Microbial & Enzyme Technology Lab
TOWARD A BIO-BASED SOCIETY
Industrial Biotechnology aims to use the power of enzymes and microorganisms to craft bioproducts, biomaterials, energy, and fuels from renewable sources. It offers environmentally friendly alternatives to traditional chemical methods.
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Our laboratory is dedicated to exploring new bacterial enzymes involved in lignocellulose degradation through comprehensive biochemical and structural characterization and enhancing their properties using protein engineering tools. We additionally focus on developing eco-friendly and cost-effective bioprocesses, looking for green methodologies and synthesis of bioactive molecules.
Current work employs a combination of enzymology, molecular biology, structural biology, and microbiology, and the research is at the crossroads of protein science and technology. We aim to expand the range of biocatalysts and are committed to uncovering the intricate relationship between enzyme function and structure and delving into the molecular mechanisms behind enzyme evolution.
Multicopper Oxidases. We are pioneers in the structural and functional characterization of bacterial laccases [Martins et 2015, 2020]. These enzymes show a broad substrate specificity, including substituted phenols, polyphenols, aromatic amines, and thiols, requiring only atmospheric oxygen as a co-substrate. Importantly, laccases are considered the most promising ligninolytic enzyme. We believe laccase-based oxidation processes will increase significantly in the lignocellulose biorefinery field in the following years. Thorough multidisciplinary investigations of wild-type and engineered variants revealed critical functional aspects of these enzymes, such as the electrostatic interactions modulating the redox potential [25, 31], the pathway of copper site assembly [30, 39, 45], and the molecular mechanisms behind the reduction of molecular oxygen to water [36, 38, 44, 46]. The studies of laccase-like enzymes from hyperthermophilic microorganisms provided the first evidence that these enzymes possess notable metal oxidase activity and an extreme intrinsic thermostability [27, 29, 32, 37]. Directed evolution strategies were optimized, paving the way to manipulate substrate specificity (i.e., expanding the range of oxidizing substrates) in a desired fashion [34, 61, 82, 96]. The molecular basis of substrate specificity and the dynamics and constraints of evolution to “unnatural” aromatic substrates was unveiled [82]. Insights were given on the role of synthetic and phenolic redox mediators that enhanced the substrate range of these biocatalysts, including in the degradation of non-phenolic lignin units [47].
A toolbox of experimental approaches was mounted to explore these biocatalysts for detoxifying industrial synthetic dyes [33, 35, 42, 78] and synthesizing heteroaromatic scaffolds from aromatic amines [50, 56, 58, 64, 68, 72, 76, 79]. We have recently developed enzymatic bioprocesses utilizing emerging lignin-derived monomeric phenolics to synthesize phenylpropanoid dimers (such as lignans, neolignans, and other dimeric structures), which serve as pharmaceutically active molecules and building blocks for bio-based polymers [108]. These findings significantly advance beyond the current state-of-the-art lignin combinatory chemistry and have important implications for sustainable lignin valorization.
Azoreductases. We have screened a small collection of microorganisms (around 150) [40] and selected a new bacterial strain, Pseudomonas putida MET94, based on its high efficiency for decolorizing a wide range of azo dyes. An azoreductase (PpAzoR) putatively involved was cloned and expressed in Escherichia coli, was purified and thoroughly characterized [40,52]. Since its low kinetic stability with a half-life of 13 min at 50 °C impaired its potential, we have applied directed evolution methodologies to improve the thermostability of PpAzoR [55]. Remarkably, the hit variant 1B6 shows a half-life of 68 h at 50 °C with increased resistance to aggregation. After mapping mutations, it becomes evident that the resistance to aggregation is mainly due to mutations that introduce surface net charges that strengthen solvent-exposed loops and inter-dimer interactions.
We showed that the coupled action of PpAzoR azoreductase and CotA-laccase from Bacillus subtilis resulted in the decolorization and detoxification of an extensive array of azo dyes, and model wastewaters [42] and their conversion into valuable compounds, quinones, phenazines, phenoxazinones, and naphthoquinones; free and immobilized whole cells allowed the development of economic bioprocess, resulting in valuable chemicals with final product yields of up to 90% [78].
DyP-peroxidases. We have investigated dye-decolorizing peroxidases (DyPs) from P. putida MET94 and B. subtilis [54]. One aspect of interest in DyPs is that they are present in bacteria and are thus valuable alternatives to fungal peroxidases, considering their enlarged temperature and pH range. Furthermore, DyPs display structural and mechanistic features unrelated to other classic peroxidases. The catalytic mechanism of a DyP was elucidated for the first time using BsDyP [62] and has provided the first kinetic evidence for the formation of a reversible enzyme-H2O2 complex (Compound 0), which was previously observed only at sub-zero temperatures using cryo-solvents. Furthermore, we have employed site-directed mutagenesis, transient and steady-state kinetics, and spectroscopic and electrochemical approaches to investigate the distinct role of the distal catalytic Asp and Arg residues in both enzymes [59, 62].
The efficiency of the bacterial PpDyP from P. putida MET94 for phenolic compounds was improved using directed evolution [66]. A variant was obtained with 100-fold enhanced catalytic efficiency for phenolic lignin-related substrates, displaying optimal pH at 8.5, an upshift of 4 units compared to the wild-type, and showing resistance to hydrogen peroxide inactivation. Structural analysis, molecular dynamics simulations, and constant-pH MD simulations shed light on their pH-dependent conformational plasticity [84]. 6E10 showed improved efficiency for producing identified syringaresinol, divanillin, and diapocynin, essential sources of structural scaffolds exploitable in medicinal chemistry, food additives, and polymers. [86]. We have recently used the web servers PROSS and FireProt to stabilize PpDyP from P. putida; the variants shifted their optimal temperatures from 15 to 25°C in wild-type to 60-70°C. The FireProt variant has a melting temperature of 73°C, while PROSS has a melting temperature of 88°C, showing a 10 and 25°C increase over the wild-type (Tm = 63°C). Applying structure-based computational protein design tools to complement laboratory engineering methodologies boosts our understanding of enzyme mechanisms and elucidates structure-activity relationships to improve thermostability: it revealed that mutations improved enthalpic interactions and resulted in more tightly bound dynamics, which could serve as a fingerprint for enhanced protein thermodynamic and kinetic stability. (paper submitted)
Pyranose oxidases. We provided the first biochemical characterization of a bacterial pyranose 2-oxidase (PsP2Ox) [65]. These enzymes catalyze the oxidation of several aldopyranoses at the C-2 position, coupling it to the reduction of dioxygen to hydrogen peroxide. P2Oxs are particularly interesting for clinical biosensors and synthetic carbohydrate chemistry. The crystal structure of PsP2Ox was solved at 2.0 Å resolution, revealing a monomeric enzyme contrasting with the tetrameric nature of fungal laccases despite having a conserved flavin-binding domain (Rossmann fold-like) and a substrate-binding domain. PsP2Ox displayed high catalytic efficiency, particularly with C-glycosides, achieving a kcat/Km value 10,000 times higher for mangiferin than D-glucose [93]. X-ray structures showed the substrate-recognition loop adopting different conformations—semi-open and open in substrate-bound forms—allowing substrates to bind at distinct positions: C2 for D-glucose and C3 for mangiferin, producing 3-keto mangiferin. Consequently, the enzyme was renamed glycose 3-oxidase (PsG3Ox) and linked to a two-step catabolic pathway for C-glycosides in soil microorganisms.
Recently, through precise optimization of reaction parameters, we achieved a highly selective synthesis of allose, a C3 epimer of D-glucose, following a chemo-enzymatic approach with excellent yields and minimal purification requirements. This method is markedly more efficient than traditional chemical processes or other tested enzymatic methods, significantly reducing costs, time, and waste [105]. This technology was submitted to a patent, and we have contacted several companies interested in the process, along with the Technology Transfer Office of ITQB NOVA.
Isoeugenol dioxygenases. We have focused our attention on the study of isoeugenol dioxygenases in the frame of the SMARTBOX project [82, 102]. These iron non-heme enzymes catalyze the one-step conversion of isoeugenol, a cheap precursor obtained from lignin depolymerization of wood biorefineries, into vanillin, a flavor and fragrance widely used in various industries. It is also essential for producing pharmaceuticals, cosmetics, and fine chemicals. However, these enzymes' structural and functional aspects remain to be elucidated. Genes coding for three enzymes were synthesized, and their expression was tested in E. coli. The enzymes were produced, purified, and characterized, and Novosphingobium aromaticivorans (NOV1), a resveratrol cleavage dioxygenase, was selected for further studies. A study integrating computational, kinetic, and structural approaches resulted in the design and characterization of an improved NOV1 variant that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for bio-based chemicals, polymers, and materials.
Using the computational tool Zymspot, we identified distal residues, constructed and selected improved variants, and demonstrated a higher immobilization efficiency (89-99%) and conversion yields (95%) than previously reported studies. Our findings provide a proof of concept for immobilizing computationally optimized enzyme variants.
Galactose oxidases. Galactose oxidases (Galox) are radical copper oxidases that catalyze the oxidation of primary alcohols to the corresponding aldehydes using galactose and a broad range of compounds, from small alcohols to polysaccharides, coupled with the reduction of O2 to hydrogen peroxide. In recent years, Galox has been used to synthesize small molecules, remove oxygen, use biosensors, and modify cell surface glycoproteins. The gene of PsGalOx was cloned from the genome of the dry-resistant bacteria. The enzyme was kinetic, biochemical, and structurally characterized, in particular (i) the role of the two carbohydrate-binding modules in modulating enzymatic affinity and activity towards galactose and galactose-containing polysaccharides and (ii) the structural determinants that govern substrate specificity within the CROs superfamily. Directed evolution was used to evolve for increased activity for benzyl alcohol and/or hydroxymethylfurfural (HMF). Benzyl alcohol is a lignin-derived alcohol that can be oxidized to one of the most critical chemicals in the aromatic aldehyde family, benzaldehyde, and a precursor of perfumes, beverages, and pharmaceutical drugs. HMF can be oxidized to 2,5-diformyfuran (DFF) and used to synthesize DFF-based chemicals such as important functional biopolymers or other biomaterials. Our findings provide new insights into the functional versatility of galactose oxidases and their potential biotechnological applications. (manuscript in preparation)
