A bifunctional cellulase-xylanase of a new Chryseobacterium strain isolated from the dung of a straw-fed cattle

Abstract

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase-xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 μmol min^-1 mg^-1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 μmol min^-1 mg^-1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5-5.0× glucose and 1.2-1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2-15.7× glucose and 3.5-4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.

Figure 1

A. Phylogenetic analysis of 16S rRNA gene sequences of Chryseobacterium sp. HT1 with 45 reference strains of Chryseobacterium genus. B. Phylogenetic analysis of AA sequences of CbGH5 with 70 putative proteins of GH5_46 from the CAZy database.

Figure 2

A. Schematic representations of predicted cbGH5 and upstream ORFs. B. Three major modules of the CbGH5 peptide.

Figure 3

A. Substrate preference of CbGH5. B. Effects of cations on the EG‐CMC (grey bar) and XYN‐bw (white bar) activities. All the cations were supplemented at a final concentration of 5 mM. The anion for the chemicals used is Cl⁻. C. Effects of chelating agents and denaturants. The final concentrations of EDTA, EGTA, SDS and urea were given in parentheses. For figures B and C, the activity without adding any cation, chelating agent or denaturant is defined as 100%. (a) significantly higher than 100%, (b) no significant difference with 100%, (c) significantly lower than 100%.

Figure 4

A. pH profiles of the EG‐CMC (solid line) and XYN‐bw (dash line) activities of CbGH5 at 60 °C (empty square) and 90 °C (solid square). B. Temperature profiles of the EG‐CMC and XYN‐bw determined at their respective optimum pH. C. Stability of the EG‐CMC and XYN‐bw activities at pH 4 (red), 8 (orange), 9 (blue) and 11 (purple). D. Stability of the EG‐CMC and XYN‐bw activities at 60 °C (purple), 70 °C (blue), 80 °C (green), 90 °C (black) and 100 °C (red). Standard deviation bars were not shown in figures C and D, for clearer visualization.

Figure 5

Homology model of CbGH5 showing the GH5 domain with (β/α)₈ TIM‐barrel fold, the linker and the CBM6 domain. The two key glutamate residues in charge of the catalytic mechanism are highlighted in red. The conserved Asn, His, Arg, Tyr and Trp residues near the catalytic centre are in other colours.

Figure 6

Influence of deleting the CBM6 domain of CbGH5 on the activity level (A), maximum reaction velocity (V _max) (B), Michaelis constant (K _m) (C) and catalytic efficiency (k _cat K _m ⁻¹) (D). Significant difference is labelled by a and b. The values of the data bars in the figures are provided in the Supporting information (Table S5). Eadie‐Hofstee plots for determination of K _m and V _max are shown in supplementary figure (Fig. S7). CMC and filter paper were used as soluble and insoluble cellulose substrates, while birchwood xylan and corncob xylan were used as soluble and insoluble xylan substrates respectively. The activity levels and kinetic parameters were all measured under respective optimum pH and temperature conditions. For the insoluble substrates filter paper and corncob xylan, measuring kinetic parameters is not applicable, because insoluble substance is unable to form homogeneous solution, which is used for making a gradient of substrate concentration requested in measurement of kinetic parameters.

Figure 7

Saccharification performance of CbGH5 and the mixed commercial cellulase+xylanase of Novozymes. Contents of lignin, cellulose, hemicellulose, glucose and xylose were measured before and after enzymatic hydrolysis, on the straws of wheat (A), rice (B), corn (C) and oilseed rape (D), as well as spent mushroom substrates made from the straws of wheat (E), rice (F), corn (G) and oilseed rape (H). White bar: raw material before hydrolysis. Light grey bar: hydrolysed by the mixed Novozymes cellulase+xylanase. Dark grey bar: hydrolysed by CbGH5. The folds of glucose and xylose released by CbGH5 compared with the mixed Novozymes cellulase+xylanase are labelled in the figures. The hydrolysis reaction was carried out for 2 h. The pH and temperature conditions were 4.8, 45 °C for the mixed Novozymes cellulase plus xylanase, and 8.5, 90 °C for CbGH5 respectively. The values of the data bars in the figures are provided in the supplementary data (Table S6).