An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts

Abstract

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose-or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

A High-Throughput Platform for Screening Milligram Quantities of Plant Biomass for Lignocellulose Digestibility

“…Some steps in the lignocellulose to ethanol pipeline have previously been automated, e.g., pretreatments, enzyme digestions, or sugar analyses [22][23][24][25][26][27], but the system described here automates all steps from tissue grinding through sugar analysis. Human intervention is necessary to load the input tubes and add the metal grinding balls; to load the racks with output tubes; and to transfer the plates three times, first, to the liquid-handling robot for addition of pretreatment solutions and enzymes, second, to the incubating oven, and, third, back to the robot for the sugar assays.…”

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

“…In vitro hydrolysis reactions were performed in 96-well flat-bottom plates (Sarstedt) corresponding to King et al 54 . Cell-free supernatants of C. reinhardtii or Chlorella kessleri cultivated in air-bubbled MM ( ± CMC) containing antibiotics (25 mg ml À 1 kasugamycin) and fungicides (5 mg ml À 1 carbendacime) were used for the in vitro hydrolysis assay.…”

“…The DNS (3,5-dinitrosalicylic acid) reducing sugar assay was performed in a 96-well microplate format according to King et al 54 , and DNS reagent was prepared as previously described 56,57 .…”

Characterization and Influence of a Multi-enzymatic Biopreparation for Biogas Yield Enhancement

“…The cellulolytic activity of the biopreparation was determined in liquid culture filtrates and based on cellulolytic activity (FPU) after 60 min of incubation at 50 °C and 37 °C (Mandels et al 1976;King et al 2009). The amount of the enzyme that liberated 1 µM of glucose (reducing sugars) during 1 min at 50 °C was assumed as a FPU activity unit (Weldesemayat 2011;Janas and Targoński 2014).…”