Journal articles: 'Rational enzyme engineering'

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Eijsink, Vincent G. H., Alexandra Bjørk, Sigrid Gåseidnes, Reidun Sirevåg, Bjørnar Synstad, Bertus van den Burg, and Gert Vriend. "Rational engineering of enzyme stability." Journal of Biotechnology 113, no. 1-3 (September 2004): 105–20. http://dx.doi.org/10.1016/j.jbiotec.2004.03.026.

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Mirzaei, Mitra, and Per Berglund. "Engineering of ωTransaminase for Effective Production of Chiral Amines." Journal of Computational and Theoretical Nanoscience 17, no. 6 (June 1, 2020): 2827–32. http://dx.doi.org/10.1166/jctn.2020.8947.

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ωTransaminases are pyridoxal-5-phosphat (PLP) dependent enzymes having the ability to catalyze the transference of an amino group to a keto compound. These enzymes are used for production of chiral amines which are important building blocks in pharmaceutical industry. There is often a need to improve enzyme properties such as enzyme stability, enzyme specificity and to decrease substrate-product inhibition. Here, protein engineering was applied to improve the enzyme activity of the enzyme from Chromobacterium violaceum Rational-design and site-directed mutagenesis were applied on position of (W60) in the active site of the enzyme. Different mutated enzyme variants such as W60H, W60F and W60Y were made. Also, the enantiopreference of the wild type enzyme was reversed to produce (R)-chiral amines. For this aim, a screening assay was followed by semi-rational approach and saturation mutagenesis in the active site of the enzyme. Creating the mutated enzyme libraries resulted to obtaining two enzyme variants. Their properties were low enantiopreference towards formations of (R)-enantiopreference and low specific constant ratio between fast and slow enantiomers (Evalue around one).

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Chen, Ridong. "Enzyme engineering: rational redesign versus directed evolution." Trends in Biotechnology 19, no. 1 (January 2001): 13–14. http://dx.doi.org/10.1016/s0167-7799(00)01522-5.

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Acebes, Sandra, Elena Fernandez-Fueyo, Emanuele Monza, M. Fatima Lucas, David Almendral, Francisco J. Ruiz-Dueñas, Henrik Lund, Angel T. Martinez, and Victor Guallar. "Rational Enzyme Engineering Through Biophysical and Biochemical Modeling." ACS Catalysis 6, no. 3 (February 5, 2016): 1624–29. http://dx.doi.org/10.1021/acscatal.6b00028.

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Steiner, Kerstin, and Helmut Schwab. "RECENT ADVANCES IN RATIONAL APPROACHES FOR ENZYME ENGINEERING." Computational and Structural Biotechnology Journal 2, no. 3 (September 2012): e201209010. http://dx.doi.org/10.5936/csbj.201209010.

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Sousa, João P. M., Pedro Ferreira, Rui P. P. Neves, Maria J. Ramos, and Pedro A. Fernandes. "The bacterial 4S pathway – an economical alternative for crude oil desulphurization that reduces CO2 emissions." Green Chemistry 22, no. 22 (2020): 7604–21. http://dx.doi.org/10.1039/d0gc02055a.

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We discuss structural and mechanistic aspects of the Dsz enzymes in the 4S pathway, with a focus on rational molecular strategies for enzyme engineering, aiming at enzyme catalytic rate and efficiency improvement to meet industrial demands.

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Russell, Alan J., and Alan R. Fersht. "Rational modification of enzyme catalysis by engineering surface charge." Nature 328, no. 6130 (August 1987): 496–500. http://dx.doi.org/10.1038/328496a0.

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Payongsri, Panwajee, David Steadman, John Strafford, Andrew MacMurray, Helen C. Hailes, and Paul A. Dalby. "Rational substrate and enzyme engineering of transketolase for aromatics." Organic & Biomolecular Chemistry 10, no. 45 (2012): 9021. http://dx.doi.org/10.1039/c2ob25751c.

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Yang, Jae-Seong, Sang Woo Seo, Sungho Jang, Gyoo Yeol Jung, and Sanguk Kim. "Rational Engineering of Enzyme Allosteric Regulation through Sequence Evolution Analysis." PLoS Computational Biology 8, no. 7 (July 12, 2012): e1002612. http://dx.doi.org/10.1371/journal.pcbi.1002612.

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Albenne, Cécile, Bart A. Van Der Veen, Gabrielle Potocki-Véronèse, Gilles Joucla, Lars Skov, Osman Mirza, Michael Gajhede, Pierre Monsan, and Magali Remaud-Simeon. "Rational and Combinatorial Engineering of the Glucan Synthesizing Enzyme Amylosucrase." Biocatalysis and Biotransformation 21, no. 4-5 (October 2003): 271–77. http://dx.doi.org/10.1080/10242420310001618537.

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Tuan, Le Quang Anh. "Rational protein design for enhancing thermal stability of industrial enzymes." ENGINEERING AND TECHNOLOGY 8, no. 1 (August 17, 2020): 3–17. http://dx.doi.org/10.46223/hcmcoujs.tech.en.8.1.340.2018.

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Enzymes possessing many excellent properties such as high selectivity, consuming less energy, and producing less side products or waste have been widely applied as biocatalysts in pharmaceutical production and many industries such as biofuel, biomaterials, biosensor, food, and environmental treatment. Although enzymes have shown its potential as biocatalysts for many industrial applications, natural enzymes were not originated for manufacturing process which requires harsh reaction conditions such as high temperature, alkaline pH, and organics solvents. It was reported that reduction of final conversion of several enzymatic reactions was declined at high temperature. Protein engineering to improve the enzymes’ thermostability is crucial to extend the use of the industrial enzymes and maximize effectiveness of the enzyme-based procesess. Various industrial enzymes with improved thermostability were produced through rational protein engineering using different strategies. This review is not aimed to cover all successful rational protein engineering studies. The review focuses on some effective strategies which have widely used to increase the thermostability of several industrial enzymes through introduction of disulfide bonds and introduction of proline.

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Bata, Zsofia, Bence Molnár, Ibolya Leveles, Andrea Varga, Csaba Paizs, László Poppe, and Beáta G. Vértessy. "Structural snapshots of multiple enzyme–ligand complexes pave the road for semi-rational enzyme engineering." Acta Crystallographica Section A Foundations and Advances 74, a2 (August 22, 2018): e37-e38. http://dx.doi.org/10.1107/s2053273318094640.

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Waltman, M. J., Z. K. Yang, P. Langan, D. E. Graham, and A. Kovalevsky. "Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design." Protein Engineering Design and Selection 27, no. 2 (January 8, 2014): 59–64. http://dx.doi.org/10.1093/protein/gzt062.

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Otten, Linda G., Frank Hollmann, and Isabel W. C. E. Arends. "Enzyme engineering for enantioselectivity: from trial-and-error to rational design?" Trends in Biotechnology 28, no. 1 (January 2010): 46–54. http://dx.doi.org/10.1016/j.tibtech.2009.10.001.

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Chica, Roberto A., Nicolas Doucet, and Joelle N. Pelletier. "Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design." Current Opinion in Biotechnology 16, no. 4 (August 2005): 378–84. http://dx.doi.org/10.1016/j.copbio.2005.06.004.

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Weng, Jing-Yi, Xu-Liang Bu, Bei-Bei He, Zhuo Cheng, Jun Xu, Lin-Tai Da, and Min-Juan Xu. "Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives." Chemical Communications 55, no. 98 (2019): 14840–43. http://dx.doi.org/10.1039/c9cc07826f.

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Naeem, Muhammad, Amjad Bajes Khalil, Zeeshan Tariq, and Mohamed Mahmoud. "A Review of Advanced Molecular Engineering Approaches to Enhance the Thermostability of Enzyme Breakers: From Prospective of Upstream Oil and Gas Industry." International Journal of Molecular Sciences 23, no. 3 (January 30, 2022): 1597. http://dx.doi.org/10.3390/ijms23031597.

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During the fracture stimulation of oil and gas wells, fracturing fluids are used to create fractures and transport the proppant into the fractured reservoirs. The fracturing fluid viscosity is responsible for proppant suspension, the viscosity can be increased through the incorporation of guar polymer and cross-linkers. After the fracturing operation, the fluid viscosity is decreased by breakers for efficient oil and gas recovery. Different types of enzyme breakers have been engineered and employed to reduce the fracturing fluid′s viscosity, but thermal stability remains the major constraint for the use of enzymes. The latest enzyme engineering approaches such as direct evolution and rational design, have great potential to increase the enzyme breakers’ thermostability against high temperatures of reservoirs. In this review article, we have reviewed recently advanced enzyme molecular engineering technologies and how these strategies could be used to enhance the thermostability of enzyme breakers in the upstream oil and gas industry.

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Farmer, Tylar Seiya, Patrick Bohse, and Dianne Kerr. "Rational Design Protein Engineering Through Crowdsourcing." Journal of Student Research 6, no. 2 (December 31, 2018): 31–38. http://dx.doi.org/10.47611/jsr.v6i2.377.

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Two popular methods exist to engineer a protein: directed evolution and rational design. Directed evolution utilizes a controlled environment to create proteins through induced mutations and selection, while rational design makes desired changes to a protein by directly manipulating its amino acids. Directed evolution is currently more commonly used, since rational design relies on structural knowledge of the protein of interest, which is often unavailable. Utilizing crowdsourcing manpower and computational power to improve protein depictions allows rational design to be more easily used to perform the manipulation of proteins. Two free programs, “Folding@home and “Foldit”, allow anyone with a computer and internet access to contribute to protein engineering. Folding@home relies on one’s computational power, while Foldit relies on user intuition to improve protein models. Rational design has allowed protein engineers to create artificial proteins that can be applied to the treatment of illnesses, research of enzyme activity in a living system, genetic engineering, and biological warfare. Starting with an overview of protein engineering, this paper discusses the methods of rational design and directed evolutions and goes on to explain how computer based programs can help in the advancement of rational design as a protein engineering method. Furthermore, this paper discusses the application of computer based programs in medicine and genetic engineering and presents some ethical issues that may arise from using such technology. The paper concludes with an analysis of whether or not computer based programs for protein engineering is worth the investment.

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Wright, Addison V., Samuel H. Sternberg, David W. Taylor, Brett T. Staahl, Jorge A. Bardales, Jack E. Kornfeld, and Jennifer A. Doudna. "Rational design of a split-Cas9 enzyme complex." Proceedings of the National Academy of Sciences 112, no. 10 (February 23, 2015): 2984–89. http://dx.doi.org/10.1073/pnas.1501698112.

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Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

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Puglia, Megan K., Mansi Malhotra, and Challa V. Kumar. "Engineering functional inorganic nanobiomaterials: controlling interactions between 2D-nanosheets and enzymes." Dalton Transactions 49, no. 13 (2020): 3917–33. http://dx.doi.org/10.1039/c9dt03893k.

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A discussion of recent advances in controlling the enzyme-nanosheet interface, and rational methods to engineer interactions at these interface to build better nanobiomaterials and biodevices is presented.

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Pratto, Bruna, Martha Suzana Rodrigues dos Santos-Rocha, Gustavo Batista, Inti Cavalcanti-Montaño, Carlos Alberto Suarez Galeano, Antonio Jose Goncalves da Cruz, and Ruy de Sousa. "Rational feeding strategies of substrate and enzymes to enzymatic hydrolysis bioreactors." Chemical Industry and Chemical Engineering Quarterly, no. 00 (2021): 30. http://dx.doi.org/10.2298/ciceq201202030p.

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Bioreactors operating in fed-batch mode improve the enzymatic hydrolysis productivity at high biomass loadings. The present work aimed to apply rational feeding strategies of substrates (pretreated sugarcane straw) and enzymes (CellicCtec2?) to achieve sugar titers at industrial levels. The instantaneous substrate concentration was kept constant at 5 % (w/v) along the fed-batch, and the enzyme dosage inside the bioreactor was adjusted so that the reaction rate was not less than a pre-defined value (a percentage of the initial reaction rate - rmin). When r reached values below rmin, enzyme pulses were applied to return the reaction rate to its initial value (r0). The optimized feeding policy indicated a reaction rate maintained at a minimum of 70 % of r0, based on the trade-off between glucose productivity and enzyme saving. Initially, it was possible to process a total of 21 % (w/v) solid load, achieving 160 g/L of glucose concentration and 80 % of glucose yield. It was verified that non-productive enzyme adsorption was the main reason for some reduction of hydrolysis yield regarding the theoretical cellulose-to-glucose conversion. An increment of 30 g/L in the final glucose concentration was achieved when a lignin-blocking additive (soybean protein) was used in the enzymatic hydrolysis.

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Liu, Yao, Yalong Cong, Chuanxi Zhang, Bohuan Fang, Yue Pan, Qiangzi Li, Chun You, et al. "Engineering the biomimetic cofactors of NMNH for cytochrome P450 BM3 based on binding conformation refinement." RSC Advances 11, no. 20 (2021): 12036–42. http://dx.doi.org/10.1039/d1ra00352f.

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23

Kamondi, Szilárd, András Szilágyi, László Barna, and Péter Závodszky. "Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling." Biochemical and Biophysical Research Communications 374, no. 4 (October 2008): 725–30. http://dx.doi.org/10.1016/j.bbrc.2008.07.095.

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24

Bailey, Constance B., Marjolein E. Pasman, and Adrian T. Keatinge-Clay. "Substrate structure–activity relationships guide rational engineering of modular polyketide synthase ketoreductases." Chemical Communications 52, no. 4 (2016): 792–95. http://dx.doi.org/10.1039/c5cc07315d.

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Structure–activity relationship studies guided stereocontrol engineering within a modular polyketide synthase ketoreductase to yield a more active enzyme whose reactivity can be explained through the Felkin–Anh model.

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Xu, Lisheng, Fangkai Han, Zeng Dong, and Zhaojun Wei. "Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli." Microorganisms 8, no. 4 (April 5, 2020): 519. http://dx.doi.org/10.3390/microorganisms8040519.

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To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

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Chow, Jeng Yeong, and Giang Kien Truc Nguyen. "Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags." International Journal of Molecular Sciences 23, no. 17 (August 23, 2022): 9515. http://dx.doi.org/10.3390/ijms23179515.

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1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.

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Li, Jian-Xu, Xin Fang, Qin Zhao, Ju-Xin Ruan, Chang-Qing Yang, Ling-Jian Wang, David J. Miller, et al. "Rational engineering of plasticity residues of sesquiterpene synthases from Artemisia annua: product specificity and catalytic efficiency." Biochemical Journal 451, no. 3 (April 12, 2013): 417–26. http://dx.doi.org/10.1042/bj20130041.

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Most TPSs (terpene synthases) contain plasticity residues that are responsible for diversified terpene products and functional evolution, which provide a potential for improving catalytic efficiency. Artemisinin, a sesquiterpene lactone from Artemisia annua L., is widely used for malaria treatment and progress has been made in engineering the production of artemisinin or its precursors. In the present paper, we report a new sesquiterpene synthase from A. annua, AaBOS (A. annua α-bisabolol synthase), which has high sequence identity with AaADS (A. annua amorpha-4,11-diene synthase), a key enzyme in artemisinin biosynthesis. Comparative analysis of the two enzymes by domain-swapping and structure-based mutagenesis led to the identification of several plasticity residues, whose alteration changed the product profile of AaBOS to include γ-humulene as the major product. To elucidate the underlying mechanisms, we solved the crystal structures of AaBOS and a γ-humulene-producing AaBOS mutant (termed AaBOS-M2). Among the plasticity residues, position 399, located in the substrate-binding pocket, is crucial for both enzymes. In AaBOS, substitution of threonine for leucine (AaBOSL339T) is required for γ-humulene production; whereas in AaADS, replacing the threonine residue with serine (AaADST399S) resulted in a substantial increase in the activity of amorpha-4,11-diene production, probably as a result of accelerated product release. The present study demonstrates that substitution of plasticity residues has potential for improving catalytic efficiency of the enzyme.

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Angelaccio, Sebastiana. "Extremophilic SHMTs: From Structure to Biotechnology." BioMed Research International 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/851428.

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Recent advances in molecular and structural biology have improved the availability of virtually any biocatalyst in large quantity and have also provided an insight into the detailed structure-function relationships of many of them. These results allowed the rational exploitation of biocatalysts for use in organic synthesis. In this context, extremophilic enzymes are extensively studied for their potential interest for many biotechnological and industrial applications, as they offer increased rates of reactions, higher substrate solubility, and/or longer enzyme half-lives at the conditions of industrial processes. Serine hydroxymethyltransferase (SHMT), for its ubiquitous nature, represents a suitable model for analyzing enzyme adaptation to extreme environments. In fact, many SHMT sequences from Eukarya, Eubacteria and Archaea are available in data banks as well as several crystal structures. In addition, SHMT is structurally conserved because of its critical metabolic role; consequently, very few structural changes have occurred during evolution. Our research group analyzed the molecular basis of SHMT adaptation to high and low temperatures, using experimental and comparativein silicoapproaches. These structural and functional studies of SHMTs purified from extremophilic organisms can help to understand the peculiarities of the enzyme activity at extreme temperatures, indicating possible strategies for rational enzyme engineering.

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Kambiré, Marius Sobamfou, Jacques Mankambou Gnanwa, David Boa, Eugène Jean P. Kouadio, and Lucien Patrice Kouamé. "Modeling of the thermal behaviour of free β-galactosidase from palm weevil, Rhynchophorus palmarum Linn. (Coleoptera: Curculionidae) larvae using Equilibrium model." International Journal of Biological and Chemical Sciences 16, no. 4 (November 1, 2022): 1765–74. http://dx.doi.org/10.4314/ijbcs.v16i4.32.

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β-galactosidases are a class of enzyme widely used as biocatalysts in the food, dairy, and fermentation industries. However, due to their biological origin, enhancement of these enzymes is generally necessary. The effect of temperature upon enzymes is a mandatory stage in rational enzyme engineering. The present work was devoted to Rhynchophorus palmarum Linn. β-galactosidase (Rpbgal) as part of the investigation of insect-derived enzymes for biotechnological applications. The thermal behaviour of Rpbgal has been studied in the temperature range 303-353 K by measuring enzymatic activities in presence of oNPG as substrate. Equilibrium model which gives complete and quantitative description of the effect of temperature on enzyme activity has been used to analyze experimental data. A satisfactorily agreement between the calculated results and the experimental data was obtained. The thermodynamic parameters provided by this model were given. Results showed that Rpbgal is relatively stable and active at 323 K. Temperatures over 330 K produce a significant decrease in the enzyme activity. In the temperature range 331 - 339 K, Rpbgal showed the best thermal stability compared to a commercial β-galactosidase from Aspergillus oryzae.

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Frazão, Cláudio J. R., Christopher M. Topham, Yoann Malbert, Jean Marie François, and Thomas Walther. "Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway." Biochemical Journal 475, no. 23 (December 12, 2018): 3887–901. http://dx.doi.org/10.1042/bcj20180765.

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A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate-binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a stepwise manner into these regions of the protein. This rational engineering led to the production of an Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (kcat) on OHB that was increased by more than 2000-fold (from 0.03 up to 65.0 s−1), which turned out to be 7-fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies (kcat/Km) between natural and synthetic OHB substrates (84 and 31 s−1 mM−1, respectively). Shake-flask cultivation of a HMS-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert HMS to OHB resulted in 89% increased DHB production as compared with our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis.

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Zhu, Dunming, and Ling Hua. "How carbonyl reductases control stereoselectivity: Approaching the goal of rational design." Pure and Applied Chemistry 82, no. 1 (January 3, 2010): 117–28. http://dx.doi.org/10.1351/pac-con-09-01-03.

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Although "Prelog’s rule" and "two hydrophobic binding pockets" model have been used to predict and explain the stereoselectivity of enzymatic ketone reduction, the molecular basis of stereorecognition by carbonyl reductases has not been well understood. The stereoselectivity is not only determined by the structures of enzymes and substrates, but also affected by the reaction conditions such as temperature and reaction medium. Structural analysis coupled with site-directed mutagenesis of stereocomplementary carbonyl reductases readily reveals the key elements of controlling stereoselectivity in these enzymes. In our studies, enzyme-substrate docking and molecular modeling have been engaged to understand the enantioselectivity diversity of the carbonyl reductase from Sporobolomyces salmonicolor (SSCR), and to guide site-saturation mutagenesis for altering the enantioselectivity of this enzyme. These studies provide valuable information for our understanding of how the residues involved in substrate binding affect the orientation of bound substrate, and thus control the reaction stereoselectivity. The in silico docking-guided semi-rational approach should be a useful methodology for discovery of new carbonyl reductases.

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Hoffmann, G., K. Bonsch, T. Greiner-Stoffele, and M. Ballschmiter. "Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering." Protein Engineering Design and Selection 24, no. 5 (January 27, 2011): 439–46. http://dx.doi.org/10.1093/protein/gzq119.

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Ferrario, Valerio, Lydia Siragusa, Cynthia Ebert, Massimo Baroni, Marco Foscato, Gabriele Cruciani, and Lucia Gardossi. "BioGPS Descriptors for Rational Engineering of Enzyme Promiscuity and Structure Based Bioinformatic Analysis." PLoS ONE 9, no. 10 (October 29, 2014): e109354. http://dx.doi.org/10.1371/journal.pone.0109354.

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Wang, Xinglong, Kangjie Xu, Yameng Tan, Song Liu, and Jingwen Zhou. "Possibilities of Using De Novo Design for Generating Diverse Functional Food Enzymes." International Journal of Molecular Sciences 24, no. 4 (February 14, 2023): 3827. http://dx.doi.org/10.3390/ijms24043827.

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Food enzymes have an important role in the improvement of certain food characteristics, such as texture improvement, elimination of toxins and allergens, production of carbohydrates, enhancing flavor/appearance characteristics. Recently, along with the development of artificial meats, food enzymes have been employed to achieve more diverse functions, especially in converting non-edible biomass to delicious foods. Reported food enzyme modifications for specific applications have highlighted the significance of enzyme engineering. However, using direct evolution or rational design showed inherent limitations due to the mutation rates, which made it difficult to satisfy the stability or specific activity needs for certain applications. Generating functional enzymes using de novo design, which highly assembles naturally existing enzymes, provides potential solutions for screening desired enzymes. Here, we describe the functions and applications of food enzymes to introduce the need for food enzymes engineering. To illustrate the possibilities of using de novo design for generating diverse functional proteins, we reviewed protein modelling and de novo design methods and their implementations. The future directions for adding structural data for de novo design model training, acquiring diversified training data, and investigating the relationship between enzyme–substrate binding and activity were highlighted as challenges to overcome for the de novo design of food enzymes.

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Der, Bryan S., David R. Edwards, and Brian Kuhlman. "Catalysis by a De Novo Zinc-Mediated Protein Interface: Implications for Natural Enzyme Evolution and Rational Enzyme Engineering." Biochemistry 51, no. 18 (April 24, 2012): 3933–40. http://dx.doi.org/10.1021/bi201881p.

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Singh, Nitu, Sunny Malik, Anvita Gupta, and Kinshuk Raj Srivastava. "Revolutionizing enzyme engineering through artificial intelligence and machine learning." Emerging Topics in Life Sciences 5, no. 1 (April 9, 2021): 113–25. http://dx.doi.org/10.1042/etls20200257.

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The combinatorial space of an enzyme sequence has astronomical possibilities and exploring it with contemporary experimental techniques is arduous and often ineffective. Multi-target objectives such as concomitantly achieving improved selectivity, solubility and activity of an enzyme have narrow plausibility under approaches of restricted mutagenesis and combinatorial search. Traditional enzyme engineering approaches have a limited scope for complex optimization due to the requirement of a priori knowledge or experimental burden of screening huge protein libraries. The recent surge in high-throughput experimental methods including Next Generation Sequencing and automated screening has flooded the field of molecular biology with big-data, which requires us to re-think our concurrent approaches towards enzyme engineering. Artificial Intelligence (AI) and Machine Learning (ML) have great potential to revolutionize smart enzyme engineering without the explicit need for a complete understanding of the underlying molecular system. Here, we portray the role and position of AI techniques in the field of enzyme engineering along with their scope and limitations. In addition, we explain how the traditional approaches of directed evolution and rational design can be extended through AI tools. Recent successful examples of AI-assisted enzyme engineering projects and their deviation from traditional approaches are highlighted. A comprehensive picture of current challenges and future avenues for AI in enzyme engineering are also discussed.

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King, Jason R., Steven Edgar, Kangjian Qiao, and Gregory Stephanopoulos. "Accessing Nature’s diversity through metabolic engineering and synthetic biology." F1000Research 5 (March 24, 2016): 397. http://dx.doi.org/10.12688/f1000research.7311.1.

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In this perspective, we highlight recent examples and trends in metabolic engineering and synthetic biology that demonstrate the synthetic potential of enzyme and pathway engineering for natural product discovery. In doing so, we introduce natural paradigms of secondary metabolism whereby simple carbon substrates are combined into complex molecules through “scaffold diversification”, and subsequent “derivatization” of these scaffolds is used to synthesize distinct complex natural products. We provide examples in which modern pathway engineering efforts including combinatorial biosynthesis and biological retrosynthesis can be coupled to directed enzyme evolution and rational enzyme engineering to allow access to the “privileged” chemical space of natural products in industry-proven microbes. Finally, we forecast the potential to produce natural product-like discovery platforms in biological systems that are amenable to single-step discovery, validation, and synthesis for streamlined discovery and production of biologically active agents.

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Aganyants, Hovsep, Pierre Weigel, Yeranuhi Hovhannisyan, Michèle Lecocq, Haykanush Koloyan, Artur Hambardzumyan, Anichka Hovsepyan, Jean-Noël Hallet, and Vehary Sakanyan. "Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)." High-Throughput 9, no. 1 (February 12, 2020): 5. http://dx.doi.org/10.3390/ht9010005.

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D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

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Rennison, Andrew, Jakob R. Winther, and Cristiano Varrone. "Rational Protein Engineering to Increase the Activity and Stability of IsPETase Using the PROSS Algorithm." Polymers 13, no. 22 (November 10, 2021): 3884. http://dx.doi.org/10.3390/polym13223884.

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Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.

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Helfrich, Eric J. N., Geng-Min Lin, Christopher A. Voigt, and Jon Clardy. "Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering." Beilstein Journal of Organic Chemistry 15 (November 29, 2019): 2889–906. http://dx.doi.org/10.3762/bjoc.15.283.

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Terpenoids are the largest and structurally most diverse class of natural products. They possess potent and specific biological activity in multiple assays and against diseases, including cancer and malaria as notable examples. Although the number of characterized terpenoid molecules is huge, our knowledge of how they are biosynthesized is limited, particularly when compared to the well-studied thiotemplate assembly lines. Bacteria have only recently been recognized as having the genetic potential to biosynthesize a large number of complex terpenoids, but our current ability to associate genetic potential with molecular structure is severely restricted. The canonical terpene biosynthetic pathway uses a single enzyme to form a cyclized hydrocarbon backbone followed by modifications with a suite of tailoring enzymes that can generate dozens of different products from a single backbone. This functional promiscuity of terpene biosynthetic pathways renders terpene biosynthesis susceptible to rational pathway engineering using the latest developments in the field of synthetic biology. These engineered pathways will not only facilitate the rational creation of both known and novel terpenoids, their development will deepen our understanding of a significant branch of biosynthesis. The biosynthetic insights gained will likely empower a greater degree of engineering proficiency for non-natural terpene biosynthetic pathways and pave the way towards the biotechnological production of high value terpenoids.

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Anobom, Cristiane D., Anderson S. Pinheiro, Rafael A. De-Andrade, Erika C. G. Aguieiras, Guilherme C. Andrade, Marcelo V. Moura, Rodrigo V. Almeida, and Denise M. Freire. "From Structure to Catalysis: Recent Developments in the Biotechnological Applications of Lipases." BioMed Research International 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/684506.

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Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.

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Asmara, W., U. Murdiyatmo, A. J. Baines, A. T. Bull, and D. J. Hardman. "Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4." Biochemical Journal 292, no. 1 (May 15, 1993): 69–74. http://dx.doi.org/10.1042/bj2920069.

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The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

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Wilding, Matthew, Nansook Hong, Matthew Spence, Ashley M. Buckle, and Colin J. Jackson. "Protein engineering: the potential of remote mutations." Biochemical Society Transactions 47, no. 2 (March 22, 2019): 701–11. http://dx.doi.org/10.1042/bst20180614.

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Abstract Engineered proteins, especially enzymes, are now commonly used in many industries owing to their catalytic power, specific binding of ligands, and properties as materials and food additives. As the number of potential uses for engineered proteins has increased, the interest in engineering or designing proteins to have greater stability, activity and specificity has increased in turn. With any rational engineering or design pursuit, the success of these endeavours relies on our fundamental understanding of the systems themselves; in the case of proteins, their structure–dynamics–function relationships. Proteins are most commonly rationally engineered by targeting the residues that we understand to be functionally important, such as enzyme active sites or ligand-binding sites. This means that the majority of the protein, i.e. regions remote from the active- or ligand-binding site, is often ignored. However, there is a growing body of literature that reports on, and rationalises, the successful engineering of proteins at remote sites. This minireview will discuss the current state of the art in protein engineering, with a particular focus on engineering regions that are remote from active- or ligand-binding sites. As the use of protein technologies expands, exploiting the potential improvements made possible through modifying remote regions will become vital if we are to realise the full potential of protein engineering and design.

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Cheng, Feng, Jianhua Yang, Ulrich Schwaneberg, and Leilei Zhu. "Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency." Biotechnology and Bioengineering 116, no. 9 (June 11, 2019): 2156–66. http://dx.doi.org/10.1002/bit.27011.

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Molina, Manon, Thomas Prévitali, Claire Moulis, Gianluca Cioci, and Magali Remaud-Siméon. "The role of the C domain in the thermostability of GH70 enzymes investigated by domain swapping." Amylase 6, no. 1 (January 1, 2022): 11–19. http://dx.doi.org/10.1515/amylase-2022-0002.

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Abstract Sucrose-active enzymes belonging to the glycoside hydrolase (GH) family 70 are attractive tools for the synthesis of oligosaccharides, polysaccharides or glycoconjugates. However, their thermostability is an important issue for the development of robust and cost-effective enzyme-based processes. Indeed, GH70 enzymes are mesophilic and no thermophilic representatives have been described so far. Furthermore, structurally guided engineering is a challenge given the size of these proteins (120 to 250 kDa) and their organization in five domains. Herein, we have investigated the possible role of the domain C in the stability of GH70 enzymes. The alternansucrase (ASR) is the most stable enzyme of the GH70 family. Structural comparison of ASR to other GH70 enzymes highlighted the compactness of its domain C. We assumed that this atypical structure might be involved in the stability of this enzyme and decided to introduce this domain in another much less stable GH70 enzyme of known three-dimensional structure, the branching sucrase GBD-CD2. The chimeric GBD-CD2 exhibited a lower specific activity on sucrose substrate but its specificity was unchanged with the enzyme remaining specific for the branching of dextran via α-1,2 linkage formation. Interestingly, the chimera showed a higher melting temperature and residual activity than the wild-type enzyme after 10 min incubation at 30 °C showing that the domain C can affect GH70 enzyme stability and could be a potential target of both random or rational mutagenesis to further improve their stability.

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Hiratake, Jun. "Enzyme inhibitors as chemical tools to study enzyme catalysis: rational design, synthesis, and applications." Chemical Record 5, no. 4 (2005): 209–28. http://dx.doi.org/10.1002/tcr.20045.

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Bashirova, Anna, Subrata Pramanik, Pavel Volkov, Aleksandra Rozhkova, Vitaly Nemashkalov, Ivan Zorov, Alexander Gusakov, Arkady Sinitsyn, Ulrich Schwaneberg, and Mehdi Davari. "Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability." International Journal of Molecular Sciences 20, no. 7 (March 30, 2019): 1602. http://dx.doi.org/10.3390/ijms20071602.

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Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

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Deweid, Lukas, Olga Avrutina, and Harald Kolmar. "Microbial transglutaminase for biotechnological and biomedical engineering." Biological Chemistry 400, no. 3 (February 25, 2019): 257–74. http://dx.doi.org/10.1515/hsz-2018-0335.

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Abstract Research on bacterial transglutaminase dates back to 1989, when the enzyme has been isolated from Streptomyces mobaraensis. Initially discovered during an extensive screening campaign to reduce costs in food manufacturing, it quickly appeared as a robust and versatile tool for biotechnological and pharmaceutical applications due to its excellent activity and simple handling. While pioneering attempts to make use of its extraordinary cross-linking ability resulted in heterogeneous polymers, currently it is applied to site-specifically ligate diverse biomolecules yielding precisely modified hybrid constructs comprising two or more components. This review covers the extensive and rapidly growing field of microbial transglutaminase-mediated bioconjugation with the focus on pharmaceutical research. In addition, engineering of the enzyme by directed evolution and rational design is highlighted. Moreover, cumbersome drawbacks of this technique mainly caused by the enzyme’s substrate indiscrimination are discussed as well as the ways to bypass these limitations.

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Chen, Fu, Le Yuan, Shaozhen Ding, Yu Tian, and Qian-Nan Hu. "Data-driven rational biosynthesis design: from molecules to cell factories." Briefings in Bioinformatics 21, no. 4 (June 26, 2019): 1238–48. http://dx.doi.org/10.1093/bib/bbz065.

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Abstract A proliferation of chemical, reaction and enzyme databases, new computational methods and software tools for data-driven rational biosynthesis design have emerged in recent years. With the coming of the era of big data, particularly in the bio-medical field, data-driven rational biosynthesis design could potentially be useful to construct target-oriented chassis organisms. Engineering the complicated metabolic systems of chassis organisms to biosynthesize target molecules from inexpensive biomass is the main goal of cell factory design. The process of data-driven cell factory design could be divided into several parts: (1) target molecule selection; (2) metabolic reaction and pathway design; (3) prediction of novel enzymes based on protein domain and structure transformation of biosynthetic reactions; (4) construction of large-scale DNA for metabolic pathways; and (5) DNA assembly methods and visualization tools. The construction of a one-stop cell factory system could achieve automated design from the molecule level to the chassis level. In this article, we outline data-driven rational biosynthesis design steps and provide an overview of related tools in individual steps.

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Ueno, Takafumi, Takahiro Ohki, and Yoshihito Watanabe. "Molecular engineering of cytochrome P450 and myoglobin for selective oxygenations." Journal of Porphyrins and Phthalocyanines 08, no. 03 (March 2004): 279–89. http://dx.doi.org/10.1142/s108842460400026x.

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Aspects of protein engineering of cytochrome P450 (P450) and myoglobin ( Mb ) to construct selective oxygenation catalysts have been described. Heme enzymes are known as biocatalysts for various oxidations but the design of substrate specificity has still remained one of the significant challenges because of dynamic nature of enzyme-substrate interactions. In particular, P450s are the most interesting targets among the heme enzymes because they are able to catalyze many types of monooxygenations such as hydroxylation, epoxidation, and sulfoxidation with high selectivity. Thus, many researchers have made efforts to convert the selectivity for natural substrates into that for unnatural substrates by several protein engineering approaches. On the other hand, we have reported a rational design of Mb to convert its oxygen carrier function into that of peroxidase or peroxygenase. The Mb mutants prepared in our work afford oxo-ferryl porphyrin radical cation (compound I) as observable species in Mb for the first time. Furthermore, some of the mutants we have constructed are useful for enantioselective oxygenations by oxygen transfer from the Mb -compound I to substrates.

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Journal articles on the topic 'Rational enzyme engineering'

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