| 摘要: |
| 以海洋微生物通过发酵制备的脂肪酶为材料，对该酶的分离纯化条件及理化性质进行了研究。在脂肪酶纯化中，采用氯仿萃取、中空纤维柱超滤及CM-Sepharose F F阳离子交换柱层析等技术对发酵制备的脂肪酶进行了纯化，结果得到达到电泳纯的脂肪酶。在脂肪酶理化性质研究中，采用SDS-PAGE电泳对该脂肪酶分子量进行测定，并在实验中以橄榄油为底物采用脂肪酶酸碱滴定测活法，对脂肪酶的最适水解条件、各种因素对脂肪酶稳定性的影响进行了研究。结果显示，该脂肪酶分子量为(38．0±1)kD，最适水解温度为35℃ ，最适pH为8．5，为一低温碱性脂肪酶。该脂肪酶可在35℃ 以下、pH =4．0—9．0范围内保持良好的稳定性，与常见金属离子、化学试剂等的配伍性较好，并且具有良好的耐盐及抗氧化性能。研究中还以p-NPL(月桂酸对硝基苯酚酯)为底物采用脂肪酶化学发光测活法，对脂肪酶进行了酶促动力学的研究。结果表明，该脂肪酶在最适条件下Km值为7.805μmol／L，Vmax 为1.2385mmol／(L?min)。通过对Zn2+抑制脂肪酶水解活性的研究，发现Zn2+ 对脂肪酶具有可逆抑制作用，从而筛选到该脂肪酶的可逆抑制剂Zn2+ 。 |
| 关键词: 海洋微生物,低温碱性脂肪酶,分离纯化,理化性质 |
| DOI： |
| 分类号: |
| 基金项目:国家“863”计划资助项目，2001AA625070号 |
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| PURIFICATION AND CHARACTERIZATION OF A LOW-TEMPERATURE ALKALINE LIPASE FROM MARINE MICROORGANISMS |
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DONG Hong-Wei1,2, SUN Mi1, WANG Yue-Jun1, YU Jian-Sheng2
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1.Yellow Sea Fisheries Research Institute，Chinese Academy of Fishery Sciences;2.College of Chemical Engineering，Qingdao University of Science and Technology
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| Abstract: |
| A novel alkaline lipase was purified from a marine microorganism and the major physical and chemical characteristics of this alkaline lipase were studied and reported in this paper.
Cell-free supernatant was prepared by centrifugation(4000r／min, 60min)of culture broth. After excess olive oil was extracted from the supernatant by the chloroform, the whole solution was centrifuged and some denatured proteins were removed from the supernatant. The supernatant was ultrafiltered using hollow fibers(100kD), the ultrafiltrate was then concentrated by ultrafiltration using a hollow fiber cartridge of 10kD cut off. The condensed lipase was purified 16.3-fold by mean s of CM Sepharose FF cationic-exchange chromatography by an elution with a linear gradient of 10mmol／L phosphate buffer(pH 3.8)containing 0.5mol／L NaCl. The purity of the purified lipase was confirmed by the presence of a single band on SDS-PAGE. The relative molecular mass of this enzyme was determined to be(38.0±1)kD.
By the titrimetric method using olive oil as the substrate, characterization of the alkaline lipase was made. The lipase showed maximum activity at pH 8.5 and 35℃ . When the temperature of the reaction system rose, there was a rapid decline of lipase activity; however, lipolytic activity dropped slowly with declining temperatures. 20% of lipase activity still remained at pH 8.5 and 5℃ . According to the data mentioned above, lipase showed cold adapted and basophilic properties. In order to determine the Km and Vmax of the lipase, the lipolytic reaction was carried out using P-NPL as the substrate. The Km and Vmax of lipase under pH 8.5 and 35℃ were 7.805μmol／L and 1.2385mmol／(L?min), respectively. The enzyme was stable between pH 4.0 and 9.0 and at temperatures up to 35℃ . In further studies, lipase showed good resistance to high salinities, the concentration of metal ions was up to 100mmol／L. Furthermore, lipase was compatible to some chemical regents. The lipase was stab le in most polyhydroxy-compounds, in line with previous reports on lipases. Good resistance to the oxidant was also observed. Tween-40 could inhibit lipase activity significantly, while surfactant SDS could activate it. 10mmol／L EDTA could inhibit lipase activity seriously, it was speculated that there would be some metal ions in the active site of the lipase. When EDTA was added to the lipase standard assay system, metal ion amount was restrained or EDTA may cover the active site, thus, inactivate the lipase. In the further studies, the effect of Zn2+ and EDTA on lipase activity was studied. The data indicated that some essential amino acid residue, which plays a key role in the catalytic mechanism, might bind with Zn2+, resulting in lipase inactivation until Zn2+ was chelated by EDTA. |
| Key words: Marine microorganism, Low-temperature alkaline lipase, Separation and purification, Characterization |
