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.     

Enzyme thermistors for process monitoring and control

In today’s biotechnology there is an increasing demand for appropriate analytical systems for process control. At present the most widely used control systems are based on measurements of pH, pO₂, and pCO₂. Such systems do not allow the direct measurement of substrates and products. To overcome this drawback sensors such as enzyme thermistors and enzyme electrodes have been designed and their development into industrial useful sensors for monitoring and controlling is the subject of active research.     

Minimalist active-site redesign: teaching old enzymes new tricks

Although nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized microenvironments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity. The prediction and control of these effects provides a promising way to access new functions. The development of methods and strategies to explore the untapped catalytic potential of natural enzyme scaffolds has been pushed by the increasing demand for industrial biocatalysts. This Review describes the use of minimal modifications at enzyme active sites to expand their catalytic repertoires, including targeted mutagenesis and the addition of new reactive functionalities. Often, a novel activity can be obtained with only a single point mutation. The many successful examples of active-site engineering through minimal mutations give useful insights into enzyme evolution and open new avenues in biocatalyst research.     

Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase: A chemistry–biology interdisciplinary study of the protein's structure–function–reaction mechanism relationships

The oxidosqualene cyclases (EC 5.4.99‐) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)‐2,3‐oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure–function relationships of the Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase‐catalyzed cyclization/rearrangement reaction. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 302–325; 2008: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.20157     

Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering

Protein design is limited by the diversity of functional groups provided by the canonical protein „building blocks“. Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split-GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3-phenylbutyrate.     

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6–12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d /l -, α/β-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.     

Creation of a lipase highly selective for trans fatty acids by protein engineering

Sorting out: Protein engineering of lipase CAL-A led to the discovery of mutants with excellent chemoselectivity for the removal of trans and saturated fatty acids from partially hydrogenated vegetable oil. These fatty acids, identified as a major risk factor for human health, can now be removed by enzyme catalysis.     

代谢工程研究进展

介绍了近几年代谢工程在降解氟氯烃等环境污染物、生产古龙酸、抗生素、丙二醇、聚羟基链烷酸及紫杉醇等重要化合物和中间体、木糖等生物质再生资源的利用、工业发酵及植物改良方面的最新进展以及基本的设计思想。     

In Vitro Evaluation of In Silico Screening Approaches in Search for Selective ACE2 Binding Chemical Probes

New models for ACE2 receptor binding, based on QSAR and docking algorithms were developed, using XRD structural data and ChEMBL 26 database hits as training sets. The selectivity of the potential ACE2-binding ligands towards Neprilysin (NEP) and ACE was evaluated. The Enamine screening collection (3.2 million compounds) was virtually screened according to the above models, in order to find possible ACE2-chemical probes, useful for the study of SARS-CoV2-induced neurological disorders. An enzymology inhibition assay for ACE2 was optimized, and the combined diversified set of predicted selective ACE2-binding molecules from QSAR modeling, docking, and ultrafast docking was screened in vitro. The in vitro hits included two novel chemotypes suitable for further optimization.     

Computer-aided Modeling of Porous Electrodes with an Immobilized Enzyme: Substrates with a Partially Regular Structure

In porous electrodes with an immobilized enzyme the substrate must have a high specific surface area, which must be accessible to landing onto it a maximum number of enzyme molecules. These demands are not easy to meet. Conceivable are limiting versions as follows: a stochastic substrate, where substrate particles (SP) are distributed in the volume randomly, and a regular substrate, where SP are distributed strictly regularly. Both versions of the organization of SP have advantages and disadvantages; therefore, in the paper studied is a third, intermediate, version, specifically, substrates with a partially regular structure. Shown is that there exists an optimum of values of fractal dimensionality for a regular base of a substrate, where, by somewhat sacrificing the amount of active enzymes, one can attain a considerable ease of the process of landing enzymes on the surface of a porous substrate. Calculations also show that of practical interest may be a porous substrate with a purely regular structure.     

Carnosine to Combat Novel Coronavirus (nCoV): Molecular Docking and Modeling to Cocrystallized Host Angiotensin-Converting Enzyme 2 (ACE2) and Viral Spike Protein

Aims: Angiotensin-converting enzyme 2 (ACE2) plays an important role in the entry of coronaviruses into host cells. The current paper described how carnosine, a naturally occurring supplement, can be an effective drug candidate for coronavirus disease (COVID-19) on the basis of molecular docking and modeling to host ACE2 cocrystallized with nCoV spike protein. Methods: First, the starting point was ACE2 inhibitors and their structure–activity relationship (SAR). Next, chemical similarity (or diversity) and PubMed searches made it possible to repurpose and assess approved or experimental drugs for COVID-19. Parallel, at all stages, the authors performed bioactivity scoring to assess potential repurposed inhibitors at ACE2. Finally, investigators performed molecular docking and modeling of the identified drug candidate to host ACE2 with nCoV spike protein. Results: Carnosine emerged as the best-known drug candidate to match ACE2 inhibitor structure. Preliminary docking was more optimal to ACE2 than the known typical angiotensin-converting enzyme 1 (ACE1) inhibitor (enalapril) and quite comparable to known or presumed ACE2 inhibitors. Viral spike protein elements binding to ACE2 were retained in the best carnosine pose in SwissDock at 1.75 Angstroms. Out of the three main areas of attachment expected to the protein–protein structure, carnosine bound with higher affinity to two compared to the known ACE2 active site. LibDock score was 92.40 for site 3, 90.88 for site 1, and inside the active site 85.49. Conclusion: Carnosine has promising inhibitory interactions with host ACE2 and nCoV spike protein and hence could offer a potential mitigating effect against the current COVID-19 pandemic.     

Enzyme immobilization strategies for the design of robust and efficient biocatalysts

Different strategies for the preparation of efficient and robust immobilized biocatalysts are here reviewed. Different physico-chemical approaches are discussed.i.- The stabilization of enzyme by any kind of immobilization on pre-existing porous supports.ii.- The stabilization of enzymes by multipoint covalent attachment on support surfaces.iii.- Additional stabilization of immobilized-stabilized enzyme by physical or chemical modification with polymers.These three strategies can be easily developed when enzymes are immobilized in pre-existing porous supports. In addition to that, these immobilized-stabilized derivatives are optimal to develop enzyme reaction engineering and reactor engineering. Stabilizations ranging between 1000 and 100,000 folds regarding diluted soluble enzymes are here reported.