Design of Artificial Enzymes Bearing Several Active Centers: New Trends, Opportunities and Problems.

Harnessing enzymes which possess several catalytic activities is a topic where intense research has been carried out, mainly coupled with the development of cascade reactions. This review tries to cover the different possibilities to reach this goal: enzymes with promiscuous activities, fusion enzymes, enzymes + metal catalysts (including metal nanoparticles or site-directed attached organometallic catalyst), enzymes bearing non-canonical amino acids + metal catalysts, design of enzymes bearing a second biological but artificial active center (plurizymes) by coupling enzyme modelling and directed mutagenesis and plurizymes that have been site directed modified in both or in just one active center with an irreversible inhibitor attached to an organometallic catalyst. Some examples of cascade reactions catalyzed by the enzymes bearing several catalytic activities are also described. Finally, some foreseen problems of the use of these multi-activity enzymes are described (mainly related to the balance of the catalytic activities, necessary in many instances, or the different operational stabilities of the different catalytic activities). The design of new multi-activity enzymes (e.g., plurizymes or modified plurizymes) seems to be a topic with unarguable interest, as this may link biological and non-biological activities to establish new combo-catalysis routes.

Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca.

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Carbohydrate-binding modules facilitate the enzymatic hydrolysis of lignocellulosic biomass: Releasing reducing sugars and dissociative lignin available for producing biofuels and chemicals.

The microbial decomposition and utilization of lignocellulosic biomass present in the plant tissues are driven by a series of carbohydrate active enzymes (CAZymes) acting in concert. As the non-catalytic domains widely found in the modular CAZymes, carbohydrate-binding modules (CBMs) are intimately associated with catalytic domains (CDs) that effect the diverse hydrolytic reactions. The CBMs function as auxiliary components for the recognition, adhesion, and depolymerization of the complex substrate mediated by the associated CDs. Therefore, CBMs are deemed as significant biotools available for enzyme engineering, especially to facilitate the enzymatic hydrolysis of dense and insoluble plant tissues to acquire more fermentable sugars. This review aims at presenting the taxonomies and biological properties of the CBMs currently curated in the CAZy database. The molecular mechanisms that CBMs use in assisting the enzymatic hydrolysis of plant polysaccharides and the regulatory factors of CBM-substrate interactions are outlined in detail. In addition, guidelines for the rational designs of CBM-fused CAZymes are proposed. Furthermore, the potential to harness CBMs for industrial applications, especially in enzymatic pretreatment of the recalcitrant lignocellulose, is evaluated. It is envisaged that the ideas outlined herein will aid in the engineering and production of novel CBM-fused enzymes to facilitate efficient degradation of lignocellulosic biomass to easily fermentable sugars for production of value-added products, including biofuels.

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases.

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

Enhance production of diterpenoids in yeast by overexpression of the fused enzyme of ERG20 and its mutant mERG20.

Yeast has been widely used for large-scale production of terpenoids. In yeast, modifications of terpenoid biosynthetic pathways have been intensively studied. tHMG1 (encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase of yeast) and UPC2-1 (the G888D mutant of UPC2 encoding a transcription factor) were integrated into yeast chromosome, and ERG9 (the squalene synthase gene of yeast) was knocked down to yield the chassis strain DH02. A F96C mutation in ERG20 (farnesyl diphosphate synthase of yeast) was conducted to obtain mERG20 which can function as a geranylgeranyl diphosphate synthase (GGPS). Then, three fused genes, including BTS1 (the yeast innate GGPS)-ERG20, ERG20-mERG20 and mERG20-ERG20, were constructed, and expressed either by the pESC-based plasmids in DH02, or by being integrated into DH02 chromosome. The highest geranylgeraniol (GGOH) content was observed in the extracts of DH12 integrated with ERG20-mERG20, corresponding to 3.2 and 2.3 folds of those of the strains integrated with BTS1 and mERG20, respectively. Finally, three genes encoding nor-copalyl diphosphate synthase (nor-CPS), ent-CPS and syn-CPS were integrated into the chromosome of DH12, respectively, to construct yeasts for producing corresponding copalyl diphosphates (CPPs). Thus, a yeast-based platform was built for characterizing all types of diterpene synthases using GGPP or various CPPs as their substrates.

Usage of GD-95 and GD-66 lipases as fusion partners leading to improved chimeric enzyme LipGD95-GD66.

Lipases are used as biocatalysts in industrial processes mainly because of their stability at broad temperature and pH range, resistance to organic solvents and wide spectrum of substrates. The usage of several lipolytic domains, each with different activity and resistance profiles, enables both the flexibility and efficiency of industrial processes. In this study, GD-95 and GD-66 lipases produced by Geobacillus sp. 95 and Geobacillus sp. 66, respectively, were used as fusion partners to create a new fused lipolytic enzyme LipGD95-GD66. Chimeric LipGD95-GD66 lipase displayed tenfold increase in activity (200 U/mg) compared to parental GD-66 lipase, improved Vmax (10 µmol/min mg-1) and catalytic efficiency (2 ∗ 105 min-1 mM-1) for p-NP palmitate as a substrate and increased activity at 70-75 °C compared to both parental lipases. All three lipases also retained >50% of their lipolytic activity after incubation with methanol, n-hexane, ethanol and DMF for longer than three weeks, highlighting a great prospect for application in industrial processes. Moreover, transesterification results revealed the capability of parental GD-95 lipase to be the most promising biocatalyst for production of methyl and ethyl esters through eco-friendly transesterification using argan oil and ethanol/methanol as acceptors of acyl group.