Asymmetric Sulfoxidations Catalyzed by Bacterial Flavin-Containing Monooxygenases.

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis (NiFMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of mFMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

Spectroelectrochemistry for determination of the redox potential in heme enzymes: Dye-decolorizing peroxidases.

Dye-decolorizing peroxidases (DyPs) are heme-containing enzymes that are structurally unrelated to other peroxidases. Some DyPs show high potential for applications in biotechnology, which critically depends on the stability and redox potential (E°') of the enzyme. Here we provide a comparative analysis of UV-Vis- and surface-enhanced resonance Raman-based spectroelectrochemical methods for determination of the E°' of DyPs from two different organisms, and their variants generated targeting E°' upshift. We show that substituting the highly conserved Arginine in the distal side of the heme pocket by hydrophobic amino acid residues impacts the heme architecture and redox potential of DyPs from the two organisms in a very distinct manner. We demonstrate the advantages and drawbacks of the used spectroelectrochemical approaches, which is relevant for other heme proteins that contain multiple heme centers or spin populations.

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase.

α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation.

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5-9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.

High-level production of industrially relevant oxidases by a two-stage fed-batch approach: overcoming catabolite repression in arabinose-inducible Escherichia coli systems.

With the growing interest in enzyme applications, there is an urgent demand for economic, affordable, and flexible enzyme production processes. In the present paper, we developed a high cell density fed-batch process for the production of two cofactor-containing oxidase, 5-hydroxymethylfurfural oxidase (HMFO) and eugenol oxidase (EUGO). The approach involved the arabinose-inducible system to drive the expression while using mineral media. In order to overcome a major drawback of arabinose-inducible promoters, carbon catabolite repression, (CCR) by glucose, we developed a high cell density culture (HCDC), two-stage fed-batch protocol allowing us to reach cell densities exceeding 70 g/L of dry cell weight (DCW) using glucose as carbon source. Then, induction was achieved by adding arabinose, while changing the carbon source to glycerol. This strategy allowed us to obtain an eightfold increase in recombinant HMFO titer when compared with a reference batch fermentation in Erlenmeyer flasks using terrific broth (TB), typically used with arabinose-inducible strains. The optimized protocol was also tested for expression of a structurally unrelated oxidase, EUGO, where a similar yield was achieved. Clearly, this two-step protocol in which a relatively cheap medium (when compared to TB) can be used reduces costs and provides a way to obtain protein production levels similar to those of IPTG-based systems. KEY POINTS: • Arabinose promoters are not well suited for HCDC production due to CCR effect. • This drawback has been overcome by using a two-stage Fed-batch protocol. • Protein yield has been increased by an eightfold factor, improving process economics.

Mechanistic and Crystallographic Studies of Azoreductase AzoA from Bacillus wakoensis A01.

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100-400 s-1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.

Development of an efficient biocatalytic system based on bacterial laccase for the oxidation of selected 1,4-dihydropyridines.

Biocatalytic oxidations mediated by laccases are gaining importance due to their versatility and beneficial environmental effects. In this study, the oxidation of 1,4-dihydropyridines has been performed using three different types of bacterial laccase-based catalysts: purified laccase from Bacillus licheniformis ATCC 9945a (BliLacc), Escherichia coli whole cells expressing this laccase, and bacterial nanocellulose (BNC) supported BliLacc catalysts. The catalysts based on bacterial laccase were compared to the commercially available Trametes versicolor laccase (TvLacc). The oxidation product of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate was obtained within 7-24 h with good yields (70-99%) with all three biocatalysts. The substrate scope was examined with five additional 1,4-dihydropyridines, one of which was oxidized in high yield. Whole-cell biocatalyst was stable when stored for up to 1-month at 4 °C. In addition, evidence has been provided that multicopper oxidase CueO from the E. coli expression host contributed to the oxidation efficiency of the whole-cell biocatalyst. The immobilized whole-cell biocatalyst showed satisfactory activity and retained 37% of its original activity after three biotransformation cycles.

Structure-Based Redesign of a Self-Sufficient Flavin-Containing Monooxygenase towards Indigo Production.

Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the kcat for indole could be improved 1.5-fold by screening a relatively small number of enzyme mutants. This project not only resulted in an improved biocatalyst but also provided an improved understanding of the structural elements that determine the activity of mFMO and provides hints for further improvement of the monooxygenase as biocatalyst.

Characterization of a thermostable flavin-containing monooxygenase from Nitrincola lacisaponensis (NiFMO).

The flavin-containing monooxygenases (FMOs) play an important role in drug metabolism but they also have a high potential in industrial biotransformations. Among the hitherto characterized FMOs, there was no thermostable representative, while such biocatalyst would be valuable for FMO-based applications. Through a targeted genome mining approach, we have identified a gene encoding for a putative FMO from Nitrincola lacisaponensis, an alkaliphilic extremophile bacterium. Herein, we report the biochemical and structural characterization of this newly discovered bacterial FMO (NiFMO). NiFMO can be expressed as active and soluble enzyme at high level in Escherichia coli (90-100 mg/L of culture). NiFMO is relatively thermostable (melting temperature (Tm) of 51 °C), displays high organic solvent tolerance, and accepts a broad range of substrates. The crystal structure of NiFMO was solved at 1.8 Å resolution, which allows future structure-based enzyme engineering. Altogether, NiFMO represents an interesting newly discovered enzyme with the appropriate features to develop into an industrially applied biocatalyst.

Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins.

Many proteins are rapidly deactivated when exposed to high or even ambient temperatures. This cannot only impede the study of a particular protein, but also is one of the major reasons why enzyme catalysis is still widely unable to compete with established chemical processes. Furthermore, differences in protein stability are a challenge in synthetic biology, when individual modules prove to be incompatible. The targeted stabilization of proteins can overcome these hurdles, and protein engineering techniques are more and more reliably supported by computational chemistry tools. Accordingly, algorithms to predict the differences in folding energy of a mutant compared to the wild-type, ΔΔGfold, are used in the highly successful FRESCO workflow. The resulting single mutant prediction library consists typically of a few hundred amino acid exchanges, and after combining the most successful hits we so far obtained stabilized mutants which exhibited increases in apparent melting temperature of 20-35°C and showed vastly increased half-lives, as well as resistance to cosolvents. Here, we report a detailed protocol to generate these mutant libraries experimentally, covering the entire workflow from primer design, through mutagenesis, protein production and screening, to mutation combination strategies. The individual parts of the method are furthermore applicable to many other scenarios besides protein stabilization, and these protocols are valuable for any project requiring individual or semi high-throughput site-directed mutagenesis, protein expression and purification, or generation of mutant combination libraries.

Creating Oxidase-Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions.

A set of bifunctional oxidase-peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low-micromolar range. The peroxidase fusions of eugenol oxidase and 5-hydroxymethylfurfural oxidase could be used for dioxygen-driven, one-pot, two-step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase-peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases.

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L(-1) was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

Catalases as biocatalysts in technical applications: current state and perspectives.

Catalases represent a class of enzymes which has found its place among industrially relevant biocatalysts due to their exceptional catalytic rate and high stability. Textile bleaching prior to the dyeing process is the main application and has been performed on a large scale for the past few decades. Their limited substrate scope has not prevented the development of various other catalase-based applications. Newly developed approaches continue to exploit their excellent catalytic potential to degrade hydrogen peroxide while (per)oxidase activity of catalases is opening a new range of possibilities as well. This review provides an overview of applications that involve heme-containing catalases that have been demonstrated in recent years.

Not so monofunctional--a case of thermostable Thermobifida fusca catalase with peroxidase activity.

Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

Bacillus amyloliquefaciens laccase--from soil bacteria to recombinant enzyme for wastewater decolorization.

One hundred wild type strains of Bacillus sp. were isolated from industrial and agricultural soil across Serbia and screened for laccase activity. Three strains showed high laccase activity temperature optimum of 65 and 80 °C towards ABTS. A new laccase gene from the strain with highest temperature optimum, namely Bacillus amyloliquefaciens 12B was cloned and expressed in Escherichia coli. Recombinant laccase degraded dye Reactive blue 52 at pH 7.0 and pH 4.0 and at elevated temperature, while fungal laccases was unable to act on this substrate at pH higher than 4.0 and was quickly inactivated at temperatures higher than 45 °C. Degradation of dye was monitored by HPLC-DAD and resulting precipitate was analyzed by FTIR spectroscopy. Single product peak without chromophore was detected in solution, while water insoluble aggregate, presumably dye polymer is formed retaining blue color.

Lignin degradation by selected fungal species.

As biological decomposition of plant biomass represents a popular alternative environmental-friendly and economically justified process, screening of ligninolytic enzyme systems of various fungal species is a topical study area. The goal of the study was to obtain clear insight into the dynamics of laccase, Mn-dependent peroxidase, and Mn-independent peroxidase activity and levels of wheat straw lignin degradation in seven wood-rotting fungi. The best laccase producers were Pleurotus ostreatus and Pleurotus eryngii. Lenzites betulinus and Fomitopsis pinicola were the best Mn-dependent peroxidase producers, and P. ostreatus the weakest one. The peak of Mn-independent peroxidase was noted in Dichomytus squalens, and the minimum value in P. ostreatus. The profiles of the three enzymes, obtained by isoelectric focusing, were variable depending on the species and cultivation period. D. squalens was the best lignin degrader (34.1% of total lignin amount), and P. ostreatus and P. eryngii the weakest ones (7.1% and 14.5%, respectively).

Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions.

Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein-metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel 'tentacle' carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0-8.0 and temperature optimum at 25°C. Immobilized PPO shows almost 100% of activity at 0°C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8h duration.

Immobilization of cell wall invertase modified with glutaraldehyde for continuous production of invert sugar.

Yeast cell wall invertase (CWI) was modified with dimethyl suberimidate, glutaraldehyde, formaldehyde, and sodium periodate. Retained activity after modification was 45% for CWI modified with formaldehyde, 77% for CWI modified with sodium periodate, 80% for CWI modified with glutaraldehyde, and 115% for CWI modified with dimethyl suberimidate. Chemically modified and native CWIs showed significantly broad pH stability (pH 3-11), whereas after incubations at 50, 60, and 70 °C, CWI modified with glutaraldehyde showed the highest thermostability. Optimum pH for CWI modified with glutaraldehyde was between 4 and 5, whereas optimum temperature was at 60 °C. Comparison to CWI modified with glutaraldehyde after immobilization within alginate beads showed broader pH optimum (4.0-5.5) as well as broader temperature optimum (55-70 °C). Column bed reactor packed with the immobilized CWI modified with glutaraldehyde was successfully used for the 95% inversion of 60% (w/w) sucrose at the flow rate of 3 bed volumes per hour, pH 4.9, and 45 °C. A 1 month productivity of 3844 kg of inverted sugar/kg of the immobilisate was obtained.