A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium.
An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 x 10⁻² U mg⁻¹, respectively. The Michaelis-Menten constant (K (m) values) and the maximal reaction velocities (V(max) values) of CelEdx16 were 47.1 μM and 9.6 x 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 x 10⁻⁵ μmol min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40 °C.
Microbe-based Fusion Technology Research Center, KRIBB, 1404 Sinjeong-dong, Jeongeup, Jeonbuk 580-185, Republic of Korea.
Kyong-Cheol
KC
Ko
Yunjon
Y
Han
Jong Hyun
JH
Choi
Geun-Joong
GJ
Kim
Seung-Goo
SG
Lee
Jae Jun
JJ
Song
D000362:Agar; D000595:Amino Acid Sequence; D000818:Animals; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D002846:Chromatography, Affinity; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics; D016000:Cluster Analysis; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D000362:Agar; D002482:Cellulose; D002480:Cellulase