Cloning and characterization of a novel mannanase from Paenibacillus sp. BME-14.
A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and 60 degrees C, respectively. The activity of Man26B was not affected by Mg(2+) and Co(2+), but was inhibited by Hg(2+), Ca(2+), Cu(2+), Mn(2+), K(+), Na(+), and beta-mercaptoethanol, and slightly enhanced by Pb(2+) and Zn(2+). EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with K(m), V(max), and k(cat) values of 3.80 mg/ml, 91.70 micromol/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at 80 degrees C and 90 degrees C for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.
Fu
Xiaoyu
X
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
Huang
Xiaoluo
X
Liu
Pengfu
P
Lin
Ling
L
Wu
Gaobing
G
Li
Chanjuan
C
Feng
Chunfang
C
Hong
Yuzhi
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002412:Cations / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D002412:Cations; D004269:DNA, Bacterial; D044902:beta-Mannosidase