Screening Of Bacteria For Producing Mannanase And Characterization Of Mannanase

Mannans are the major constituents of plant hemicelluloses, and Amorphophallus konjac is the major plant which contains highly pure mannans in nature. The market of konjak products is enlarging year by year. Beta-mannanases which could hydrolyze mannans into mannooligosaccharides have been the hotspot in biomaterial industry and health care products. Also, B-mannanases have been widely used in food, pharmaceutical, paper, textile, printing, dyeing and petroleum industry. Thermostable mannanases from alkaliphic bacteria provide potential advantages for the applications in industries that require extreme conditions such as biopulping and sewage deterging. Also, they play an important role in fundamental research such as saccharide engineering, analysis of saccharide structure and polysaccharide hydrolyzing.In this paper, an alkaliphic mannanase-producing strain which grow optimally at pH 10.0, was isolated from decayed konjak vermicelli with konjak powder as the sole carbon source. It was identified as Paenibacillus dendritiformis by 16s rDNA sequence and classic morphology. It was defined as Paenibacillus dendritiformis P411 and its genbank accession number is HM071942.Effects of different carbon sources, nitrogen sources, metal ions, pH, temperature, volume of medium, rotation and incubation time on mannanase production by Paenibacillus dendritiformis P411 was studied. Maximum mannanase activity of 103.8 IU/ml was achieved with medium pH 10.0, konjak powder 1.0%, peptone 0.5%, NaCl 0.5%, yeast extract 0.2%, FeSO4 0.3mM, at the conditions of 34℃,180 rpm shaker using 250 ml Erlenmeyer flask containing 25 ml medium for 48h incubation.The supernatant, Paenibacillus dendritiformis culture centrifugated at 8,000xg and 4℃for 30min, was collected as crude enzyme. After ammonium sulphate fractional precipitation, dialysis desalination and Sepharose Q-FF chromatography (5 ml, GE) by AKTA explorer 100, the mannanase was purified and identified as a single band by SDS-PAGE, and the subunit molecular weight was about 30.0KD. The enzyme specific activity was about 5238IU/mg and the purification fold was 15.6.The product of hydrolyzation detected by TLC demonstrates that the mannanase produced by Paenibacillus dendritiformis P411 has endo-mannanase activity. Optimum pH of the enzyme is 8.0 and the enzyme is stable at pH range of 6.5-9.0, optimum temperature of the enzyme is 60℃and the enzyme is stable below 60℃. The enzyme was significantly activated by Mn2+, Cu2+and Fe2+(5mM), but inhibited 75%with 5mM Mg2+and Zn2+. Comparatively lower Michaelis-Menten constant (9.75mg/ml) and higher maximum reaction velocity (764.5IU/ml) demonstrate the specific substrate of this mannanase for konjak polysaccharide.