) in liter-scale reactor, suggesting that it is a potential candidate for industrial PET depolymerization processes. The molecular dynamic simulations revealed that the distal substitutions stabilized the loops around the active sites and transmitted the stabilization effect to the active sites through enhancing inter-loop interactions network.     

Developments in Directed Evolution for Improving Enzyme Functions

The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature’s recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages. Submitted to Applied Biochemistry and Biotechnology     

金属巯蛋白与人体内铜锌元素的代谢

金属巯蛋白是一类富含金属的化学结构极为特殊的内源性蛋白。它构成机体内金属元素的新陈代谢中快速应答的缓冲体系。尤其在铜、锌两种元素的吸收、转运、排泄方面发挥着重要作用。     

急性腹泻患儿血清锌、铜及锌／铜比值的初步探讨

对息性腹泻息儿血清锌、铜水平及锌／铜比值关系进行了探讨．认为锌／铜比值变化具有临床意义．     

LaCl3对大豆Cu, Zn-SOD活性影响研究

研究证明0～100μmol·L-1的LaCl3对于大豆Cu,Zn SOD的NBT光还原活性抑制作用具有明显影响,即受La3+作用,大豆Cu,Zn SOD活性动力学曲线为S形,同时受La3+作用Cu,Zn SOD的热稳定性明显提高。大豆Cu,Zn SOD受La3+作用的阈值浓度和饱和浓度分别为cLa3+=18μmol·L-1,cLa3+=65μmol·L-1。根据Hill系数3.9和荧光光谱分析,大豆Cu,Zn SOD分子中存在4个La3+的强结合位点。证明了La3+对Cu,Zn SOD具有明显的正协同效应。     

Directed Evolution of Enzymes

On the way to a combinatorial biotechnology? The directed evolution of enzymes promises a rapid access to effective biocatalysts. New molecular biology techniques for random mutagenesis in combination with high-throughput screening might revolutionize the creation of enzymes with new and improved properties.     

卵巢癌患者血清中铜、锌含量及铜/锌比值分析

为了解微量元素与卵巢癌的关系，对21例卵巢癌患者进行了血清Cu、Zn含量及Cu/Zn比值分析，并以30例健康女性为对照。结果表明，卵巢癌患者血清中Zn含量明显低于对照组(P＜0．01)，而Cu含量明显高于对照组(P＜0．01)，Cu/Zn比值也比对照组高。提示卵巢癌的发生与体内微量元素异常有关。     

千名孕妇发Zn,Cu分布及相互关系

测定了１０００名孕妇发中Ｚｎ、Ｃｕ含量，发现低Ｚｎ率为１９．８％，Ｃｕ不足占５２．９％，可能与环境、饮水、膳食等因素有关。统计分析表明，两种元素均呈不对称分布，Ｚｎ与Ｃｕ呈正相关，Ｚｎ与Ｚｎ／Ｃｕ比值为负相关。     

火焰原子吸收法连续测定茶叶中铅、铜、锌、镉含量

采用火焰原子吸收方法连续测定了茶叶中铜，铜，锌，镉的含量，方法灵敏，准确。测定时无需富集，萃取，操作简便，回收率93．0％－98．1％，相对标准偏差不超过5．3％。     

Directed evolution of enantioselective hybrid catalysts: a novel concept in asymmetric catalysis

The concept of directed evolution of enantioselective hybrid catalysts was proposed in 2001/2002 and implemented experimentally for the first time in a proof-of-concept study in 2006. The idea is based on directed evolution, which comprises repeating cycles of random gene mutagenesis/expression/screening in a Darwinistic sense for the purpose of improving the catalytic profile of enzymes. In the case of hybrid catalysts, mutagenesis/expression of a protein is first performed with formation of a library of mutants, which are then modified chemically en masse with the introduction of an appropriate achiral ligand system harboring a transition metal. Screening these mutant hybrid catalysts in a given transition metal-catalyzed reaction then leads to an improved catalyst, so that the corresponding gene can be used to start another evolutionary cycle. This process can be repeated as often as needed until the desired catalytic profile has been reached, e.g., enhanced enantioselectivity.     

Cu/Zn、Cu/Zn/Ni催化剂甲醇部分氧化制氢

研究了甲醇在Cu/Zn及Cu/Zn/Ni催化剂上部分氧化热耦合裂解制氢的反应,系 统地考察了不同O2/CH3OH比及反应温度下催化剂性能.当O2/CH3OH=0.2时,催化剂的性能最 佳.在同样条件下, Cu/Zn催化剂对CO的选择性较Cu/Zn/Ni催化剂低,更具优势. Cu/Zn催化 剂用于甲醇部分氧化反应时,甲醇转化率在150 h寿命实验中基本保持在90％左右. XRD谱图 表明Cu/Zn合金的生成是导致Cu/Zn系催化剂在甲醇裂解反应中快速失活的主要原因,而在部 分氧化反应中, O2的存在可抑制Cu/Zn合金的生成,使Cu/Zn催化剂表现出高度的稳定性.     

健康老年人血清锌,铜,镁含量的调查

应用原子吸收分光光度法对济南市健康老年人血清中铜、锌、镁含量进行了测定。结果表明，健康老年人血液铜、锌水平低，但铜、锌比值正常，而镁的含量则较高。提示这组健康老年人未患心、脑血管疾病及糖尿病的原因可能与铜、锌、镁等微量元素有关。     

火焰原子吸收光谱法测定红花中的Cu 、Fe、Zn

采用火焰原子吸收光谱法和表面增敏的方法测定了红花中Cu、Fe、Zn的含量。实验表明,使用表面活性剂十二烷基硫酸钠(SDS),可使Zn的吸光度增感34.91%,使Cu增感37.39%,Fe增感232.10%。该法的检测限分别为Zn1.85×10-3μg/mL;Fe1.50×10-2μg/mL;Cu3.50×10-2μg/mL,加标回收率在99.37%~104.23%之间。该法灵敏度高,结果准确,可用于红花等中草药中Cu、Fe、Zn的同时测定。     

冠心病患者全血微量元素锌、铜、铁的分析

用原子吸收法测定５０例健康人、５２例冠心病患者全血微量元素锌、铜、铁的含量，并对５２例冠心病患者全血微量元素锌、铜、铁的含量及锌／铜比值，与其血脂总胆固醇（ＣＨ）、甘油三酯（ＴＧ）、高密度脂蛋白（ＨＤＬ）、低密度脂蛋白（ＬＤＬ）的浓度分别作线性相关分析。结果表明：（１）冠心病患者全血锌、铁、锌／铜比值均高于健康人（Ｐ＜０．０５），全血铜低于健康人（Ｐ＜０．０５）。（２）全血锌与ＨＤＬ呈负相关（Ｐ＜０．０１）、锌／铜比值与总胆固醇呈正相关（Ｐ＜０．０５），铁与ＬＤＬ呈正相关（Ｐ＜０．０１）．提示锌／铜比值增高及体内铁储存增加均可能是冠心病易患因素．