title: A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria.
abstract: Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The Michaelis-Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 μM and 1.55 × 10(-)(4 )μmol min(-1), respectively, and the enzyme was optimally active at pH 5.0 and 37°C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg(-1)), CMC (105.9 U mg(-1)), birch wood xylan (132.3 U mg(-1)), oat spelt xylan (67.9 U mg(-1)), and 2-hydroxyethyl-cellulose (26.3 U mg(-1)). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.
authors.affiliation: Biorefinery Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeonbuk 580-185, Republic of Korea.
authors.forename: Kyong-Cheol
authors.initials: KC
authors.lastname: Ko
authors.forename: Ju Hee
authors.initials: JH
authors.lastname: Lee
authors.forename: Yunjon
authors.initials: Y
authors.lastname: Han
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Choi
authors.forename: Jae Jun
authors.initials: JJ
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle / Q000382:microbiology*; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli; D057166:High-Throughput Screening Assays; D056186:Metagenomics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D056910:Republic of Korea; D012371:Robotics; D012417:Rumen / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D011994:Recombinant Proteins; D044602:Cellulases
keywords: Cellulolytic enzyme; Enzyme assay; Metagenome; Multifunctional enzyme