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KMID : 0545120100200030518
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 3 p.518 ~ p.524
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Cloning and Characterization of a novel Mannanase from Paenibacillus sp. BME-14
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Fu Xiao-Yu
Huang Xiao-Luo
Liu Peng-Fu
Lin Ling
Wu Gao-Bing
Li Chan-Juan
Feng Chun-Fang
Hong Yu-Zhi
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Abstract
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A mannanase gene (man26B) was obtained from the sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1428 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 60ºC, 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 Km, Vmax and kcat values were 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 enzymes stability while enabling it to retain 80% and 60% of its initial activity after treatment at 80?C and 90?C for 30min, respectively. This finding will be useful in industrial application of Man26B because of the harsh circumstances associated with such processes.
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KEYWORD
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Paenibacillus sp., mannanase, glycosyl hydrolase family 26, carbohydrate binding module 6, thermal stability
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