Viruses, variants and vaccines.

The current SARS-CoV-2 pandemic has brought a number of major global clinical, sociological and economic issues into sharp focus. We address some of these issues, focusing on short-term factors such as virus mutations and vaccine efficacy, and also considering the longer-term implications of the current pandemic. We discuss societal responses to the presence of a pathogen that will probably remain in circulation for decades or longer, and to future new emergent viruses.

Research in COVID-19 times: The way forward.

The COVID-19 pandemic has had a major impact on research at universities. Universities around the world, including in South Africa, have been or are still closed as part of national lockdown strategies. Students have not been attending classes or doing hands-on experimental work, and students and academics have been working from home. Many thousands of students have had their university education interrupted, and for them, the resumption of learning programmes online, and where possible in research laboratories, is critically important. There is no question that as we emerge from lockdown we will not be entering a world that resembles a 'norm' as lived in the pre-COVID-19 era, and many changes will be required. Here we discuss the importance of research, the urgency to get things up and running again, and strategies that will need to be implemented to ensure that research activities continue. At the same time, it is necessary to ensure that students and staff are not exposed to risk in their research endeavours, which will require the development and implementation of risk management plans.

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

AIMS: To optimize 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 optimized 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 8·0) 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 and 400 ml culture volumes resulted in decreased peroxidase production (0·53 ± 0·10 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 optimized 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.

Assessment of temporal and spatial evolution of bacterial communities in a biological sand filter mesocosm treating winery wastewater.

AIMS: To assess the impact of winery wastewater (WW) on biological sand filter (BSF) bacterial community structures, and to evaluate whether BSFs can constitute alternative and valuable treatment- processes to remediate WW. METHODS AND RESULTS: During 112 days, WW was used to contaminate a BSF mesocosm (length 173 cm/width 106 cm/depth 30 cm). The effect of WW on bacterial communities of four BSF microenvironments (surface/deep, inlet/outlet) was investigated using terminal-restriction fragment length polymorphism (T-RFLP). BSF achieved high Na (95·1%), complete Cl and almost complete chemical oxygen demand (COD) (98·0%) and phenolic (99·2%) removals. T-RFLP analysis combined with anosim revealed that WW significantly modified the surface and deep BSF bacterial communities. CONCLUSIONS: BSF provided high COD, phenolic and salt removals throughout the experiment. WW-selected bacterial communities were thus able to tolerate and/or degrade WW, suggesting that community composition does not alter BSF performances. However, biomass increased significantly in the WW-impacted surface sediments, which could later lead to system clogging and should thus be monitored. SIGNIFICANCE AND IMPACT OF THE STUDY: BSFs constitute alternatives to constructed wetlands to treat agri effluents such as WW. To our knowledge, this study is the first unravelling the responses of BSF bacterial communities to contamination and suggests that WW-selected BSF communities maintained high removal performances.

Treatment of high ethanol concentration wastewater by biological sand filters: enhanced COD removal and bacterial community dynamics.

Winery wastewater is characterized by its high chemical oxygen demand (COD), seasonal occurrence and variable composition, including periodic high ethanol concentrations. In addition, winery wastewater may contain insufficient inorganic nutrients for optimal biodegradation of organic constituents. Two pilot-scale biological sand filters (BSFs) were used to treat artificial wastewater: the first was amended with ethanol and the second with ethanol, inorganic nitrogen (N) and phosphorus (P). A number of biochemical parameters involved in the removal of pollutants through BSF systems were monitored, including effluent chemistry and bacterial community structures. The nutrient supplemented BSF showed efficient COD, N and P removal. Comparison of the COD removal efficiencies of the two BSFs showed that N and P addition enhanced COD removal efficiency by up to 16%. Molecular fingerprinting of BSF sediment samples using denaturing gradient gel electrophoresis (DGGE) showed that amendment with high concentrations of ethanol destabilized the microbial community structure, but that nutrient supplementation countered this effect.

Phenolic removal processes in biological sand filters, sand columns and microcosms.

This study evaluated the removal processes involved in the removal of the phenolic component of winery wastewater in biological sand filters, sand columns and sand microcosms. It was found that at low influent phenolic concentrations, complete organic removal was accomplished, but at high concentrations, there was incomplete substrate removal and an accumulation of potentially toxic metabolites, including catechol. The sand provided a suitable substrate for the treatment of phenolic-laden waste, and both biotic (48%) and abiotic (52%) removal mechanisms effected the removal of model phenolics. Prior acclimation of microbial communities increased the biodegradation rate of phenolic acids significantly.

A novel recombinant ethyl ferulate esterase from Burkholderia multivorans.

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity.

An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0 degrees C, respectively. The amidase exhibited high thermal stability at 50 and 60 degrees C, with half-lives greater than 5 h at both temperatures. At 70 and 80 degrees C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with D: -selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4 degrees C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25 degrees C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70 degrees C and 30 min at 80 degrees C. The amidase has potential for application under high temperature conditions as a biocatalyst for D: -selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.

Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S.

The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding D-amino acids or L-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase ( N-carbamoylase). Pseudomonas putida strain RU-KM3S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and L-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and beta-ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not beta-ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or beta-ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3S cells.

Metagenomics, gene discovery and the ideal biocatalyst.

With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the "ideal biocatalyst" for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of "sequence space" that is not addressed by traditional screening.

Optimization of catechol production by membrane-immobilized polyphenol oxidase: a modeling approach.

Although previous research has focused on phenol removal efficiencies using polyphenol oxidase in nonimmobilized and immobilized forms, there has been little consideration of the use of polyphenol oxidase in a biotransformation system for the production of catechols. In this study, polyphenol oxidase was successfully immobilized on various synthetic membranes and used to convert phenolic substrates to catechol products. A neural network model was developed and used to model the rates of substrate utilization and catechol production for both nonimmobilized and immobilized polyphenol oxidase. The results indicate that the biotransformation of the phenols to their corresponding catechols was strongly influenced by the immobilization support, resulting in differing yields of catechols. Hydrophilic membranes were found to be the most suitable immobilization supports for catechol production. The successful biocatalytic production of 3-methylcatechol, 4-methylcatechol, catechol, and 4-chlorocatechol is demonstrated.

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens.

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.

Activity of mushroom polyphenol oxidase in organic medium.

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max) values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o-quinones were measured by a novel method: (1)H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water-sensitive, unstable o-quinones.