底物特异性的生物催化与酶设计改造

随着生物产业的发展,生物酶催化发挥着越来越重要的作用。然而,部分酶在应用过程中仍然存在诸多问题,影响了生物催化的进一步发展。本文以酶的底物特异性为切入点,回顾了酶的专一性、高效性和环保性;介绍了酶在药物合成和天然产物改性领域的应用以及所遇到的问题;综述了酶的底物特异性改造过程中各种方法的应用,包括化学修饰、非理性和理性设计。化学修饰作为一种直观的修饰方法,通过化学反应对酶分子进行改造;非理性设计是利用易错PCR和DNA Shuffling等手段获得底物特异性提高的突变体;理性设计是基于序列和结构信息对酶分子进行改造。本文从重塑活性口袋提高酶的底物特异性和重塑活性口袋改变酶促反应类型两个方面出发,详述了理性设计改变酶的底物特异性的方法,为酶的特异性改造提供借鉴。     

关于酶的专一性概念的探讨

准确地讲,酶的专一性是指酶催化某一种或者某一类底物发生特定的反应生成特定的产物这个性质,而不能将之简单地表述为酶作用于某一种或者某一类底物的性质。因为,同样的底物完全可能经不同的酶催化而生成不同的产物。     

气相中的生物酶催化反应

在气相中用生物酶催化底物反应对生物反应器设计来说有许多优于液相反应的优点.迄今为止,在气相中进行酶催化的工作还很有限,需要更多的工作以研究其反应学和产率.     

新型化学敏感器的合成及性能研究

分子识别 (MolecularRecognition)是超分子科学研究中的一个基本问题 .在生命科学中酶的特殊催化功能不仅表现为其催化能力的高效性 ,更重要的是在于它能特征性的对某些化合物分子发生专一性的反应 .这种能力的产生是建立在酶对于底物分子识别基础上的 ,因此设计和合成有光谱响     

酶催化区域选择性合成蔗糖酯的研究进展

详细综述了非水相酶催化合成蔗糖酯的研究进展。主要介绍了酶的种类、反应介质、底物性质、外加辅助手段等因素对蔗糖酯产率以及对酶区域选择性的影响。酶催化区域选择性合成蔗糖酯的难点在于蔗糖具有多个可酰化羟基及其与酰基供体的不相溶性,提出通过介质工程、底物修饰改性、外加物理场如微波和超声波辅助等改善底物相溶性,通过筛选酶、蔗糖修饰等可以得到高区域选择性的蔗糖酯。最后指出酶催化合成蔗糖酯存在的问题、发展前景等。     

海藻糖合酶的分离纯化及部分酶学性质

用硫酸铵沉淀、凝胶过滤层析、离子交换层析对一株芽孢杆菌SH-110产生的海藻糖合酶粗酶液进行纯化,用PAGE电泳逐步分析纯化的结果,用SDS-PAGE电泳证明该酶的分子量为62.4 kD。酶反应最适pH值为7.0,最适作用温度为35℃,金属离子Zn2+、K+、Na+对酶活性有激活作用,Ba2+、Ca2+、Mn2+对酶活性有抑制作用。酶底物专一性初步分析结果表明,该酶确实是以麦芽糖为专一性底物来转化生成海藻糖的海藻糖合酶的一种新酶。     

生物转化在硫醇类香料化合物制备中的应用

介绍了两种通过生物转化制备硫醇类香料化合物的方法。一种是通过脂肪酶催化硫代醋酸酯水解。比较了各种脂肪酶的催化活性,分析了pH值、温度、溶剂对反应的影响。另一种是通过β-裂合酶催化半胱氨酸加合物的裂解。具有β-裂合酶活性的细菌E.limosum萃取物具有较高的催化活性和较低的底物专一性,面包酵母也具有一定的β-裂合酶催化活性,是一类有潜力的制备硫醇的生物催化剂。酶催化方法将成为制备手性硫醇类香料非常重要的途径。     

高效液相色谱-固定化酶反应器

酶是一种具有特殊的三度空间构象的蛋白质,它能催化构成生命活动的许多化学反应。有些酶需要具备一种或多种称为辅助因子的非蛋白组分方能显示其活性,这种辅助因子,可以是称作辅酶的有机物分子或金属离子,这类具有催化活性的酶-辅助因子复合物叫做全酶。酶能催化某一底物进行特异性反应,生成特有的反应产物。1860年Emil Fisher提出酶和底物的反应是“一把钥匙开一个锁”的机理,即一种酶对应于一个反应,因此,酶的催化反应具有高度的专一性。例如葡糖氧化酶只催化β-D-葡葡糖,使其氧化生成葡糖酸和过氧化氢。这就是最常用的葡萄糖分析法。酶试剂通常是较贵的,作为催化剂虽然理论上酶自身并不消耗,但在溶液中,酶很容易变性而失活。如果将酶固定到某一载体上,就不需要在分析前配置各种浓度的酶溶液,还能提高其热稳定性,增加可储存的时间,使得一次性应用的酶试剂变为可重复使用而不会降低其功效。1916年,Nelson和Griffin首先报导     

分子印迹技术及其在催化领域中的应用

分子印迹技术是制备具有分子识别功能聚合物的一种技术,广泛应用于分离分析和催化领域中,尤其是在模拟酶催化中表现出很高的催化活性和专一性.分子印迹聚合物有多种制备方法,如原位聚合法、悬浮聚合法、表面印迹法、电化学聚合法等.分子印迹催化剂主要以过渡态类似物、底物类似物、产物类似物为模板,以及利用辅酶因子来制备.主要应用在化学反应和生物酶的催化等方面.     

新型硫脲类小分子催化剂的合成与应用

设计合成了一类多氢键供体的硫脲类小分子催化剂。该催化剂对丙二腈与硝基苯乙烯的不对称Michael加成反应具有一定的催化性能,收率可达79%,ee值为11%。该催化剂在催化不对称Michael加成反应时表现出酶催化的特性,对反应底物具有一定的专一性,且催化剂中手性中心构型一致时才具有催化性能。     

酶催化剂的固定化研究及其在催化反应中的应用

酶催化剂具有高效性,多样性,底物专一性,区域选择性、化学选择性、对映选择性以及反应条件温和的特点。而酶的固定化后除了保持原有的特点外,易与反应物和产物分离,可回收重复使用,降低生产成本。本文对酶催化剂的固定化方法以及在有机催化反应中的应用作了部分简述。并对固定化方法进行了比较和评价。     

Engineered human carboxypeptidase B enzymes that hydrolyse hippuryl-L- glutamic acid: reversed-polarity mutants

Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C- terminal acidic amino acid residues instead of the normal C-terminal basic residues. This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side- chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn. These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer.      

Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes

It is widely agreed that new enzymes evolve from existing ones through the duplication of genes encoding existing enzymes followed by sequence divergence. While evolution is an inherently random process, studies of divergently related enzymes have shown that the evolution of new enzymes follows one of three general routes in which the substrate specificity, reaction mechanism, or active site architecture of the progenitor enzyme is reused in the new enzyme. Recent developments in structural biology relating to divergently related (beta/alpha)8 enzymes have brought new insight into these processes and have revealed that conserved structural elements play an important role in divergent evolution. These studies have shown that, although evolution occurs as a series of random mutations, stable folds such as the (beta/alpha)8 barrel and structural features of the active sites of enzymes are frequently reused in evolution and adapted for new catalytic purposes.     

金属-酶协同催化动态动力学拆分反应研究进展

金属催化和酶催化在很长时间被认为是两个不同的领域,动态动力学拆分反应是金属-酶协同催化的成功应用。如何有效地协同金属催化的消旋化与酶催化的动力学拆分是其中的关键问题。本文对金属-酶协同催化动态动力学拆分醇和胺类化合物进行了综述,重点介绍了动态动力学拆分中常用的金属催化剂,各种金属与酶的协同,并对其前景进行了展望,指出提高酶在反应体系中的稳定性是未来的发展方向。     

Rational Design of a β‐Glycosidase with High Regiospecificity for Triterpenoid Tailoring

Triterpenoids with desired glycosylation patterns have attracted considerable attention as potential therapeutics for inflammatory diseases and various types of cancer. Sugar‐hydrolyzing enzymes with high substrate specificity would be far more efficient than other methods for the synthesis of such specialty triterpenoids, but they are yet to be developed. Here we present a strategy to rationally design a β‐glycosidase with high regiospecificity for triterpenoids. A β‐glycosidase with broad substrate specificity was isolated, and its crystal structure was determined at 2.0 Å resolution. Based on the product profiles and substrate docking simulations, we modeled the substrate binding modes of the enzyme. From the model, the substrate binding cleft of the enzyme was redesigned in a manner that preferentially hydrolyzes glycans at specific glycosylation sites of triterpenoids. The designed mutants were shown to produce a variety of specialty triterpenoids with high purity.     

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.     

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C₄–C₈) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94–96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.     

Physical and biological stability of dehydro-thermally crosslinked α-amylase–poly(vinyl alcohol) blends

A dehydro-thermal treatment has been used to crosslink α-amylase (EC 3.2.1.1)–poly(vinyl alcohol) blends in order to reduce their water solubility and to favour their potential use as bio-artificial polymeric materials based on enzymes. The influence of a thermal treatment on the thermal and biological stability of the enzyme blended with a synthetic polymer was carefully studied by means of calorimetry and biological assays. The values of the kinetic parameters obtained from the biological assays indicate that the enzyme specificity for the substrate in all the blends examined is not affected by the thermal treatment. In addition, the results of the releasing tests showed that the thermally crosslinked α-amylase–poly(vinyl alcohol) blends can be considered suitable for manifacturing delivery systems able to release enzymes. © 1998 Society of Chemical Industry     

Modified substrate specificity of L-hydroxyisocaproate dehydrogenase derived from structure-based protein engineering

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad substrate specificity and utilizes a wide range of 2-oxo acids branched at the C4 atom. Modifications have been made to the sequence of the NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and alter the region of substrate specificity towards various 2- oxocarbonic acids. All variations were based on a 3D-structure model of the enzyme using the X-ray coordinates of the functionally related L- lactate dehydrogenase (L-LDH) from dogfish as a template. This protein displays only 23% sequence identity to L-HicDH. The active site of L- HicDH was modelled by homology to the L-LDH based on the conservation of catalytically essential residues. Substitutions of the active site residues Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify their unique participation in substrate recognition and orientation. The kinetic properties of the L239A, L239M, L236V and T245A enzyme variants confirmed the structural model of the active site of L-HicDH. The substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate, phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the active site of the subsequently refined model. In order to design dehydrogenases with an improved substrate specificity towards keto acids branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236 and Thr245 were introduced and resulted in mutant enzymes with completely different substrate specificities. The substitution T245A resulted in a relative shift of substrate specificity for keto-tert-leucine of more than 17000 compared with the 2-oxocaproate (kcat/KM). For the substrates branched at C4 a relative shift of up to 500 was obtained for several enzyme variants. A total of nine mutations were introduced and the kinetic data for the set of six substrates were determined for each of the resulting mutant enzymes. These were compared with those of the wild-type enzyme and rationalized by the active site model of L-HicDH. An analysis of the enzyme variants provided new insight into the residues involved in substrate binding and residues of importance for the differences between LDHs and HicDH. After the protein design project was complete the X-ray structure of the enzyme was solved in our group. A comparison between the model and the experimental 3D structure proved the quality of the model. All the variants were designed, expressed and tested before the 3D structure became available.