Screening New Xylanase Biocatalysts from the Mangrove Soil Diversity

Abstract

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia marina and Rhizophora stylosa trees during the wet and dry seasons and at two different sediment depths. GH11 xylanase diversity varied more according to tree species and season, than with respect to depth. One complete cDNA was selected (OFU29) and expressed in Pichia pastoris. The corresponding enzyme (called Xyn11-29) was biochemically characterized, revealing an optimal activity at 40-50 °C and at a pH of 5.5. Xyn11-29 was stable for 48 h at 35 °C, with a half-life of 1 h at 40 °C and in the pH range of 5.5-6. Xyn11-29 exhibited a high hydrolysis capacity on destarched wheat bran, with 40% and 16% of xylose and arabinose released after 24 h hydrolysis. Its activity on wheat straw was lower, with a release of 2.8% and 6.9% of xylose and arabinose, respectively. As the protein was isolated from mangrove sediments, the effect of sea salt on its activity was studied and discussed.

Keywords: biomass degradation; heterologous expression; lignocellulose degrading enzymes; mangrove; marine fungus; salt adaptation; xylanases.

Figure 1

Diversity of expressed genes encoding xylanases in surface and deeper sediments collected beneath two tree species (Avicennia marina and Rhizophora stylosa) during the wet (March) and dry (November) seasons. (A) Diversity estimated with the Shannon index and (B) distribution of the different operational functional units (OFUs).

Figure 2

Sequence alignment of Xyn11-29 with the biochemically characterized xylanase XynMF13A isolated the mangrove fungus Phoma sp. (GenBank accession number AVV62005.1). The highly conserved glutamate catalytic residues are marked with blue arrows. Alignment was produced with Clustal Omega, and symbols below the sequences indicate full conservation of the same (asterisk) or equivalent residues (colon) and partial residue conservation (dot).

Figure 3

Effect of pH and temperature on enzyme activity: (A) Optimal pH in 50 mM sodium tartrate buffer from pH 2 to 4 and citrate-phosphate buffer from pH 5 to 7 at 30 °C; (B) Optimal temperature for beechwood xylan hydrolysis (0.5%, w/v) in the range 20–70 °C at pH 5.5. Activity values were calculated as a percentage of maximum activity (set to 100%) at optimum temperature and pH. Each data point (mean +/− standard deviation) is the result of triplicate experiments.

Figure 4

pH and temperature stability. (A) pH stability in 50 mM citrate-phosphate buffer from pH 5 to 6 at 35 °C; (B) Temperature stability from 35 °C to 50 °C at pH 5.5. Activity values were calculated as a percentage of maximum activity (set to 100%) at optimum temperature and pH. Each data point (mean +/− standard deviation) is the result of triplicate experiments.

Figure 5

Enzymatic hydrolysis capacity on destarched wheat bran (A,B) and wheat straw (C,D) of Xyn11-29 xylanase (red bars) compared with T. xylanolyticus xylanase, Tx-Xyn11 (gray bars) after 0, 6, 24, and 48 h incubation at pH 5.5 at 35 °C and at pH 5.8 at 60°C for Xyn11-29 and Tx-Xyn11, respectively. Results are expressed as a percentage of released sugars (arabinose and xylose). Each data point (mean +/− standard deviation) is the result of duplicate experiments.

Figure 6

Sea salt effect on Xyn11-29 xylanase activity and ratios of negatively to positively charged amino acids for Xyn11-29, T. xylanolyticus, Tx-Xyn11, and Phoma sp. xylanase, XynMF13A. (A) Sea salt was used at concentrations of 1% and 5%. Activities are expressed as a percentage of the activity without sea salt (set to 100%). Each data point (mean +/− standard deviation) is the result of triplicate experiments. (B) Negatively charged (D + E) to positively charged (R + K) amino acid ratios.