J. Susan van Dyk

Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment.

Pesticides are released intentionally into the environment and, through various processes, contaminate the environment. Three of the main classes of pesticides that pose a serious problem are organochlorines, organophosphates and carbamates. While pesticides are associated with many health effects, there is a lack of monitoring data on these contaminants. Traditional chromatographic methods are effective for the analysis of pesticides in the environment, but have limitations and prevent adequate monitoring. Enzymatic methods have been promoted for many years as an alternative method of detection of these pesticides. The main enzymes that have been utilised in this regard have been acetylcholinesterase, butyrylcholinesterase, alkaline phosphatase, organophosphorus hydrolase and tyrosinase. The enzymatic methods are based on the activation or inhibition of the enzyme by a pesticide which is proportional to the concentration of the pesticide. Research on enzymatic methods of detection, as well as some of the problems and challenges associated with these methods, is extensively discussed in this review. These methods can serve as a tool for screening large samples which can be followed up with the more traditional chromatographic methods of analysis.

A review of the enzymatic hydrolysis of mannans and synergistic interactions between β-mannanase, β-mannosidase and α-galactosidase

Mannan is an important polysaccharide found in softwoods and many other plant sources. Mannans from various sources display large differences in composition, structure and complexity. To hydrolyse mannan into its monomer sugars requires a number of enzymes working in synergy. This review examines mannan structure and the enzymes required for its hydrolysis. Several studies have investigated the effect of supplementing β-mannanases with β-mannosidases and α-galactosidases in binary and ternary combinations. Synergistic enhancement of hydrolysis has been found in some, but not all cases. In the case of mannosidases, they sometimes display an anti-synergistic effect with mannanases, most likely due to competition for binding sites. Most importantly, in the case of α-galactosidases, the same enzyme from different families display differences in synergistic interactions due to different specificities. An improved understanding of enzyme interactions will aid in achieving enhanced hydrolysis of mannans and higher sugar yields. This review highlights areas which require further research in order to gain a better understanding of mannan hydrolysis and utilisation. Such knowledge is very important as this can be used in the optimisation of commercial or purified enzyme mixtures to improve the economic viability of the conversion of high mannan-containing biomass such as softwoods into fermentable sugars for bioethanol production.

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-D-glucurono-D-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-L-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.

Time dependence of enzyme synergism during the degradation of model and natural lignocellulosic substrates

Cellulosic ethanol production relies on the biochemical (enzymatic) conversion of lignocellulose to fermentable sugars and ultimately to bioethanol. However, the cost of lignocellulolytic enzymes is a limiting factor in the commercialisation of this technology. This therefore necessitates the optimisation of lignocellulolytic enzyme cocktails through the elucidation of synergistic interactions between enzymes so as to improve lignocellulose hydrolysis and also lower protein loadings in these reactions. However, many factors affect the synergism that occurs between these lignocellulolytic enzymes, such as enzyme ratios, substrate characteristics, substrate loadings, enzyme loadings and time. This review examines the effect of time on the synergistic dynamics between lignocellulolytic enzymes during the hydrolysis of both complex (true) lignocellulosic substrates and model substrates. The effect of sequential and simultaneous application of the lignocellulolytic enzymes on the synergistic dynamics during the hydrolysis of these substrates is also explored in this review. Finally, approaches are further proposed for efficient and synergistic hydrolysis of both complex lignocellulosic substrates and model substrates. With respect to the synergistic enzymatic hydrolysis of lignocellulosic biomass, this review exposed knowledge gaps that should be covered in future work in order to fully understand how enzyme synergism works: e.g. elucidating protein to protein interactions that exist between these enzymes in establishing synergy; and the effect of lignocellulose degradation products of one enzyme on the behaviour of the other enzyme and ultimately their synergistic relationship.

Chapter 7 – Challenges and Opportunities for the Conversion Technologies Used to Make Forest Bioenergy

Most of the forest biomass utilised around the world is employed to provide bioenergy, with the remainder being used to produce conventional forest products such as pulp and paper, sawnwood, etc. Forest bioenergy is dominated by traditional, developing-world applications such as cooking and charcoal production, but developed-world applications such as combined heat and power (CHP), pellet combustion, etc., continue to expand. More modern bioenergy applications are expected to play an increasing role in the world’s future renewable energy consumption. Forest biomass is already widely employed throughout the energy sector in heat/power generation, as part of stand-alone facilities, or integrated within industrial processes, residential heating (modern and traditional) and transportation. Although several conversion technologies, such as, gasification, pyrolysis and cellulosic ethanol production, are being pursued, combustion will likely remain the most prevalent bioenergy process used to generate heat and power for the immediate future. Even after densification processes such as pelletisation or torrefaction, forest biomass has a low energy density relative to fossil fuels. Therefore, it is likely that most forest-derived biomass will continue to be used locally because transportation over long distances is economically challenging. However, in some cases, upgrading technologies such as pelletisation, pyrolysis, gasification and biochemical conversion can create sufficiently energy-dense, adaptable and carbon-friendly fuels to be attractive for import. The drivers of forest-based bioenergy consumption are not entirely based on direct cost comparisons with fossil fuels. Other priorities, such as climate change mitigation, energy security, greenhouse gas reduction, rural employment, etc., also influence government support for bioenergy/biofuels. The different approaches used to encourage bioenergy/biofuel production and consumption in each country is a product of government policies, programmes and underlying political motivations. Modern biomass conversion technologies offer enormous opportunities, in both developing and developed countries, through better use of forest- and mill-derived residues, improved efficiency of biomass conversions, reduced energy-related carbon emissions and enhanced energy security.

Enzyme Synergy for Enhanced Degradation of Lignocellulosic Waste

Lignocellulosic substrates are very complex with a number of different components associated within a three-dimensional network. The complexity of the substrate, as well as the close association between components, makes the substrate recalcitrant to degradation. Such substrates require a large number of hydrolytic enzymes working in synergy to achieve complete degradation. The use of synergy studies allows us to determine whether enzymes display cooperation in hydrolysis of lignocellulosic substrates. This allows us to evaluate which enzymes should possibly be included in designer enzyme cocktails for lignocellulose hydrolysis. It also provides us with insight into areas of substrate recalcitrance, including obstacles such as physical association between components which prevent access of enzymes to their substrate.