Construction of a Metagenomic Library from Compost and Screening of Cellulase- and Xylanase-positive Clones

Abstract

Metagenomic library was constructed from compost made with pig manure and mushroom cultural waste using fosmid vector. Composting was carried out using moving roller system with intermittent mixing and forced aeration, and samples were taken from 7th- and 14th-day spots, which represent the mid and late stages of the process, respectively. DNAs of about 40 kb were obtained by preparative electrophoresis on 0.4% low-melting agarose gel to fractionate DNA fragments and to remove humic substances. Total 12,380 fosmid clones were obtained. Restriction analysis of randomly selected clones showed that most clones had different inserts, and average size of inserts was about 35 kb. Two cellulase-positive and five xylanase-positive clones were selected from metagenomic library. Cellulase of clone C1 showed maximal activity at 50°C and pH 6.0, and retained its original activity after 30 min of heat treatment at 60°C. Optimum temperature for xylanases of clones X1, X2, X3, and X4 was 50°C, and that of clone X5 was 55°C. Thermostabilities of xylanases were in the order of X4>X5>X1, X2, and X3. Optimum pH of xylanases of X1, X2, and X3 was 6.0, that of X4 was 5.5, and that of X5 was 5.5∼8.0. Xylanasepositive clones could be divided into three groups, X1/X2/X3, X4, and X5, based on influences of temperature and pH on enzyme activity. Sequence analysis of positive subclone of clone C1 showed cellulase, Cel6H, had the highest similarity of 64% to that of Cellulomonas fimi (P07984), suggesting cellulase and xylanase from metagenomic library are novel enzymes.