Study On Isolation And Identification Of A Alkaline Phosphatase-producing Bacterium And Its Enzyme Production Conditions

Alkaline Phosphatase (EC3. 1. 3. 1) is a lower substrate-specificity phosphomonosterase that degrade organophosphorus compounds to release free inorganic phosphate. Alkaline Phosphatase is used wide application in enzyme-linked immunosorbent assay (ELISA) systems, biological sensor, nonisotopic probing, blotting and sequencing systems. In molecular biology, alkaline Phosphatase can breakdown extreme phosphate, and is a useful tool enzyme in genetic engineering. Alkaline Phosphatase also plays an important role in organophosphorus pesticides biodegradation.Alkaline Phosphatase exists in human, animal, plant and microorganism. So far, commercial alkaline Phosphatase comes from mainly animal (calf intestinal) or plant (wheat germ). However, alkaline Phosphatase of animal or plant usefulness is limited by their inherently low thermostability and low productivity. In order to get ALP with high productive radio and good thermostability, a strain of bacteria capable of producing alkaline Phosphatase was isolated from 32 strains of bacteria. And was identified subsequently. The conditions of the enzyme fermentation were carried out in this study. The results were showed as follows:1. The isolation and screening of the producing alkaline Phosphatase strain:With micro well plats method, we selected three strains HB1, 1#, B7 among 32 bacteria strains (12 strains were isolated from soil, other 20 strains got from our strains bank), which showed higher alkaline Phosphatase activities. From three strains, strain HB1 had the highest alkaline Phosphatase activities by the second screening test. It was found that strain HB1 excreted extracellular alkaline Phosphatase with good thermostability.2. The identification of the producing alkaline Phosphatase strain: On the basis of strain HB1 morphological and physiological characteristics, and G+Cmol% of DNA as well as 16S rDNA sequence and Blast analysis, this new isolate belongs to Bacillus sp.3. Optimal conditions for alkaline Phosphatase produced by strain HB1:Based on the studies in the conditions of enzyme production, the optimal fermentation condition was established. The compositions of the medium were: glucose 6g, yeast powder 8g, MgSO₄ 0.5g, NaCl 1g, H₂O 1000ml and pH 7.5. When the cells of stain HB1 was inoculated in a 100 ml flask containing 25 ml medium and incubated on a shaker of 120 rpm at 30℃for 24 h, the alkaline Phosphatase activity in its cell-free supernatant peaked at 2.25u/ml.In this study, a strain HB1 of bacteria capable of producing alkaline Phosphatase was isolated and supplies microbial resources to enzymatic industry. The result of the fermentation conditions supplies laboratory parameters for industry production of alkaline Phosphatase.