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。     

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     

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.     

New Concepts for Increasing the Efficiency in Directed Evolution of Stereoselective Enzymes

Directed evolution of stereo‐ and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10‐residue site, the other also employs only one but possibly different amino acid at each position of a 9‐residue site. The small but smart libraries provide highly enantioselective epoxide hydrolase or lipase mutants, respectively.     

Stereodivergent Evolution of Artificial Enzymes for the Michael Reaction

Enzymes are valuable biocatalysts for asymmetric synthesis due to their exacting stereocontrol. Changing the selectivity of an existing catalyst for new applications is, however, challenging. Here we show that, in contrast, the stereoselectivity of an artificial enzyme created by design and directed evolution is readily tunable. We engineered a promiscuous artificial retro‐aldolase into four stereocomplementary catalysts for the Michael addition of a tertiary carbanion to an unsaturated ketone. Notably, this selectivity is also preserved with alternative Michael nucleophiles. Complete stereodiversification of other designer enzymes should similarly be possible by extension of these approaches.     

Catalytic Activity of Enzymes Altered at Their Active Sites

Protein engineering has as its goals the design and construction of new peptides and proteins with novel binding and catalytic properties. In one approach to protein engineering, new active sites have been introduced into naturally occurring proteins either by site-directed mutagenesis or by chemical modification. Providing that important changes in the tertiary structures do not result from such alterations, at least a portion of the binding site of the original protein should be available for the formation of complexes between the altered enzyme and its substrates. Many examples of active-site mutations have been described, including the generation by us of a cysteine mutant of alkaline phosphatase. A fundamental limitation of the site-directed mutagenesis methodology is that replacements of residues are restricted to the twenty naturally occurring amino acids. The alternative, chemical modification, is difficult to carry out for the specific replacement of one amino acid by another. However, we have shown that through such modification coenzyme analogues can be introduced covalently into appropriate positions in proteins, allowing us to produce semisynthetic enzymes with catalytic activities radically altered from those of their precursor proteins. In another approach to protein engineering efforts have focused on the construction of systems where, as a first approximation, folding can be neglected and the preparation of secondary structural units is the target. Examples of the successful design of biologically active peptides and proteins along such lines, taken from our own work, include molecules mimicking apolipoproteins, toxins, and many hormones. In recent studies we have progressed to the stage where we are starting to combine the two general approaches to protein engineering we have described and are able to construct small enzymes like ribonuclease T₁ and its structural analogues.     

酶分子在试管内的定向进化及其在手性有机物合成中的应用

本文扼要介绍了当今改进酶分子性能的有效手段———分子定向进化技术的基本方法如易错PCR和DNA洗牌,以及派生的一些新技术的原理与进展,并列举了在手性有机物合成中一些成功的实例,为该领域的研究与应用提供了一些新的信息。     

酶分子在试管内的定向进化及其在手性有机物合成中的应用

本文扼要介绍了当今改进酶分子性能的有效手段——分子定向进化技术的基本方法如易错PCR和DNA洗牌,以及派生的一暨新技术的原理与进展,并列举了在手性有机物合成中一些成功的实例,为该领域的研究与应用提供了一些新的信息。     

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long‐standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non‐ending source of robust and selective catalysts for a variety of useful applications.     

Enzymes as Catalysts in Synthetic Organic Chemistry [New Synthetic Methods (53)]

Enzymes have great potential as catalysts for use in synthetic organic chemistry. Applications of enzymes in synthesis have so far been limited to a relatively small number of largescale hydrolytic processes used in industry, and to a large number of small-scale syntheses of materials used in analytical procedures and in research. Changes in the technology for production of enzymes (in part attributable to improved methods from classical microbiology, and in part to the promise of genetic engineering) and for their stabilization and manipulation now make these catalysts practical for wider use in large-scale synthetic organic chemistry. This paper reviews the status of the rapidly developing field of enzyme-catalyzed organic synthesis, and outlines both present opportunities and probable future developments in this field.     

The Biochemical Reactions of Organometallics with Enzymes and Proteins

Most specialists doing organometallic chemistry have little understanding of what modern biochemistry is. On the other hand, most biochemists believe that organometallic chemistry stands much apart from the problems they study. But the real distance, if any, between these magnificent pyramids of modern science is progressively decreasing. Their interaction has given birth to a new branch of science, organometallic biochemistry, the general aspects of which are discussed here.     

A Method for the Selection of Catalytic Activity Using Phage Display and Proximity Coupling

Phage display has been used extensively for the selection of proteins with binding sites for ligands. Here, as illustrated with the example of DNA polymerase, the use of phage display for selection according to catalytic activity is described. Active enzymes are selected by binding of the reaction product P (see the scheme) cross-linked in the proximity of the enzyme E that catalyzed the reaction with the substrate S.     

A Phospholipase with a Novel Catalytic Triad

Catalytic triads are a long-standing paradigm of enzyme catalysis. By using a “matched mutation” approach, that is, a simultaneous exchange of the protein (through mutagenesis) and the substrate (through substitution of oxygen atoms by sulfur atoms) followed by enzyme kinetic analysis, a novel catalytic triad (see figure) with an unusual amino acid composition is now proposed for a phospholipase that fulfills a dual function in catalysis.     

Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate‐Utilizing Enzymes

Chorismate‐utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free‐living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.     

Directed Evolution Methods for Enzyme Engineering

Enzymes underpin the processes required for most biotransformations. However, natural enzymes are often not optimal for biotechnological uses and must be engineered for improved activity, specificity and stability. A rich and growing variety of wet-lab methods have been developed by researchers over decades to accomplish this goal. In this review such methods and their specific attributes are examined.     

Lipases: Interfacial Enzymes with Attractive Applications

Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols—for example, in the stomach and intestine during digestion of dietary fat—and various synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio- and enantioselectivity. Thus, the Ca²⁺ antagonist diltiazem ( 1 ) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.     

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.