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1.
Biotechnol Bioeng ; 119(2): 493-503, 2022 02.
Article in English | MEDLINE | ID: mdl-34796477

ABSTRACT

Lignin valorization may offer a sustainable approach to achieve a chemical industry that is not completely dependent on fossil resources for the production of aromatics. However, lignin is a recalcitrant, heterogeneous, and complex polymeric compound for which only very few catalysts can act in a predictable and reproducible manner. Laccase is one of those catalysts and has often been referred to as an ideal "green" catalyst, as it is able to oxidize various linkages within lignin to release aromatic products, with the use of molecular oxygen and formation of water as the only side product. The extent and rate of laccase-catalyzed lignin conversion were measured using the label-free analytical technique isothermal titration calorimetry (ITC). IITC provides the molar enthalpy of the reaction, which reflects the extent of conversion and the time-dependent power trace, which reflects the rate of the reaction. Calorimetric assessment of the lignin conversion brought about by various fungal and bacterial laccases in the absence of mediators showed marked differences in the extent and rate of conversion for the different enzymes. Kraft lignin conversion by Trametes versicolor laccase followed Michaelis-Menten kinetics and was characterized by the following thermodynamic and kinetic parameters ΔHITC = -(2.06 ± 0.06)·103 kJ mol-1 , KM = 6.6 ± 1.2 µM and Vmax = 0.30 ± 0.02 U/mg at 25°C and pH 6.5. We envision calorimetric techniques as important tools for the development of enzymatic lignin valorization strategies.


Subject(s)
Calorimetry/methods , Laccase , Lignin , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Kinetics , Laccase/chemistry , Laccase/metabolism , Lignin/analysis , Lignin/chemistry , Lignin/metabolism , Polyporaceae/enzymology , Polyporaceae/genetics
2.
Water Res ; 202: 117398, 2021 Sep 01.
Article in English | MEDLINE | ID: mdl-34252865

ABSTRACT

Hydrolysis is considered to be the rate-limiting step in anaerobic digestion of waste activated sludge (WAS). In this study, an innovative 4 stages cascade anaerobic digestion system was researched to (1) comprehensively clarify whether cascading configuration enhances WAS hydrolysis, and to (2) better understand the governing hydrolysis kinetics in this system. The cascade system consisted of three 2.2 L ultra-short solids retention times (SRT) continuous stirred tank reactors (CSTRs) and one 15.4 L CSTR. The cascade system was compared with a reference conventional CSTR digester (22 L) in terms of process performance, hydrolytic enzyme activities and microbial community dynamics under mesophilic conditions (35 °C). The results showed that the cascade system achieved a high and stable total chemical oxygen demand (tCOD) reduction efficiency of 40-42%, even at 12 days total SRT that corresponded to only 1.2 days SRT each in the first three reactors of the cascade. The reference-CSTR converted only 31% tCOD into biogas and suffered process deterioration at the applied low SRTs. Calculated specific hydrolysis rates in the first reactors of the cascade system were significantly higher compared to the reference-CSTR, especially at the lowest applied SRTs. The activities of several hydrolytic enzymes produced in the different stages revealed that protease, cellulase, amino peptidases, and most of the tested glycosyl-hydrolases had significantly higher activities in the first three small digesters of the cascade system, compared to the reference-CSTR. This increase in hydrolytic enzyme production by far exceeded the increase in specific hydrolysis rate, indicating that hydrolysis was limited by solids-surface availability for enzymatic attack. Correspondingly, high relative abundances of hydrolytic-fermentative bacteria and hydrogenotrophic methanogens as well as the presence of syntrophic bacteria were found in the first three digesters of the cascade system. However, in the fourth reactor, acetoclastic methanogens dominated, similarly as in the reference-CSTR. Overall, the results concluded that using multiple CSTRs that are operated at low SRTs in a cascade mode of operation significantly improved the enzymatic hydrolysis rate and extend in anaerobic WAS digestion. Moreover, the governing hydrolysis kinetics in the cascading reactors were far more complex than the generally assumed simplified first-order kinetics.


Subject(s)
Bioreactors , Sewage , Anaerobiosis , Hydrolysis , Kinetics , Methane
3.
Org Process Res Dev ; 25(6): 1414-1418, 2021 Jun 18.
Article in English | MEDLINE | ID: mdl-34168423

ABSTRACT

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L-1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

4.
RSC Adv ; 11(35): 21857-21861, 2021 Jun 15.
Article in English | MEDLINE | ID: mdl-35478789

ABSTRACT

A new immobilization strategy using compartmentalized nanoreactors is herein reported for two biocatalytic processes: (1) N-acetylneuraminate lyase (NAL) is internalized in NAL-c-CLEnAs and used in a continuous flow aldol condensation of N-acetyl-d-mannosamine with sodium pyruvate to N-acetylneuraminic acid; (2) two hydroxysteroid dehydrogenases (HSDH) 7α- and 7ß-HSDH are incorporated in c-CLEnAs and used in a two-step cascade batch synthesis of ursodeoxycholic acid (UDCA). The versatile use of c-CLEnA demonstrates that this immobilization methodology is a valuable addition to the toolbox of synthetic chemists.

5.
Biochim Biophys Acta Gen Subj ; 1865(1): 129766, 2021 01.
Article in English | MEDLINE | ID: mdl-33069831

ABSTRACT

BACKGROUND: Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site. METHODS: We first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed. RESULTS: MD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity. CONCLUSIONS: These findings reveal how MD can predict how changes in the "second shell" residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities. GENERAL SIGNIFICANCE: This study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.


Subject(s)
Adenosine Triphosphate/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , Binding Sites , Mitochondrial Proton-Translocating ATPases/chemistry , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism
6.
Appl Microbiol Biotechnol ; 104(13): 5801-5812, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32358760

ABSTRACT

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.


Subject(s)
Bacterial Proteins/metabolism , Hydro-Lyases/metabolism , Oleic Acid/metabolism , Rhodococcus/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/metabolism , Genome, Bacterial/genetics , Hydro-Lyases/chemistry , Hydro-Lyases/genetics , Hydrogen-Ion Concentration , Kinetics , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/classification , Rhodococcus/genetics , Substrate Specificity , Temperature
7.
Nat Commun ; 11(1): 2258, 2020 05 07.
Article in English | MEDLINE | ID: mdl-32382158

ABSTRACT

En route to a bio-based chemical industry, the conversion of fatty acids into building blocks is of particular interest. Enzymatic routes, occurring under mild conditions and excelling by intrinsic selectivity, are particularly attractive. Here we report photoenzymatic cascade reactions to transform unsaturated fatty acids into enantiomerically pure secondary fatty alcohols. In a first step the C=C-double bond is stereoselectively hydrated using oleate hydratases from Lactobacillus reuteri or Stenotrophomonas maltophilia. Also, dihydroxylation mediated by the 5,8-diol synthase from Aspergillus nidulans is demonstrated. The second step comprises decarboxylation of the intermediate hydroxy acids by the photoactivated decarboxylase from Chlorella variabilis NC64A. A broad range of (poly)unsaturated fatty acids can be transformed into enantiomerically pure fatty alcohols in a simple one-pot approach.


Subject(s)
Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/metabolism , Fatty Alcohols/chemistry , Fatty Alcohols/metabolism , Biological Products/chemistry , Biological Products/metabolism , Limosilactobacillus reuteri/metabolism , Stenotrophomonas maltophilia/metabolism , Substrate Specificity
8.
Chem Sci ; 11(10): 2765-2769, 2020 Feb 07.
Article in English | MEDLINE | ID: mdl-34084336

ABSTRACT

Nano-sized enzyme aggregates, which preserve their catalytic activity are of great interest for flow processes, as these catalytic species show minimal diffusional issues, and are still sizeable enough to be effectively separated from the formed product. The realization of such catalysts is however far from trivial. The stable formation of a micro-to millimeter-sized enzyme aggregate is feasible via the formation of a cross-linked enzyme aggregate (CLEA); however, such a process leads to a rather broad size distribution, which is not always compatible with microflow conditions. Here, we present the design of a compartmentalized templated CLEA (c-CLEnA), inside the nano-cavity of bowl-shaped polymer vesicles, coined stomatocytes. Due to the enzyme preorganization and concentration in the cavity, cross-linking could be performed with substantially lower amount of cross-linking agents, which was highly beneficial for the residual enzyme activity. Our methodology is generally applicable, as demonstrated by using two different cross-linkers (glutaraldehyde and genipin). Moreover, c-CLEnA nanoreactors were designed with Candida antarctica Lipase B (CalB) and Porcine Liver Esterase (PLE), as well as a mixture of glucose oxidase (GOx) and horseradish peroxidase (HRP). Interestingly, when genipin was used as cross-linker, all enzymes preserved their initial activity. Furthermore, as proof of principle, we demonstrated the successful implementation of different c-CLEnAs in a flow reactor in which the c-CLEnA nanoreactors retained their full catalytic function even after ten runs. Such a c-CLEnA nanoreactor represents a significant step forward in the area of in-flow biocatalysis.

9.
RSC Adv ; 9(42): 24039-24042, 2019 Aug 02.
Article in English | MEDLINE | ID: mdl-35527883

ABSTRACT

We report the use of commercial laundry powder as a biocatalyst for a range of lipase-catalysed reactions including (trans)esterification, ester hydrolysis and chemoenzymatic epoxidation reactions. The enzymatic laundry powder exhibited excellent stability and recyclability, making it a readily available and cheap biocatalyst for chemical transformations.

10.
ChemSusChem ; 12(13): 3192-3203, 2019 Jul 05.
Article in English | MEDLINE | ID: mdl-30265441

ABSTRACT

Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α- and 7ß-HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α-OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7ß-OH). With a view to developing robust and active biocatalysts, novel NADH-active 7ß-HSDH species are necessary to enable a solely NAD+ -dependent redox-neutral cascade for UDCA production. A wild-type NADH-dependent 7ß-HSDH from Lactobacillus spicheri (Ls7ß-HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD+ -dependent 7ß-HSDH enzyme in combination with 7α-HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD+ , resulting in high yields (>90 %) of UCA and UDCA.


Subject(s)
Chenodeoxycholic Acid/metabolism , Cholic Acid/metabolism , Hydroxysteroid Dehydrogenases/metabolism , NAD/metabolism , Biocatalysis , Biotransformation , Clostridium/enzymology , Hydrogen-Ion Concentration , Kinetics , Lactobacillus/enzymology , Oxidation-Reduction , Stenotrophomonas maltophilia/enzymology , Temperature
11.
Appl Microbiol Biotechnol ; 102(24): 10579-10588, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30302519

ABSTRACT

Peroxidases are well-known biocatalysts produced by all organisms, especially microorganisms, and used in a number of biotechnological applications. The enzyme DypB from the lignin-degrading bacterium Rhodococcus jostii was recently shown to degrade solvent-obtained fractions of a Kraft lignin. In order to promote the practical use, the N246A variant of DypB, named Rh_DypB, was overexpressed in E. coli using a designed synthetic gene: by employing optimized conditions, the enzyme was fully produced as folded holoenzyme, thus avoiding the need for a further time-consuming and expensive reconstitution step. By a single chromatographic purification step, > 100 mg enzyme/L fermentation broth with a > 90% purity was produced. Rh_DypB shows a classical peroxidase activity which is significantly increased by adding Mn2+ ions: kinetic parameters for H2O2, Mn2+, ABTS, and 2,6-DMP were determined. The recombinant enzyme shows a good thermostability (melting temperature of 63-65 °C), is stable at pH 6-7, and maintains a large part of the starting activity following incubation for 24 h at 25-37 °C. Rh_DypB activity is not affected by 1 M NaCl, 10% DMSO, and 5% Tween-80, i.e., compounds used for dye decolorization or lignin-solubilization processes. The enzyme shows broad dye-decolorization activity, especially in the presence of Mn2+, oxidizes various aromatic monomers from lignin, and cleaves the guaiacylglycerol-ß-guaiacyl ether (GGE), i.e., the Cα-Cß bond of the dimeric lignin model molecule of ß-O-4 linkages. Under optimized conditions, 2 mM GGE was fully cleaved by recombinant Rh_DypB, generating guaiacol in only 10 min, at a rate of 12.5 µmol/min mg enzyme.


Subject(s)
Bacterial Proteins/metabolism , Coloring Agents/chemistry , Guaifenesin/analogs & derivatives , Manganese/metabolism , Peroxidases/metabolism , Anthraquinones/chemistry , Anthraquinones/metabolism , Azure Stains/chemistry , Azure Stains/metabolism , Bacterial Proteins/genetics , Coloring Agents/metabolism , Dimethyl Sulfoxide/chemistry , Escherichia coli/genetics , Guaifenesin/metabolism , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Kinetics , Naphthalenesulfonates/chemistry , Naphthalenesulfonates/metabolism , Oxidation-Reduction , Peroxidases/genetics , Polysorbates/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Rhodococcus/genetics , Temperature
12.
Angew Chem Int Ed Engl ; 57(41): 13648-13651, 2018 10 08.
Article in English | MEDLINE | ID: mdl-30106504

ABSTRACT

The photoenzymatic decarboxylation of fatty acids to alkanes is proposed as an alternative approach for the synthesis of biodiesel. By using a recently discovered photodecarboxylase from Chlorella variabilis NC64A (CvFAP) we demonstrate the irreversible preparation of alkanes from fatty acids and triglycerides. Several fatty acids and their triglycerides are converted by CvFAP in near-quantitative yield and exclusive selectivity upon illumination with blue light. Very promising turnover numbers of up to 8000 were achieved in this proof-of-concept study.

13.
Beilstein J Org Chem ; 14: 470-483, 2018.
Article in English | MEDLINE | ID: mdl-29520309

ABSTRACT

Ursodeoxycholic acid (UDCA) is a pharmaceutical ingredient widely used in clinics. As bile acid it solubilizes cholesterol gallstones and improves the liver function in case of cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. The now available chemical routes for the obtainment of UDCA yield about 30% of final product. For these syntheses several protection and deprotection steps requiring toxic and dangerous reagents have to be performed, leading to the production of a series of waste products. In many cases the cholic acid itself first needs to be prepared from its taurinated and glycilated derivatives in the bile, thus adding to the complexity and multitude of steps involved of the synthetic process. For these reasons, several studies have been performed towards the development of microbial transformations or chemoenzymatic procedures for the synthesis of UDCA starting from CA or chenodeoxycholic acid (CDCA). This promising approach led several research groups to focus their attention on the development of biotransformations with non-pathogenic, easy-to-manage microorganisms, and their enzymes. In particular, the enzymatic reactions involved are selective hydrolysis, epimerization of the hydroxy functions (by oxidation and subsequent reduction) and the specific hydroxylation and dehydroxylation of suitable positions in the steroid rings. In this minireview, we critically analyze the state of the art of the production of UDCA by several chemical, chemoenzymatic and enzymatic routes reported, highlighting the bottlenecks of each production step. Particular attention is placed on the precursors availability as well as the substrate loading in the process. Potential new routes and recent developments are discussed, in particular on the employment of flow-reactors. The latter technology allows to develop processes with shorter reaction times and lower costs for the chemical and enzymatic reactions involved.

14.
Appl Microbiol Biotechnol ; 102(5): 2425-2439, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29380032

ABSTRACT

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75 °C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Gene Expression , Geobacter/enzymology , Laccase/chemistry , Laccase/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Geobacter/chemistry , Geobacter/genetics , Hot Temperature , Hydrogen-Ion Concentration , Laccase/genetics , Laccase/metabolism , Substrate Specificity
15.
Biotechnol Rep (Amst) ; 13: 49-57, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28352563

ABSTRACT

Degradation of lignin constitutes a key step in processing biomass to become useful monomers but it remains challenging. Compared to fungi, bacteria are much less characterized with respect to their lignin metabolism, although it is reported that many soil bacteria, especially actinomycetes, attack and solubilize lignin. In this work, we screened 43 filamentous actinomycetes by assaying their activity on chemically different substrates including a soluble and semi-degraded lignin derivative (known as alkali lignin or Kraft lignin), and we discovered a novel and valuable peroxidase activity produced by the recently classified actinomycete Nonomuraea gerenzanensis. Compared to known fungal manganese and versatile peroxidases, the stability of N. gerenzanensis peroxidase activity at alkaline pHs and its thermostability are significantly higher. From a kinetic point of view, N. gerenzanensis peroxidase activity shows a Km for H2O2 similar to that of Phanerochaete chrysosporium and Bjerkandera enzymes and a lower affinity for Mn2+, whereas it differs from the six Pleurotus ostreatus manganese peroxidase isoenzymes described in the literature. Additionally, N. gerenzanensis peroxidase shows a remarkable dye-decolorizing activity that expands its substrate range and paves the way for an industrial use of this enzyme. These results confirm that by exploring new bacterial diversity, we may be able to discover and exploit alternative biological tools putatively involved in lignin modification and degradation.

16.
Enzyme Microb Technol ; 96: 143-150, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27871375

ABSTRACT

Enzymatic lignin degradation represents a key challenge for integrated biorefineries. Notwithstanding the rich content in aromatic compounds, lignin's complex structure has hampered identification of an effective and cost-efficient enzymatic procedure to transform it into less complex product families. Advancements in enzymatically modifying or degrading lignin require a simple and reliable analytical method to quickly screen diverse lignin samples by employing different enzymes and conditions. Here, we report on a novel, rapid, and economic colorimetric assay for lignin oxidation based on the reaction of 2,4-dinitrophenylhydrazine with the carbonyl groups generated by enzymatic oxidation. The assay was validated on monomeric and dimeric lignin model compounds by comparison with HPLC analysis. The colorimetric method was used to investigate the activity of ten laccases and eight peroxidases on three technical lignins under different experimental conditions (e.g., by altering pH and mediator used). The colorimetric method was also coupled to a size-exclusion chromatographic separation of the lignin sample obtained after the enzymatic treatment in order to verify whether the enzymatic treatment resulted in lignin depolymerization, too. On the basis of this novel procedure, appropriate enzymatic treatments can now be identified to generate valuable lignin product streams.


Subject(s)
Colorimetry/methods , Lignin/metabolism , Biotechnology , Chromatography, Gel , Chromatography, High Pressure Liquid , High-Throughput Screening Assays , Laccase/metabolism , Lignin/chemistry , Oxidation-Reduction , Peroxidases/metabolism , Substrate Specificity
17.
Protein Expr Purif ; 123: 60-9, 2016 07.
Article in English | MEDLINE | ID: mdl-27050199

ABSTRACT

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.


Subject(s)
Cloning, Molecular , Laccase/metabolism , Marinomonas/enzymology , Catechol Oxidase/chemistry , Catechol Oxidase/genetics , Catechol Oxidase/metabolism , Catechols/metabolism , Enzyme Stability , Escherichia coli/genetics , Kinetics , Laccase/chemistry , Laccase/genetics , Lignin/metabolism , Marinomonas/chemistry , Marinomonas/genetics , Marinomonas/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , Substrate Specificity , Temperature
18.
N Biotechnol ; 33(3): 387-98, 2016 May 25.
Article in English | MEDLINE | ID: mdl-26844639

ABSTRACT

Laccases from different sources are employed in a number of biotechnological processes, each characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In order to avoid the bias generated by different assay methodologies, in this work we investigated the main properties of ten laccases from fungi and bacteria under identical conditions. As a general rule, the optimal activity was apparent at pH 3-4 and was lost at pH≥7.0 (all laccases were stable at pH≥7.0); enzymes active at neutral pH values were also identified. For all tested laccases, activity increased with temperature up to 80°C and stability was good at 25°C. Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO (up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin degradation processes requiring solvents. The available laccases are versatile and satisfy requirements related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably compares with the tested enzymes, indicating that it is well suited for different biotechnological applications.


Subject(s)
Bacteria/enzymology , Fungi/enzymology , Industrial Microbiology , Laccase/metabolism , Benzothiazoles/chemistry , Dimethyl Sulfoxide/pharmacology , Hydrogen-Ion Concentration , Kinetics , Laccase/isolation & purification , Polysorbates/pharmacology , Sodium Chloride/pharmacology , Sulfonic Acids/chemistry , Temperature
19.
FEBS J ; 282(7): 1190-213, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25649492

ABSTRACT

A main goal of green biotechnology is to reduce our dependence on fossil reserves and to increase the use of renewable materials. For this, lignocellulose, which is composed of cellulose, hemicellulose and lignin, represents the most promising feedstock. The latter is a complex aromatic heteropolymer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxyphenyl units linked by ß-aryl ether linkages, biphenyl bonds and heterocyclic linkages. Accordingly, lignin appears to be a potentially valuable renewable aromatic chemical, thus representing a main pillar in future biorefinery. The resistance of lignin to breakdown is the main bottleneck in this process, although a variety of white-rot fungi, as well as bacteria, have been reported to degrade lignin by employing different enzymes and catabolic pathways. Here, recent investigations have expanded the range of natural biocatalysts involved in lignin degradation/modification and significant progress related to enzyme engineering and recombinant expression has been made. The present review is focused primarily on recent trends in ligninolytic green biotechnology to suggest the potential (industrial) application of ligninolytic enzymes. Future perspectives could include synergy between natural enzymes from different sources (as well as those obtained by protein engineering) and other pretreatment methods that may be required for optimal results in enzyme-based, environmentally friendly, technologies.


Subject(s)
Laccase/chemistry , Lignin/chemistry , Peroxidases/chemistry , Bacterial Proteins/chemistry , Biocatalysis , Fungal Proteins/chemistry , Kinetics , Oxidation-Reduction , Protein Engineering
20.
Appl Microbiol Biotechnol ; 98(2): 739-48, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23995223

ABSTRACT

Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7 mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis.


Subject(s)
Biosensing Techniques/methods , Directed Molecular Evolution , Food Analysis/methods , Histamine/analysis , Meat , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Animals , Arthrobacter/enzymology , Arthrobacter/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/genetics , Fishes , Gene Expression , Molecular Sequence Data , Sensitivity and Specificity , Sequence Analysis, DNA
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