High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal β-glucosidase production

Abstract

Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS)-based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for β-glucosidase production under various growth conditions. Media that produced high β-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC), Medium 84 + rolled oats, and M9TE + MCC at 45°C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45°C than at all other temperatures. While T. bispora is reported to grow optimally at 60°C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45°C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.

Keywords: NIMS; enzymatic activity screening; high throughput; microbial communities; β-glucosidase.

FIGURE 1

Schematic of high throughput NIMS for screening growth conditions and method validation. (A) Samples generated from various growth conditions were acoustically printed and analyzed by NIMS approach. (B) Spectrum of 1 μM substrate (0.5 μL sample was manually deposited, 500 fmoles), which is the limit of quantification (LOQ) in this method (S/N ratio >10). (C) Spectrum of an assay mixture that produced 55% conversion. The lowest concentration of substrate (α) is detected with S/N ratio >5 in this assay. The low coefficient of variance (CV) we found to be <20%.

FIGURE 2

Analysis of β-glucosidase production from cell culture supernatants grown in various media and at different temperatures. Enzyme activities for the various growth conditions were compared using a cellobiose probe incubated at 50°C for 1 h.

FIGURE 3

Direct comparison of supernatants from cell cultures grown in media containing the recommended energy source after 24 h. Squares, I/S + 1% MCC; circles, Medium 65 + 10 mM glucose; triangles, Medium 84 + rolled oats; diamonds, M9TE + 1% MCC.

FIGURE 4

Optimization of assay conditions with cellobiose substrate under growth conditions identified by high throughput NIMS screening. Supernatants of cell culture grown in I/S + 10 mM glucose at 50°C for 24 h were incubated with the probe at various temperatures (A) or pH buffers (B). Error bars represent standard deviation from the mean of triplicate technical replicates. Significance indicated by: ^αp< 0.05 versus activity at 45°C, ^βp > 0.05 versus activity at 50°C, ^δp > 0.05 versus activity at pH5.