高通量筛选技术在多相催化研究中的应用

通过对组合催化研究的关键因素－－催化剂高通量筛选技术的评价分析认为：在对已有的催化体系和催化剂优化的同时，如何高校研制开发新筛选检测技术，诸如反应器设计和检测器的选择，是组合催化研究的主导思想，对此的深刻理解和运用将增强实验室的创新能力。     

生物催化剂的高通量筛选方法

生物催化技术是工业可持续发展最有希望的技术，生物催化剂有着很好的应用前景。介绍了高通量筛选生物催化剂的常用方法。     

FTIR技术结合代谢物组学分析方法在菌株高通量筛选中的应用

为了寻找菌株高通量筛选的新方法，尝试运用代谢物组学的技术及其分析方法来筛选菌株。通过运用FTIR技术结合PCA、HCA，很好地区分了处于不同发酵时间的利迪链霉菌AS 4.2501，并找到了利迪链霉菌合成利迪链菌素时可能的生物标志物;并尝试使用FTIR技术结合ANN较好地预测了具有不同利迪链菌素合成能力的利迪链霉菌的突变株。实验结果表明基于代谢物组分析的FTIR技术和化学计量学数据处理方法的结合，有望用于菌株诱变后的高通量筛选。     

高通量筛选技术在农用杀菌剂创制研究中的应用

高通量筛选技术是发现创新药物的重要技术手段之一,在医药研究领域已经得到了广泛的应用,但在农药研究领域还处于初始阶段。综述了近年来高通量筛选技术在农用杀菌剂创制中的应用,并展望了其发展前景。     

高通量测序技术辅助筛选脱硫菌

将脱硫菌的筛选与新一代高通量测序技术相结合,探索脱硫菌筛选的新方法,为生物甲烷的高值化利用提供技术支持。实验将富含硫细菌的环境土样及经过培养基富集培养后的液样分别进行16S rDNA V6区的高通量测序,并分析了物种组成和丰度、Alpha多样性和菌群结构。结果表明,高通量测序的平均数据利用率达99.177%,测序量能够充分反映样品在该区域细菌的群落组成和结构。取自环境的土壤样品物种组成丰富,含有Pseudomonadales(假单胞菌目)、Rhizobiales(根瘤菌目)、Desulfuromonadales(除硫单孢菌目)、Desulfobacterales(脱硫杆菌目)、Acidithiobacillales(酸硫杆状菌目)等脱硫菌目。经过培养基富集后,菌群多样性显著降低,主要组成菌为Pseudomonadales(假单胞菌目)和Rhizobiales(根瘤菌目)。通过高通量测序技术,能够在筛选脱硫菌之前就了解实验样品的菌群结构和组成,为有针对性地设计筛选脱硫菌实验提供了一种新的方法。     

高通量测序技术辅助筛选脱硫菌

将脱硫菌的筛选与新一代高通量测序技术相结合,探索脱硫菌筛选的新方法,为生物甲烷的高值化利用提供技术支持。实验将富含硫细菌的环境土样及经过培养基富集培养后的液样分别进行16S rDNA V6区的高通量测序,并分析了物种组成和丰度、Alpha多样性和菌群结构。结果表明,高通量测序的平均数据利用率达99.177%,测序量能够充分反映样品在该区域细菌的群落组成和结构。取自环境的土壤样品物种组成丰富,含有Pseudomonadales（假单胞菌目）、Rhizobiales（根瘤菌目）、Desulfuromonadales（除硫单孢菌目）、Desulfobacterales（脱硫杆菌目）、Acidithiobacillales（酸硫杆状菌目）等脱硫菌目。经过培养基富集后,菌群多样性显著降低,主要组成菌为Pseudomonadales（假单胞菌目）和Rhizobiales（根瘤菌目）。通过高通量测序技术,能够在筛选脱硫菌之前就了解实验样品的菌群结构和组成,为有针对性地设计筛选脱硫菌实验提供了一种新的方法。     

有机垃圾堆肥过程中的菌种筛选

从农田稻草堆放处的腐粒稻草中分离、筛选出了四株分解能力较强的菌株,并用其对有机垃圾堆肥中的稻草进行了发酵试验,其对纤维素有较好的毁解率。     

高通量合成与筛选在新农药创制中的应用

高通量筛选与组合合成技术相结合是发现创新药物的重要技术手段之一。这极大地提高了化合物库的建立速度及对目标分子、活性物质以及先导药物的筛选速度,在农药创制过程中可以缩短新农药分子的研发周期,提高成功率。     

有机垃圾堆肥过程中的菌种筛选

从农田稻草堆放处的腐烂稻草中分离、筛选出了四株分解纤维素能力较强的菌株 ,并用其对有机垃圾堆肥中的稻草进行了发酵试验 ,其对纤维素有较好的毁解率。     

头孢菌素C乙酰化酶高通量筛选方法的改进

目的改进头孢菌素C(cephalosporin C,CPC)乙酰化酶高通量筛选方法,获得催化活性显著提高的突变体。方法基于对二甲氨基苯甲醛(p-dimethylaminobenzaldehyde,p-DAB)与7-氨基头孢烷酸(7-amino cephalosporanic acid,7-ACA)的复合物在405 nm有最大吸收值的原理,采用无菌96孔深孔板对重组菌进行低温低浓度诱导剂诱导表达,再以含溶菌酶和DNase的缓冲液进行菌体裂解,最后以96孔板进行底物的水解和反应终止,经p-DAB显色后,利用酶标仪进行产物的快速定量,以实现CPC乙酰化酶突变体的高通量筛选。结果利用改进的p-DAB比色法从先前建立的突变文库中筛选到2个在13℃下比酶活明显提高的突变子1A10和2G8,其比酶活分别为1.7和2.4 U/mg,较原酶的0.85 U/mg显著提高。在优化的筛选条件下,A405的变化能够准确反映酶活性的大小,确保了该方法筛选结果的可信度。结论改进的p-DAB比色法具有筛选快速,操作简便,筛选通量大及准确度高等优点,能够提高CPC乙酰化酶改造及筛选的效率,为低温CPC乙酰化酶的创制提供了技术支持。     

Strategies for directed and adapted evolution as part of microbial strain engineering

The industrial production of chemicals by microorganisms usually requires improvements to the enzymes, pathways, and strain that go beyond the capacity of innate enzymes. To achieve these phenotypes and overcome our limited capacity to de novo design these parts, directed and adaptive evolutionary approaches are used to explore new functions. This review highlights the recent advances in both sequence diversity generation and selection strategies from traditional in vitro mutagenesis to novel in vivo continuous evolution applications. The focus here is on comparison of the different gene diversity methods in an attempt to distinguish the best strategy for protein or strain engineering for a given goal. Furthermore, the important role that screening and selection can play in advancing directed and adapted evolution is discussed. © 2018 Society of Chemical Industry     

基于微孔过滤板的蛋白吸附高通量筛选方法

针对色谱分离过程优化,建立了基于微孔过滤板的蛋白吸附高通量筛选方法,用于介质初筛、吸附性能考察、吸附等温线和吸附动力学测定、吸附和洗脱条件优化等。首先优化了96孔过滤板的操作参数,以2种离子交换介质和2种混合模式介质为典型代表,采用微孔过滤板方法考察了不同介质和液相条件下牛血清白蛋白的吸附,得到结合载量分布图,确定了合适的蛋白吸附和解吸条件。进一步测定了4种介质在特定吸附条件下的吸附等温线和吸附动力学曲线,获得吸附相关参数。最后,采用微孔过滤板进行了洗脱条件优化,并与填充柱色谱分离进行比较,验证了方法的可靠性。结果表明,基于微孔过滤板的蛋白吸附高通量筛选是切实可行的,可以快速筛选色谱介质和液相,优化蛋白分离条件,具有资源消耗小、实验通量大、研发周期短、适用性广、稳定性高的特点,是蛋白色谱分离过程优化的一种新方法。     

Directed Evolution of Enzymes for Industrial Biocatalysis

Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost‐effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment.     

木聚糖酶高产菌筛选研究进展

从自然界筛选具有工业化应用潜力的高酶活产酶菌株,对于满足科学研究和实际生产需要都具有重要的意义。本文按照不同酶活梯度与采样点的关系,总结含有高酶活菌株样品的一些特征,如：富含腐烂木质纤维素的土样,该采样点的地势较平坦等。此外,对筛选到的高活力菌株的微生物种类及发酵优化中碳源的重要性进行了概括,提出最佳碳源的正确选择对最终的发酵优化工艺具有极大影响作用。对木聚糖酶高产菌株的筛选及发酵优化提供了有益的参考。     

用于工业杀菌剂评估的高通量筛选方法的建立

为建立一种工业杀菌剂高通量筛选方法,采用96孔板培养法,以大肠杆菌作为待测菌,利用显色剂显色反应对杀菌剂的抑菌能力进行评价。结果显示,采用1~4 d培养菌液,稀释至10%或菌液吸光度值A=0.038,显色剂TTC(2,3,5-氯化三苯基四氮唑)加入量20 mg/kg,显色18~24 h可得到与试管法一致的结果。该方法操作简捷,微量省力,结果易观察,成本低,可用于工业杀菌剂的大量筛选和应用评估。     

Evolutionary engineering of Phaffia rhodozyma for astaxanthin-overproducing strain

Evolutionary engineering is a novel whole-genome wide engineering strategy inspired by natural evolution for strain improvement. Astaxanthin has been widely used in cosmetics, pharmaceutical and health care food due to its capability of quenching active oxygen. Strain improvement of Phaffia rhodozyma, one of the main sources for natural astaxanthin, is of commercial interest for astaxanthin production. In this study a selection procedure was developed for adaptive evolution of P. rhodozyma strains under endogenetic selective pressure induced by additive in environmental niches. Six agents, which can induce active oxygen in cells, were added to the culture medium respectively to produce selective pressure in process of evolution. The initial strain, P. rhodozyma AS2-1557, was mutagenized to acquire the initial strain population, which was then cultivated for 550 h at selective pressure and the culture was transferred every 48h. Finally, six evolved strains were selected after 150 generations of evolution. The evolved strains produced up to 48.2% more astaxanthin than the initial strain. Our procedure may provide a promising alternative for improvement of high-production strain.     

Focused directed evolution of pentaerythritol tetranitrate reductase by using automated anaerobic kinetic screening of site-saturated libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96‐well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site‐saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)‐2‐aryl‐1‐nitropropene and α‐methyl‐trans‐cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β‐unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.     

高性能COF材料的高通量筛选策略：己烷异构体分离

己烷异构体中双支链异构体的分离可以提高汽油的辛烷值从而减少发动机的爆震现象。针对传统的蒸馏方法耗能高和新型吸附剂金属有机框架成本高、工作能力低、稳定性差的缺点,采用高通量计算筛选方法研究了688种共价有机框架(COFs)对己烷异构体的分离性能。首先计算了所有COF的几何结构描述符,通过限制孔径(PLD) 6.2~15 Å的范围筛选出209个可容纳所有己烷异构体的COF,再利用巨正则Monte Carlo (GCMC)方法模拟433 K下上述COF对己烷异构体的吸附解吸过程。对再生能力R>80%且吸附性能分值(APS)最高的COF进行排序,筛选出具有最高APS值的COF-DL229 2-fold,APS值为23.36 mol/kg,R为99.38%。分析了6个几何结构描述符与APS的相关性,发现对于COF来说较高的孔隙率(VF)、较高的孔隙体积(PV)、较低的密度(\rho)可提高COF的APS值。最后基于PV、VF、\rho利用决策树算法设计出高APS值COF的筛选路径,研究工作对今后设计用于己烷异构体吸附分离的COF具有一定的指导意义。