Sustainability and oxidase biocatalysis – An overview

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Biotechnological Potential of Oxidative Enzymes from Actinobacteria

Oxidative enzymes are often considered for use in industrial processes because of the variety of reactions they are able to catalyse. In the past, most of these oxidative enzymes were obtained from fungi. However, in recent years, it has become evident that these enzymes are also produced by bacteria, including actinobacterial strains, which can therefore be considered as an underexploited resource of oxidative enzymes with potential for application in various industries. This chapter will focus on selected oxidative enzymes found in actinobacteria, their potential for application in industrial processes and how we can access and improve these enzymes to suit the required bioprocess conditions

Flavonoid and phenolic acid profiles of dehulled and whole vigna subterranea (l.) Verdc seeds commonly consumed in South Africa

Bambara groundnut (BGN) is an underexploited crop with a rich nutrient content and is used in traditional medicine, but limited information is available on the quantitative characterization of its flavonoids and phenolic acids. We investigated the phenolic profile of whole seeds and cotyledons of five BGN varieties consumed in South Africa using UPLC-qTOF-MS and GC-MS. Twenty-six phenolic compounds were detected/quantified in whole seeds and twenty-four in cotyledon, with six unidentified compounds

Multicopper oxidases : modular structure, sequence space, and evolutionary relationships

Multicopper oxidases (MCOs) use copper ions as cofactors to oxidize a variety of substrates while reducing oxygen to water. MCOs have been identified in various taxa, with notable occurrences in fungi. The role of these fungal MCOs in lignin degradation sparked an interest due to their potential for application in biofuel production and various other industries. MCOs consist of different protein domains, which led to their classification into two‐, three‐, and six‐domain MCOs. The previously established Laccase and Multicopper Oxidase Engineering Database (https://lcced.biocatnet.de) was updated and now includes 51 058 sequences and 229 structures of MCOs. Sequences and structures of all MCOs were systematically compared. All MCOs consist of cupredoxin‐like domains. Two‐domain MCOs are formed by the N‐ and C‐terminal domain (domain N and C), while three‐domain MCOs have an additional domain (M) in between, homologous to domain C. The six‐domain MCOs consist of alternating domains N and C, each three times. Two standard numbering schemes were developed for the copper‐binding domains N and C, which facilitated the identification of conserved positions and a comparison to previously reported results from mutagenesis studies. Two sequence motifs for the copper binding sites were identified per domain. Their modularity, depending on the placement of the T1‐copper binding site, was demonstrated. Protein sequence networks showed relationships between two‐ and three‐domain MCOs, allowing for family‐specific annotation and inference of evolutionary relationships.Federal Ministry of Education and Research, GermanyNational Research Foundation, South Afric

Expansin Engineering Database : a navigation and classification tool for expansins and homologues

Expansins have the remarkable ability to loosen plant cell walls and cellulose material without showing catalytic activity and therefore have potential applications in biomass degradation. To support the study of sequence‐structure‐function relationships and the search for novel expansins, the Expansin Engineering Database (ExED, https://exed.biocatnet.de) collected sequence and structure data on expansins from Bacteria, Fungi, and Viridiplantae, and expansin‐like homologues such as carbohydrate binding modules, glycoside hydrolases, loosenins, swollenins, cerato‐platanins, and EXPNs. Based on global sequence alignment and protein sequence network analysis, the sequences are highly diverse. However, many similarities were found between the expansin domains. Newly created profile hidden Markov models of the two expansin domains enable standard numbering schemes, comprehensive conservation analyses, and genome annotation. Conserved key amino acids in the expansin domains were identified, a refined classification of expansins and carbohydrate binding modules was proposed, and new sequence motifs facilitate the search of novel candidate genes and the engineering of expansins.Federal Ministry of Education and Research, GermanyNational Research Foundation, South AfricaProjekt DEA

Increasing the scale of peroxidase production by Streptomyces sp. strain BSII#1

AIMS : To optimise peroxidase production by Streptomyces sp. strain BSII#1, up to 3 l culture volumes. METHODS AND RESULTS : Peroxidase production by Streptomyces sp. strain BSII#1 was optimised in terms of production temperature and pH and the use of lignin-based model chemical inducers. The highest peroxidase activity (1.30±0.04 U ml-1) in 10 ml culture volume was achieved in a complex production medium (pH 8) at 37°C in the presence of 0.1 mmol l-1 veratryl alcohol, which was greater than those reported previously. Scale-up to 100 ml and 400 ml culture volumes resulted in decreased peroxidase production (0.53±0.10 U ml- 1 and 0.26±0.08 U ml-1 respectively). However, increased aeration improved peroxidase production with the highest production achieved using an airlift bioreactor (4.76±0.46 U ml-1 in 3 l culture volume). CONCLUSIONS : Veratryl alcohol (0.1 mmol l-1) is an effective inducer of peroxidase production by Streptomyces sp. strain BSII#1. However, improved aeration increased peroxidase production in larger volumes without the use of an inducer, surpassing induced yields in an optimised small-scale process. SIGNIFICANCE AND IMPACT OF THE STUDY : Only a limited number of reports in literature have focused on the up-scaling of bacterial peroxidase production. There remains opportunity for feasible large-scale production of bacterial peroxidases with potentially novel biocatalytic properties.National Research Foundation (NRF) of South Africa,Water Research Commission.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-2672hb201

Partial purification and characterisation of two actinomycete tyrosinases and their application in cross-linking reactions

Actinomycetes are a ubiquitous group of bacteria, and are hypothesised to produce tyrosinases for pro-tection against the potential toxic effect of phenolic compounds and for the production of melanin. In thisstudy, tyrosinase production by Streptomyces pharetrae CZA14T(CZA14Tyr) and Streptomyces polyantibi-oticus SPRT(SPRTyr) was optimised. The enzymes were partially purified and biochemically characterisedto determine their suitability for industrial applications. SPRTyr was stable up to 40◦C and at pH 4.5–10.0,while CZA14Tyr was stable up to 40◦C and at pH 6.5–10.0. The enzymes showed variable stability in thepresence of water-miscible organic solvents and were able to oxidize l-DOPA in the presence of these sol-vents. A limited inhibitory effect was observed with arbutin, EDTA, sodium chloride and sodium dodecylsulphate, while both enzymes were strongly inhibited by the reducing agents used in this study. Inhibi-tion of enzyme activity was observed in the presence of 1 mM Cu2+and 5 mM Co2+for SPRTyr, and 5 mMFe2+and 5 mM Zn2+for CZA14Tyr. When applied in various cross-linking reactions both tyrosinases wereable to cross-link casein and gelatine in the absence of a phenolic compound, showing potential forapplication in the food industry and for the production of biomaterials.National Research Foundation (NRF) of South Africa for project funding [Grant No. 73691] and Cape Peninsula University of Technology (CPUT) University Research Funding.http://www.elsevier.com/locate/molcatb2016-12-31hb201

Enzymatic modification of 2,6-dimethoxyphenol for the synthesis of dimers with high antioxidant capacity

2,6-Dimethoxyphenol is a phenolic compound that is extensively used for the measurement of laccase activity, but is often not exploited for its potential as an antioxidant compound. Since laccase can be used to modify phenolic antioxidants as a way of improving their activities, the present study investigated the laccase-mediated oxidation of 2,6-dimethoxyphenol in biphasic or homogenous aqueous-organic media for the production of compounds with higher antioxidant capacity than the starting substrate. The main product was a dimer (m/z 305.0672), which was further characterized as a symmetrical C-C linked 3,3’,5,5’-tetramethoxy biphenyl-4,4’-diol. In the monophasic system, the dimer was preferentially formed when acetone was used as co34 solvent, while in the biphasic system, formation of the dimer increased as the concentration of ethyl acetate was increased from 50 to 90 %. The dimer showed higher antioxidant capacity than the substrate (≈ 2×) as demonstrated by standard antioxidant assays (DPPH and FRAP). These results demonstrate that a product of the laccase-catalysed oxidation of 2,6-dimethoxyphenol can find useful application as a bioactive compound.National Research Foundation (South Africa)http://www .elsevier.com/locate/procbiohb2013ai201

Minor differences in sand physicochemistry lead to major differences in bacterial community structure and function after exposure to synthetic acid mine drainage

The formation of environmentally toxic acidic waste from mining activities is a world-wide problem. Neutralization of this waste can be accomplished by physicochemical and/or biological means. In this short-term study, synthetic acid mine drainage was added to sandfilled mesocosms containing silica-dominated (quartz) sand. Glucose was added as a carbon source for microbial iron and/or sulphate reduction. Replicates contained two separate batches of sand obtained from the same quarry site. The investigations used to assess and compare the chemical and biological functioning of the replicates included system hydraulic conductivity measurements, sand chemistry, effluent chemistry and bacterial community fingerprinting. Minor differences in composition of the sand, including the levels of available nutrients and micronutrients, resulted in major differences in measured parameters. Significant differences in effluent chemistry were found in systems containing different batches of sand. It was demonstrated that the characteristics of the sand and the presence of acid mine drainage impacted the bacterial community structure and function. The importance of the physical substrate on the selection of functional microbial communities in systems remediating AMD should not be under-estimated. The physical substrate should be carefully selected and it may be prudent to include small-scale comparative studies in each particular setting prior to full-scale implementation.Water Research Commission, Cape Peninsula University of Technology and the National Research Foundation of South Africahttp://link.springer.com/journal/12257hb201

Selection of Clostridium spp. in biological sand filters neutralizing synthetic acid mine drainage

In this study, three biological sand filter (BSF) were contaminated with a synthetic iron- [1500 mg L-1 Fe(II), 500 mg L-1 Fe(III)] and sulphate-rich (6000 mg L-1 SO2/4-) acid mine drainage (AMD) (pH = 2), for 24 days, to assess the remediation capacity and the evolution of autochthonous bacterial communities (monitored by T-RFLP and 16S rRNA gene clone libraries). To stimulate BSF bioremediation involving sulphate-reducing bacteria, a readily degradable carbon source (glucose, 8000 mg L-1) was incorporated into the influent AMD. Complete neutralization and average removal efficiencies of 81.5 (±5.6)%, 95.8 (±1.2)% and 32.8 (±14.0)% for Fe(II), Fe(III) and sulphate were observed, respectively. Our results suggest that microbial iron reduction and sulphate reduction associated with iron precipitation were the main processes contributing to AMD neutralization. The effect of AMD on BSF sediment bacterial communities was highly reproducible. There was a decrease in diversity, and notably a single dominant operational taxonomic unit (OTU), closely related to Clostridium beijerinckii, which represented up to 65% of the total community at the end of the study period.Web of Scienc