生物转化技术的前景

生物技术快速发展，已经成为合成新药最重要的途径之一。2010年，约20％的化工产品将由生物技术工艺生产，其销售价值将达到3100亿美元。生物技术工艺应用潜力最大的领域是精细化学品，预计2010年其销售值的60％将来自于生物技术。     

生物转化中新酶应用研究的最新进展

目前,在生物转化中出现了越来越多与传统工艺大不相同的新工艺,特别是生物酶在生物转化中的应用。硫酸酯酶可以立体选择性和对映体选择性地催化水解仲烷基硫酸酯,并通过改变其构象从而产生纯手性的化合物;卤代醇脱卤酶可以催化多种非天然亲核性底物;在水介质中,裂解酶可以催化偶姻或苯偶姻反应从而产生非对称的碳-碳键;最近还出现了用酶催化生产纯手性α-L-氨基酸的新方法。     

酶定向进化技术及其在化学品生物转化中的应用

定向进化可有目的地按照需要改造蛋白质分子中的氨基酸残基或结构域,从而定向改造蛋白质的性质,使其成为具有人们预期功能的新型蛋白质,应用于不同化学品生物转化反应中。本文简要的介绍了酶定向进化技术及其在化学品生物转化中的应用。     

生物催化与生物转化的研究进展

生物催化与生物转化是生物学、化学、过程工程科学的交叉领域,其核心目标是大规模采用微生物或酶为催化剂生产化学品、医药、能源、材料等。本文指出生物催化与生物转化研究重新崛起,并已成为发达国家的重要科技与产业发展战略,概述了生物催化与生物转化技术的发展现状与趋势,介绍了我国重大基础研究项目生物催化与生物转化的研究动态,该项目的关键问题及主要研究方向是：(1)生物催化多样性理论及其实现方法;(2)催化剂改造的方法学;(3)生物系统催化的理论和方法;(4)生物催化剂适应性原理和方法问题;(5)重要生物催化体系的催化机理。     

反胶束用于蛋白质纯化和生物转化的研究进展

反胶束是表面活性剂分子在非极性有机溶剂中自发形成的具有热力学稳定性的纳米级聚集体,其内部可溶解水和其他亲水性分子,包括多种生物活性物质,在生物技术领域应用广泛。综述了反胶束中蛋白质纯化和生物转化的研究进展,对存在的问题进行了评述,并对其应用前景作了展望。     

煤生物转化研究新进展

自然风化或经氧化处理的低品位煤为真菌作用所溶(降)解,溶解产率取决于煤氧化程序、菌种和培养条件等。高氧化煤在适宜的真菌固相表面培养作用下,溶解产率高达80～90%,液体培养的无细胞滤液也有明显的溶解作用。提出了酶溶解、碱溶解和金属螯合致溶解等机理。溶解产物分子量为数干至数万,具有与原煤类似的化学结构。溶解产物继续通过生化作用转化为分子量较小的化学产物或气化燃料。化学产物具有某种官能基团表面活性及螯合作用等,可在环保,农业和工矿业生产中获得应用。     

新型异亮氨酸双加氧酶及其重组大肠杆菌合成羟基异亮氨酸

异亮氨酸双加氧酶（IDO）可特异性的转化底物L-异亮氨酸（L-Ile）生成4-羟基-L-异亮氨酸（4-HIL）,该产物具有促进胰岛素分泌的功能,可用于抗糖尿病、降胆固醇等。本研究结合了酶标显色和薄层层析（TLC）的方法从自然界中筛到了具有IDO活性的菌株,并将该菌株中的目的基因ido克隆到大肠杆菌中,获得重组表达菌株,并且验证该菌具有IDO的转化功能。本研究优化了转化反应体系和条件,同时通过30℃过夜温育菌体细胞的方法,使该菌株全细胞转化合成4-HIL的产率达到85%以上。     

褐煤生物转化及其研究展望

综述了褐煤生物转化的3种方式:解聚、溶解和利用,以及目前对转化方式的机理的研究结果和作用菌种.褐煤的解聚主要是酶的作用,如木质素过氧化物酶、漆酶等; 褐煤的溶解已知有碱作用、螯合剂作用和水解酶作用等.作用的微生物主要是属于担子菌、子囊菌和半知菌的真菌,大部分俗称白腐真菌.虽已经取得不少研究成果,但该领域的研究仍处在初始阶段,离工业利用尚远.     

离子液介质中生物催化与生物转化的研究进展

Ionic liquids have negligibly low vapor pressure, high stability and polarity. They are regarded as green solvents. Enzymes, especially lipases, as well as whole-cell of microbe, are catalytically active in ionic liquids or aqueous-ionic liquid biphasic systems. Up to date, there have been many reports on enzyme-exhibited features and enzyme-mediated reactions in ionic liquids. In many cases, remarkable results with respect to yield, catalytic activity, stability and (enantio-, regio-) selectivity were obtained in ionic liquids in comparison with those observed in conventional media. Accordingly, ionic liquids provide new possibilities for the application of new type of solvent in biocatalytic reactions.     

离子液介质中生物催化与生物转化的研究进展

Ionic liquids have negligibly low vapor pressure, high stability and polarity. They are regarded as green solvents. Enzymes, especially lipases, as well as whole-cell of microbe, are catalytically active in ionic liquids or aqueous-ionic liquid biphasic systems. Up to date, there have been many reports on enzyme-exhibited features and enzyme-mediated reactions in ionic liquids. In many cases, remarkable results with respect to yield, catalytic activity, stability and (enantio-, regio-) selectivity were obtained in ionic liquids in comparison with those observed in conventional media. Accordingly, ionic liquids provide new possibilities for the application of new type of solvent in biocatalytic reactions.     

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.     

HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye

HaloTag is a small self-labeling protein that is frequently used for creating fluorescent reporters in living cells. The small-molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino-styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second-order bioconjugation rate constants could be improved up to 4-fold and 42-fold, respectively. One of the top variants, HT-SP5 , enabled reliable imaging in mammalian cells, with a 113-fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self-labeling proteins for alternative fluorophores.     

Biosystems design by directed evolution

Through iterative rounds of genetic diversification and screening or selection, directed evolution has been widely used to engineer relatively simple biosystems such as nucleic acids and proteins with desired functions. In addition, directed evolution has played an important role in engineering more complex biosystems such as pathways and genomes. Since 2013, directed evolution has been further explored for biosystems design with numerous newly developed techniques that have enabled design and engineering of proteins, pathways, and genomes in a much more effective manner. In this review, we will highlight the abiotic biotransformations arisen from directed evolution and novel strategies for continuous evolution in vivo and ultrahigh-throughput screening. We will also discuss the future challenges and opportunities of applying directed evolution for biosystems design.     

Engineered pH-inducible intein Mtu ΔI-CM variants with markedly reduced premature cleavage activity

Inteins have been widely exploited for the purification of tagless proteins. Among them, pH-inducible C-terminal-cleavage inteins enable the preparation of proteins and peptides with an authentic N-terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu ΔI-CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu ΔI-CM intein, when used in conjunction with a cleavable self-assembling tag. This work suggests that more controllable pH-inducible inteins can be obtained by manipulating the residues in the self-cleavage sites and provide better performance for tag-based protein preparation strategies.     

Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding

DnaK is a member of the Hsp70 family of molecular chaperones. This molecular machine couples the binding and hydrolysis of ATP to binding and release of substrate proteins. The switches that are involved in allosteric communication within this multidomain protein are mostly unknown. Previous insights were largely obtained by mutants, which displayed either wild-type activity or reduced folding assistance of substrate proteins. With a directed evolution approach for improved folding assistance we selected a DnaK variant characterized by a glycine to alanine substitution at position 384 (G384A); this resulted in a 2.5-fold higher chaperone activity in an in vitro DnaK-assisted firefly luciferase refolding assay. Quantitative biochemical characterization revealed several changes of key kinetic parameters compared to the wild type. Most pronounced is a 13-fold reduced rate constant for substrate release in the ATP-bound state, which we assume, in conjunction with the resulting increase in substrate affinity, to be related to improved chaperone activity. As the underlying mechanistic reason for this change we propose an altered interface of allosteric communication of mutant G384A, which is notably located at a hinge position between nucleotide and substrate binding domain.     

Second‐Generation Engineering of a Thermostable Transketolase (TKGst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

The transketolase from Geobacillus stearothermophilus (TK_Gst) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non‐α‐hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TK_Gst based on double‐site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TK_Gst for various applications in asymmetric carboligation.     

Protein Engineering of the Antitumor Enzyme PpADI for Improved Thermal Resistance

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas. Studies in human lymphatic leukemia cell lines have confirmed the anti‐angiogenic activity of ADI. Activity and thermal resistance limit the efficacy of ADI in treatment of auxotrophic tumors. Previously, we reengineered ADI from Pseudomonas plecoglossicida (PpADI) for improved activity under physiological conditions (37 °C, PBS buffer, pH 7.4) by two rounds of directed evolution and combination of beneficial substitutions through site‐directed mutagenesis. The best variant, PpADI M6 (K5T/D38H/D44E/A128T/E296K/H404R), showed a 64.7‐fold improvement in k_cat value and a 37.6 % decreased S_0.5 value under physiological conditions. However, M6 lost rapidly its activity (half‐life of ～2 days at 37 °C). Here we report the re‐engineering of PpADI M6 for improved thermal resistance by directed evolution in order to increase its half‐life under physiological conditions. Directed evolution and recombination of the two most beneficial positions yielded variant PpADI M9 (K5T/D38H/D44E/A128T/V140L/E296K/F325L/H404R), for which the T_m value increased from 47 (M6) to 54 °C (M9); this corresponds to an increased half‐life from ～2 days (M6) to ～3.5 days (M9) under physiological conditions. Structure analysis of the homology model of M9 showed that the beneficial substitutions V140L and F325L likely promote the formation of tetrameric PpADI, which has greater thermal resistance than dimeric PpADI.