【作者】 庄百川；

【导师】 贾小明；

【作者基本信息】 浙江大学 ， 微生物学， 2006， 硕士

【摘要】 碱性磷酸酶（Alkaline phosphatase，EC3.1.3.1，简称ALP）是一种底物专一性较低的、裂解有机磷化合物释放无机磷的磷酸单脂酶。碱性磷酸酶在酶联免疫测定、生物传感器、非同位素探针、杂交和测序方面有广泛应用。在分子生物学方面，碱性磷酸酶分解核酸的末端磷酸盐，是遗传工程的有用工具酶。碱性磷酸酶还在有机磷农药降解中起到非常重要的作用。碱性磷酸酶主要存在于人体、动物、植物和微生物中。目前商业碱性磷酸酶大多产自动物体（小牛肠碱性磷酸酶）或植物体（麦芽），产率低，价格昂贵；有些热稳定性差，使它的使用受到了限制。为了获得具有自主产权的产率高、热稳定性好的碱性磷酸酶，本研究从32株细菌中分离筛选到一株热稳定性较好的产碱性磷酸酶的菌株HB1，对其进行了鉴定，并进一步研究了该菌株的产酶条件，主要实验结果如下：1．产碱性磷酸酶菌株的分离、筛选从土壤分离的（12株）和浙江大学应用微生物学研究室已有保存菌株（20株）共32株菌中用微孔板法初筛，获得三株碱性磷酸酶活性较高的菌株HB1、1#、B7。磷酸苯二纳法测酶活复筛后确定菌株HB1产酶活性最高。该菌株主要产胞外碱性磷酸酶，并发现该酶具有较好的热稳定性。2．产酶菌株的鉴定通过对菌株HB1的形态学观察，生理生化特性测定，DNA中G+C mol％含量，16S rDNA测序和比对分析，确定菌株HB1属于芽胞杆菌属（Bacillus．sp）的一个种。3．菌株HB1产酶发酵条件的优化用摇瓶批量发酵法测得HB1发酵的最佳培养基为：葡萄糖，6g；酵母粉，8g；MgSO₄·7H₂O，0.5g；NaCl，1g；水，1000mL；pH 7.5。最适培养条件为：培养温度30℃，装液量为100mL三角瓶装25mL培养液，于120rpm转速下培养24h。在此条件下，发酵上清液酶活性可达2.25U／mL。本研究分离到的产酶菌株为发酵生产碱性磷酸酶提供了微生物资源；优化发酵试验结果为利用该菌发酵生产碱性磷酸酶提供了实验室小试参数。更多还原

【Abstract】 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.更多还原