A fosmid library constructed with DNA from a naturally heated (67 °C), alkaline (pH 9.3) sample from the
crater of the Avachinsky volcano in the Kamchatka region was screened functionally for cellulase, xylanase and
β‐glucosidase activities. Sequence analyses of the fosmids conferring xylanase and cellulase activity led to the
identification of an ORF coding for a putative xylanase and an ORF coding for a putative cellulase, Xyn10K and Cel5K.
Both enzymes were produced in E. coli and characterized.
Purified recombinant Xyn10K (50 kDa) was active against various xylans, with beech wood xylan being the preferred
substrate. Cel5K (38 kDa) showed activity against CM cellulose (CMC), lichenan and barley beta-glucan. Hydrolysis
product analyses indicated that Xyn10K acts as an endoxylanase whereas Cel5K exhibits endocellulase activity. Xyn10K
was active over a pH range of 6-8 and the pH optimum of Cel5K was at pH 7. The maximum hydrolytic activity of Cel5K
and Xyn10K under the assay conditions employed was found at 96 °C and 95 °C respectively. Cel5K is the first
characterized endoglucanase from a metagenomic library with a temperature optimum above 90 °C. Also, its exceptional
resistance against thermoinactivation at temperatures above 80 °C, with a half life of 8h at 86 °C, sets this enzyme apart
from previously reported metagenomic cellulases. The xylanase Xyn10K, to our knowledge the first extreme thermostable
xylanase from a metagenome study, also displayed a remarkable long term-stability against thermal inactivation, with a
half life of 22 h at 85 °C. Both enzymes displayed, partially, a high hydrolytic activity and stability in different ionic
liquids. In conclusion, the properties of the metagenome-derived (hemi)cellulolytic enzymes reported here indicate
robustness and suitability for applications in biotechnology.