Genetic and functional characterization of an extracellular modular GH6 endo-β-1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13.
The gene (1608-bp) encoding a GH6 endo-β-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley β-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including β-1,3-D-glucan, β-1,4-D-galactomannan, and β-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley β-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.
Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Republic of Korea.
Do Young
DY
Kim
Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Republic of Korea.
Min Ji
MJ
Lee
Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Republic of Korea.
Han-Young
HY
Cho
Gyeonggi Bio-Center, Gyeonggi Institute of Science & Technology Promotion, Suwon, 443-270, Republic of Korea.
Jong Suk
JS
Lee
Division of Metabolism and Functionality Research, Korea Food Research Institute (KFRI), Sungnam, 463-746, Republic of Korea.
Mi-Hwa
MH
Lee
Department of Biological Sciences, Andong National University, Andong, 760-749, Republic of Korea.
Chung Wook
CW
Chung
Insect Biotech Co. Ltd., Daejeon, 305-811, Republic of Korea.
Dong-Ha
DH
Shin
Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon, 305-764, Republic of Korea.
Young Ha
YH
Rhee
Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Republic of Korea. sonkh@kribb.re.kr.
Kwang-Hee
KH
Son
Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Republic of Korea. hypark@kribb.re.kr.
Ho-Yong
HY
Park
D000595:Amino Acid Sequence; D000818:Animals; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008031:Lignin / Q000378:metabolism; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000382:microbiology*; D011509:Proteoglycans; D014990:Xylans / Q000378:metabolism; D047071:beta-Glucans / Q000378:metabolism
D008351:Mannans; D011509:Proteoglycans; D014990:Xylans; D047071:beta-Glucans; D002686:Chitin; D002475:Cellobiose; C010770:polysaccharide-K; D008031:Lignin; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium
Cellulosimicrobium funkei HY-13; Earthworm-symbiotic bacterium; Endo-β-1,4-glucanase; GH family 6