Characterization of a GH10 extremely thermophilic xylanase from the metagenome of hot spring for prebiotic production

Abstract

A xylanase gene (named xyngmqa) was identified from the metagenomic data of the Gumingquan hot spring (92.5 °C, pH 9.2) in Tengchong City, Yunnan Province, southwest China. It showed the highest amino acid sequence identity (82.70%) to endo-1,4-beta-xylanase from Thermotoga caldifontis. A constitutive expression plasmid (denominated pSHY211) and double-layer plate (DLP) method were constructed for cloning, expression, and identification of the XynGMQA gene. The XynGMQA gene was synthesized and successfully expressed in Escherichia coli DH5α. XynGMQA exhibited optimal activity at 90 °C and pH 4.6, being thermostable by maintaining 100% of its activity after 2 h incubated at 80 °C. Interestingly, its enzyme activity was enhanced by high temperatures (70 and 80 °C) and low pH (3.0-6.0). About 150% enzyme activity was detected after incubation at 70 °C for 20 to 60 min or 80 °C for 10 to 40 min, and more than 140% enzyme activity after incubation at pH 3.0 to 6.0 for 12 h. Hydrolytic products of beechwood xylan with XynGMQA were xylooligosaccharides, including xylobiose (X2), xylotriose (X3), and xylotetraose (X4). These properties suggest that XynGMQA as an extremely thermophilic xylanase, may be exploited for biofuel and prebiotic production from lignocellulosic biomass.

Figure 1

Construction process of constitutive expression plasmid pSHY211.

Figure 2

Phylogenetic dendrogram obtained by maximum likelihood analysis based on amino acid sequences showing the phylogenetic position of XynGMQA with related xylanase. Bootstrap values (expressed as a percentage of 1000 replications) are given at nodes.

Figure 3

Screening of xylanase active clones (E. coli DH5α/pSHY211-XynGMQA) by double-layer plate method in E. coli DH5α. Complete agarose gel and plate images were shown in Supplementary Figure S4 and Supplementary Figure S5, respectively.

Figure 4

The tertiary structure (a) and SDS-PAGE analysis of XynGMQA (b). Lane 1, protein molecular weight marker, mass indicated on the left; lane 2, total protein of E. coli DH5α/pSHY211-XynGMQA; lane 3, purified XynGMQA. The recombinant XynGMQA were 42.73 kDa. Complete SDS-PAGE image was shown in Supplementary Figure S6.

Figure 5

Effects of temperature and pH on the activity and stability of the recombinant XynGMQA. (a) Temperature effect on the activity of XynGMQA. (b) pH effect on the activity of XynGMQA. (c) The effect of temperature on stability at different temperatures (70, 80, and 90 °C) for 0, 20, 40, 60, 80, 100, and 120 min. (d) The effect of pH on stability. The primary activity was taken as 100%. Each value in the Figure represents the mean ± SD (n = 3). 100% = 3.4 ± 0.3 U/mg.

Figure 6

Thin-Layer Chromatography (TLC) of hydrolyzation products of xylooligosaccharides by XynGMQA. Lane 1, standards: X1 (xylose), X2 (xylobiose), X3 (xylotriose), and X4 (xylotetraose); lane 2, beechwood xylan without enzyme; lane 3, beechwood xylan hydrolysis by purified XynGMQA.