【作者】 徐美娟；

【导师】 方慧英；

【作者基本信息】 江南大学 ， 微生物学， 2008， 硕士

【摘要】 二羟丙酮(DHA)是一种重要的化工原料,在精细化工、制药、食品等方面具有广泛的应用前景。本研究从自然界筛选出21株能够以甘油为唯一碳源产二羟丙酮的菌株,经初步发酵测定发酵液中DHA含量,其中菌株6-8 DHA产量最高达6.2 g/L。对其进行常规生理生化鉴定实验,并结合16S rDNA基因分析,比对结果表明,菌株6-8与Acinetobacter sp. B8-22相似性最高,达99.7%,在细菌分类学上属于假单胞菌目莫拉菌科不动杆菌属,将其命名为Acinetobacter sp.6-8。在研究Acinetobacter sp. 6-8在LB培养基中的生长曲线的基础上,考察了培养时间与培养温度对Acinetobacter sp. 6-8发酵DHA的影响,最终确定37℃时发酵72 h即可获得较高的DHA产量。在摇瓶水平上,分别考察了碳源和氮源对Acinetobacter sp. 6-8发酵产DHA的影响,并对碳氮源综合水平进行了正交实验优化,最终确定采用甘油10.0%、山梨醇2.0%、酵母膏0.5%、玉米浆0.2%作为发酵培养基的碳氮源浓度,以此条件发酵,DHA产量为7.21 g/L,比未优化时DHA产量提高了9.8%;建立了简单且准确测定二羟丙酮(DHA)的高效液相色谱(HPLC)方法,以Alltima C18(5μm,250×4.6 mm)为分离柱,5%甲醇水溶液(0.05%H3PO4调pH至3.0)为流动相,用紫外检测器在203 nm处检测DHA。结果测得DHA标准样品在203nm的保留时间为6.2 min,并测得DHA的线性范围为0.1～10.0 g/L,用HPLC法测得以甘油为底物发酵产DHA发酵液中DHA含量与显色法测得的DHA含量一致。以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶(GDH)基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coli BL21(DE3)中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni2+柱纯化具有表达活性的甘油脱氢酶(GDH),纯化后比酶活达到156 U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质:酶反应的最适pH为11.0,在pH7.0～12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25℃～45℃;Mg2+、Cu2+、Zn2+、K+对酶促反应有一定的激活作用;酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49μmol/(mL·min),说明了此酶对甘油的亲和性较高。本实验采用了生物全细胞转化的方法,将重组大肠杆菌培养至对数生长后期,经IPTG诱导收集菌体,将其加入转化液中,利用全细胞转化法只需12 h甘油转化为DHA达1.4 g/L。为利用基因工程菌的工业化应用打下了良好的基础。更多还原

【Abstract】 Dihydroxyacetone is a kind of important chemical materials, which has been widely applied in chemistry, pharmacy, food and so on. More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6-8 reached 6.2 g/L. In addition to general morphological and biochemical characteristics, the strain 6-8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. B8-22 suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp. The experiment was used to optimiaze parameters of the fermentation condition of Acinetobacter sp. 6-8. The maximal yield of DHA (7.21 g/L) could be obtained when the concentrations of glycerol, mannitol, yeast extract, and corn steep liquor were set at 10.0%, 2.0%, 0.5%, and 0.2%. In this condition the production of DHA was enhanced by 9.8% than that of the prior to optimization.A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on an Alltima C18( 5μm,250×4.6 mm ). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 mL/min and the detective wavelength was 203 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC method, which was in agreement with the result by spectrophotometric method. The method in this paper was simple, rapid and applicable for DHA determination in the fermentation broth. The method was applicable for DHA determination in the fermentation process.The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified and inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and introduced into Escherichia coli BL21 (DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the result showed a single band about 39 kDa on SDS-PAGE gel, and the specified activity was about 156 U/mg, the special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little change when incubated in the buffer of pH 7.0~11.0. The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃~45℃. The Km value was 0.54 mmol/L and Vmax was 0.49μmol/(mL·min) in the glycerol. The production of dihydroxyacetone from glycerol was 1.4 g/L by whole cell catalysed conversion of recombinant E.coli BL21.更多还原