Outlook for cellulase improvement: screening and selection strategies.

A fluorimetric microplate enzyme assay has been developed to study enzyme diversity in soil as an approach to understanding functional diversity. The microplate assay allows a large number of soil samples and/or enzymes to be analysed in a short time. The substrates used are conjugates of the highly fluorescent compounds 4-methylumbelliferone (MUB) and 7-amino-4-methyl coumarin (AMC). The main advantage of using fluorimetrically-labelled substrates is that product formation can be measured directly in the microplate without previous extraction and purification of the product. A detailed protocol for this new technology is presented and some potential applications are outlined. A comparative study was carried out between the new microplate fluorimetric assay and a standard colorimetric enzyme assay based on p-nitrophenyl substrates. The kinetics of β-glucosidase and phosphatase activities were investigated in soils with different fertilizer backgrounds. Both methods generated similar values for Vmax (maximum rate of activity) whereas the affinity of β-glucosidase and phosphatase for their respective substrates (as indicated by Km values, Michaelis–Menten constant) was up to two orders of magnitude greater for the 4-methylumbelliferyl substrates compared to the p-nitrophenyl substrates.

A microplate fluorimetric assay for the study of enzyme diversity in soils

https://andrewsforest.oregonstate.edu/pubs/pdf/pub4056.pdf

Enzyme activities as a component of soil biodiversity: A review

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to...

Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

Current soil enzyme methods measure potential enzyme activities, which are indicative of overall enzyme concentrations. However, they do not provide insight in the actual rates of enzymatically catalyzed reactions under natural in situ conditions. The objectives of this review are to (1) clarify what is being measured by current standard soil enzymology methods; (2) present an overview of the factors that control in situ activities of soil enzymes; and (3) evaluate how emerging technologies and modeling approaches could enhance our understanding of in situ extracellular enzyme activity (EEA). Genomic studies targeting functional genes coding for extracellular enzymes can identify the genetic potential of microbial communities to produce enzymes. Microbial regulation of enzyme production can be assessed with transcriptomic studies of mRNA. Emerging proteomic tools could be used assess the pool sizes, diversity, and microbial source of soil enzymes. New mass-spectrometry approaches can be used to quantify the products of enzymatic degradation. The insights gathered from these approaches will foster improved models of decomposition that explicitly include enzymes and microbial species or functional groups. A comprehensive approach to measuring in situ activity and elucidating the regulation of enzyme production and stabilization is required to advance our understanding of the biochemistry of decomposition.

Emerging tools for measuring and modeling the in situ activity of soil extracellular enzymes

A new approach, based on the application of soluble, dye-labelled and acid-precipitable polysaccharide derivatives, is introduced for the sensitive assay of polysaccharide endo-hydrolases extracted from soil. An extraction procedure involving sodium acetate-acetic acid buffer (pH 5, 0.5M; 5ml g−1 soil) and an assay system adapted to microtitre plates were developed for routine determinations of endo -acting cellulase, xylanase, chitinase, 1,3-β-glucanase and amylase activities. An Acid Brown Earth under a mature beech forest (Fagus sylvatica L., humusform: typical Moder) was studied with respect to distinct, clearly-developed soil horizons (L, F, H, Ahh, Aeh). Enzymes purified by (NH4)2SO4-precipitation and dialysis were assayed for pH and temperature activity profiles. pH optima were determined in the range of 4.5–5.5, temperature optima were in the range of 40–55°C, revealing stable and fairly similar characteristics of these enzymes in the horizons under study. In routine investigations, highest enzyme activities were determined in the L and F horizons. Significantly decreasing activities with increasing soil depth and decreasing organic matter content were determined in the H, Ahh and Aeh horizons, respectively. Cellulase, xylanase and chitinase activities were highly correlated with total-C content of the soil horizons under study 0.871 ⩽r2⩽ 0.954).

S.J. Wirth

Papers

Micro-plate colourimetric assay for Endo -acting cellulase, xylanase, chitinase, 1,3-β-glucanase and amylase extracted from forest soil horizons