Isolation and characterization of a novel thermostable and catalytically efficient laccase from Peniophora sp. strain UD4

Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date

Spherezymes: A novel structured self-immobilisation enzyme technology

<p>Abstract</p> <p>Background</p> <p>Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.</p> <p>Results</p> <p>Through addition of protein cross-linking agents to a water-in-oil emulsion of an aqueous enzyme solution, structured self-immobilised spherical enzyme particles of <it>Pseudomonas fluorescens </it>lipase were formed. The particles could be recovered from the emulsion, and activity in aqueous and organic solvents was successfully demonstrated. Preliminary data indicates that the lipase tended to collect at the interface.</p> <p>Conclusion</p> <p>The immobilised particles provide a number of advantages. The individual spherical particles had a diameter of between 0.5–10 μm, but tended to form aggregates with an average particle volume distribution of 100 μm. The size could be controlled through addition of surfactant and variations in protein concentration. The particles were robust enough to be recovered by centrifugation and filtration, and to be recycled for further reactions. They present lipase enzymes with the active sites selectively orientated towards the exterior of the particle. Co-immobilisation with other enzymes, or other proteins such as albumin, was also demonstrated. Moreover, higher activity for small ester molecules could be achieved by the immobilised enzyme particles than for free enzyme, presumably because the lipase conformation required for catalysis had been locked in place during immobilisation. The immobilised enzymes also demonstrated superior activity in organic solvent compared to the original free enzyme. This type of self-immobilised enzyme particle has been named spherezymes.</p

Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in the removal of radical NO produced during respiratory metabolism, and applications in bioremediation and biocatalysis have been identified. However, a rapid, accurate, and sensitive enzyme assay has not yet been developed for this enzyme family. In this study, we optimized reaction conditions for the development of a spectrophotometric NOR activity microassay using NOC-5 for the provision of NO in solution. We also demonstrate that the assay is suitable for the quantification and characterization of P450-type NOR. The Km and kcat kinetic constants obtained by this assay were comparable to the values determined by gas chromatography, but with improved convenience and cost efficiency, effectively by miniaturization. To our knowledge, this is the first study to present the quantification of NOR activity in a kinetic microassay format.A CSIR parliamentary grant (Pretoria, South Africa)http://www.elsevier.com/locate/yabiohb201

Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.

The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSynTM polymer microspheres with varying functional group densities. The successful immobilisation ofthe enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 mol g−1 displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg−1 and 6.75 U mg−1 for GDH and NOD with respective loading capacities of 51% (0.51 mg mg−1) and 129% (1.29 mg mg−1). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSynTM A during temperature incubation at 65 ◦C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 ◦C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH

Sustainable production of nucleoside analogues by a high-efficient purine 2'-deoxyribosyltransferase immobilized onto Ni 2+ chelate magnetic microparticles

The present work aims to develop a magnetic biocatalyst for customized production of nucleoside analogues using mutant His-tagged purine 2'-deoxyribosyltransferase from Trypanosoma brucei (TbPDTV11S) immobilized onto Ni2+ chelate magnetic iron oxide porous microparticles (MTbPDTV11S). Biochemical characterization revealed MTbPDTV11S5 as optimal candidate for further studies (10,552 IU g-1; retained activity 54% at 50 °C and pH 6.5). Interestingly, MTbPDTV11S5 displayed the highest activity value described up to date for an immobilized NDT. Moreover, MTbPDTV11S5 was successfully employed in the one-pot, one-step production of different therapeutic nucleoside analogues, such as cladribine or 2'-deoxy-2-fluoroadenosine, among others. Finally, MTbPDTV11S5 proved to be stable when stored at 50 °C for 8 h and pH 6.0 and reusable up to 10 times without negligible loss of activity in the enzymatic production of the antitumor prodrug 2'-deoxy-2-fluoroadenosine.Sin financiación7.539 JCR (2019) Q1, 1/13 Agricultural Engineering2.430 SJR (2019) Q1, 15/193 BioengineeringNo data IDR 2019UE

Enzymatic synthesis of therapeutic nucleosides using a highly versatile purine nucleoside 2’-Deoxyribosyl transferase from Trypanosoma brucei

11 pags, 5 figs, 7 tabsThe use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo-, regio- and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2’-deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50–70 °C), pH (4–7) and ionic strength (0–500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8–10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure-guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8-fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde-activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for >25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.This work was supported by grants SAN151610 from the Santander Foundation and 2016/UEM08 from the European University of Madrid to JFL and SAF2015-64629-C2-2-R from the Spanish Ministerio de Economía y Competitividad to F

Enzymatic Synthesis of Therapeutic Nucleosides using a Highly Versatile Purine Nucleoside 2’‐DeoxyribosylTransferase from Trypanosoma brucei

The use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo‐, regio‐ and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2’‐deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50–70 °C), pH (4–7) and ionic strength (0–500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8–10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure‐guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8‐fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde‐activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for >25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.Sin financiación4.495 JCR (2018) Q2, 43/148 Chemistry, Physical1.472 SJR (2018) Q1, 12/60 Catalysis, 10/72 Inorganic Chemistry, 19/185 Organic Chemistry, 22/166 Physical and Theoretical ChemistryNo data IDR 2018UE

Optical microscope photographs of spheres stained with comassie blue (left) and scanning electron microscope image (right) of 20% lipase/80% albumin spheres

<p><b>Copyright information:</b></p><p>Taken from "Spherezymes: A novel structured self-immobilisation enzyme technology"</p><p>http://www.biomedcentral.com/1472-6750/8/8</p><p>BMC Biotechnology 2008;8():8-8.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2266724.</p><p></p

Spherezyme particles consisting of various percentage combinations of lipase and BSA

Lipase specific activity (on -nitrophenyl palmitate) is represented as a function of lipase mass as opposed to total protein mass. Data was generated from particles that were formed using 40 μl glutaraldehyde-EDA mix as a cross-linker and nonoxynol-4 as the surfactant.<p><b>Copyright information:</b></p><p>Taken from "Spherezymes: A novel structured self-immobilisation enzyme technology"</p><p>http://www.biomedcentral.com/1472-6750/8/8</p><p>BMC Biotechnology 2008;8():8-8.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2266724.</p><p></p

Activity of a spherezyme particles comprised of a combination of enzymes (lipase and Denilitelaccase) cross-linked with glutaraldehyde: PNPB – -nitrophenyl butyrate; PNPP – -nitrophenyl palmitate (A)

The ratio of -nitrophenyl palmitate to -nitrophenyl butyrate activity of these combined enzyme particles can be compared to free lipase and lipase spherezyme particles (B).<p><b>Copyright information:</b></p><p>Taken from "Spherezymes: A novel structured self-immobilisation enzyme technology"</p><p>http://www.biomedcentral.com/1472-6750/8/8</p><p>BMC Biotechnology 2008;8():8-8.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2266724.</p><p></p