Characterization of a novel bifunctional enzyme from buffalo rumen metagenome and its effect on in vitro ruminal fermentation and microbial community composition

Abstract

To efficiently use lignocellulosic materials in ruminants, it is crucial to explore effective enzymes, especially bifunctional enzymes. In this study, a novel stable bifunctional cellulase-xylanase protein from buffalo rumen metagenome was expressed and characterized, CelXyn2. The enzyme displayed optimal activity at pH 6.0 and 45 °C. The residual endoglucanase and xylanase activities were 90.6% and 86.4% after a 60-min pre-incubation at 55 °C. Hydrolysis of rice straw, wheat straw, sheepgrass and sugar beet pulp by CelXyn2 showed its ability to degrade both cellulose and hemicellulose polymers. Treatment with CelXyn2 improved the hydrolysis of agricultural residues with an evident increase in production of total gas, lactate and volatile fatty acids. The results of 16S rRNA and real-time PCR showed that the effect on in vitro ruminal microbial community depended on fermentation substrates. This study demonstrated that CelXyn2 could strengthen lignocellulose hydrolysis and in vitro ruminal fermentation. These characteristics of CelXyn2 distinguish it as a promising candidate for agricultural application.

Keywords: Bifunctional enzyme; In vitro ruminal fermentation; Lignocellulose; Microbial community.

Fig. 1

Structural analysis of CelXyn2. (A) The conserved domain of CelXyn2 using NCBI Conserved Search. Structural superposition of (B) cellulase (cyan) with PDB code 1CEC (orange), and (C) xylanase (cyan) with PDB code 2JEP (orange) using PyMOL program (Bramucci et al., 2012).

Fig. 2

Analysis of purity and functional activity of CelXyn2. (A) 1.2% agarose gel electrophoresis and 12% SDS-PAGE analysis of gene fragment and purified enzyme. Lanes: 1, DNA or protein marker; 2, The PCR product of the gene celxyn2 or purified protein CelXyn2. (B) Enzymatic activities tested against carboxymethylcellulose (CMC) and beechwood xylan. Tre.: activated CelXyn2, Con.: inactivated CelXyn2.

Fig. 3

Effect of pH, temperature and storage on CelXyn2 activity. (A) pH profile of CelXyn2 at pH 2.0 to 10.0. (B) pH stability. Temperature profile of CelXyn2 in (C) 100 mM Citric acid-Na₂HPO₄ buffer (pH 6.0) and (D) 4 M NaCl solution at 30 to 60 °C. Storage stability of CelXyn2 by incubating the protein for 60 min at (E) 50 and (F) 55 °C. The T_1/2 of CelXyn2 at (G) 50 and (H) 55 °C. Data is shown as mean ± standard deviation.

Fig. 4

Effect of metal ions and chemicals on (A) cellulase and (B) xylanase activities of CelXyn2. Data is shown as mean ± standard deviation. PMSF = phenylmethylsulfonyl fluoride; EDTA = ethylenediaminetetraacetic acid; SDS = sodium dodecyl sulfate.

Fig. 5

Impact of salt on CelXyn2 activity. Effect of different concentrations (1 to 5 M) of NaCl solution on (A) cellulase and (B) xylanase activities of CelXyn2. Thermostability of CelXyn2 under the influence of 4 M NaCl during 60 min incubation at (C) 50 and (D) 55 °C. The (E) cellulase and (F) xylanase activities of CelXyn2 in artificial seawater compared with that in 4 M NaCl and salt-free control conditions. The CelXyn2 activity at 45 °C in a salt-free buffer was set as 100%. Data is shown as mean ± standard deviation.

Fig. 6

Hydrolysis of sugar beet pulp, wheat straw, rice straw and sheepgrass by CelXyn2 after 168 h at 45 °C. Data is shown as mean ± standard deviation.

Fig. 7

The PCoA plot of samples from in vitro ruminal incubation of rice straw, wheat straw and sheepgrass based on Bray–Curtis distances. The PCoA analysis was performed using the OTUs obtained. PCoA = principal coordinate analysis; RS = rice straw; WS = wheat straw; SG = sheepgrass.

Fig. 8

Distribution of the predominant rumen bacteria at (A) phylum level and (B) genus level.

Fig. 9

The STAMP analysis displaying the ruminal bacteria change between the control group and CelXyn2 group in vitro ruminal incubation of (A) rice straw, (B) wheat straw and (C) sheepgrass.

Fig. 10

Analysis of microbial community. (A) Effect of CelXyn2 on 16S rRNA gene copy numbers of total bacteria. The relative abundances of (B) F. succinogenes, (C) R. flavefaciens and (D) R. albus. RS = rice straw; WS = wheat straw; SG = sheepgrass. (∗ 0.01 < P < 0.05).