Characterisation of novel biomass degradation enzymes from the genome of Cellulomonas fimi

Abstract

Recent analyses of genome sequences belonging to cellulolytic bacteria have revealed many genes potentially coding for cellulosic biomass degradation enzymes. Annotation of these genes however, is based on few biochemically characterised examples. Here we present a simple strategy based on BioBricks for the rapid screening of candidate genes expressed in Escherichia coli. As proof of principle we identified over 70 putative biomass degrading genes from bacterium Cellulomonas fimi, expressing a subset of these in BioBrick format. Six novel genes showed activity in E. coli. Four interesting enzymes were characterised further. α-l-arabinofuranosidase AfsB, β-xylosidases BxyF and BxyH and multi-functional β-cellobiosidase/xylosidase XynF were partially purified to determine their optimum pH, temperature and kinetic parameters. One of these enzymes, BxyH, was unexpectedly found to be highly active at strong alkaline pH and at temperatures as high as 100 °C. This report demonstrates a simple method of quickly screening and characterising putative genes as BioBricks.

Keywords: Arabinofuranosidase; C. fimi; Multifunctional endoxylanase; Thermotolerant xylosidase.

Fig. 1

Initial screening data for genes expressed in E. coli with A) PNPA, ONPC and ONPX as substrates. Negative (−ve) control for all substrates, plasmid only; Positive (+ve) control for ONPC and ONPX only was Cex. B) Biological repeats of cell lysates displaying positive activity. Mean acitivities of n = 2 independent biological repeats and ± standard error are shown.

Fig. 2

Temperature optima of the purified C. fimi enzymes AfsB (A), BxyF (B), BxyH (C), XynF (D). Mean activities (n = 2) of independent biological repeats are shown with ±SE displayed.

Fig. 3

Mean activities (n = 2) of independent biological repeats of enzymes under optimal assay conditions in the presence of varying buffer supplements, all of 1 mM concentration. Enz only is the purified enzyme under assay conditions with no supplement. ±standard error is shown.

Fig. 4

Eadie-Hofstee plots of enzyme velocity (U/mg) as a factor of substrate concentration (mM). A) AfsB, PNPA; B) BxyF, ONPX; C) XynF, ONPC. Error bars of ±SE, n = 2 independent biological repeats.