Isolation of highly thermostable ?-xylosidases from a hot spring soil microbial community using a metagenomic approach.

ABSTRACT: The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable ?-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs of?>500?bp in length. Similarity search for ?-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for ?-xylosidase activity. A total of 82 proteins exhibited ?-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.

SUBMITTER: Sato M 

PROVIDER: S-EPMC5726482 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature