Cloning and Characterization of a Novel Mannanase from Paenibacillus sp. BME-14
2010
Fu, Xiaoyu, Huazhong Agricultural University, Wuhan, People's Republic of China | Huang, Xiaoluo, Huazhong Agricultural University, Wuhan, People's Republic of China | Liu, Pengfu, Huazhong Agricultural University, Wuhan, People's Republic of China | Lin, Ling, Huazhong Agricultural University, Wuhan, People's Republic of China | Wu, Gaobing, Huazhong Agricultural University, Wuhan, People's Republic of China | Li, Chanjuan, Huazhong Agricultural University, Wuhan, People's Republic of China | Feng, Chunfang, Huazhong Agricultural University, Wuhan, People's Republic of China | Hong, Yuzhi, Huazhong Agricultural University, Wuhan, People's Republic of China
A mannanase gene (mun26B) was obtained from a sea bacterium, Puenibacillus 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℃, respectively. The activity of Man26B was not affected by Mg²+ and Co²+, but was inhibited by Hg²+, Ca²+, Cu²+, Mn²+, K+, Na+, and β-mercaptoethanol, and slightly enhanced by Pb²+ and Zn²+. 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 μmol/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℃ and 90℃ for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.
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