一株棘孢曲霉的鉴定及其柚苷酶合成规律

Identification and characterization of Aspergillus aculeatus JMUdb058 for naringinase production

摘要:

摘要:【目的】对一株新分离的柚苷酶产生菌株JMUdb058进行鉴定，并研究该菌株柚苷酶合成的基本规律。【方法】利用形态观察、28S rDNA序列分析对JMUdb058进行鉴定;通过反相高效液相色谱测定JMUdb058粗酶液对柚皮苷的水解作用，鉴定其柚苷酶活性;分别用11种碳源和6种氮源进行摇瓶发酵，研究碳源、氮源对该菌株分泌柚苷酶的影响;在固态和液态条件下分别进行发酵，测定该菌株合成柚苷酶的能力。【结果】菌株JMUdb058的菌落形态和显微形态符合曲霉属黑色组的典型形态特征，其28S rDNA序列与棘孢曲霉(Aspergillus aculeatus)的相似度达100%;用液态和固态两种发酵方法制得的粗酶液均可将标准溶液中的柚皮苷水解产生普鲁宁和柚皮素，还可有效地水解琯溪蜜柚汁中的柚皮苷;用橘皮苷、柚皮苷、芸香苷和鼠李糖为碳源，胰蛋白胨和豆饼粉等有机物为氮源时可分泌柚苷酶;该菌种在固态发酵中表现出很强的柚苷酶发酵能力，用HPLC法测定的α-L-鼠李糖苷酶和柚苷酶活力分别达到5903和1939 U/gds，用Davis法测定的柚苷酶活力达到72232 U/gds。【结论】本研究首次发现棘孢曲霉能够分泌柚苷酶，含鼠李糖基团的物质可作为其产柚苷酶的诱导物。棘孢曲霉JMUdb058固态发酵产柚苷酶的能力突出，是一种高产柚苷酶的微生物新资源。

Abstract:

Abstract:［Objective］ A new naringinase-producing strain，JMUdb058 was identified and characterized. ［Methods］The strain was identified by morphological observation and 28S rDNA homogeneous analysis． Naringinase was identified by monitoring the hydrolysis of naringin to prunin and naringenin using a reversed phase High Performance Liquid Chromatography (HPLC)．The regulation of naringinase expression was studied by measuring naringinase activity of 11 different carbon sources and 7 nitrogen sources in shaking cultivation． The naringinase-producing capacity was investigated in both solid-state fermentation and submerged fermentation．［Results］The macro-morphology and micro-morphology of JMUdb058 corresponded to the characteristics of Aspergillus section Nigri Gams，and the 28S rDNA sequences showed homogeneity at 100% to Aspergillus aculeatus． Crude enzymes prepared by both submerged fermentation and solid-state fermentation could hydrolyze naringin to prunin and naringenin．In addition，the enzyme could remove naringin from citrus juice effectively． Carbon resources，including hesperidin，naringin，rutin and rhamnose，and organic nitrogen resources，i.e.，tryptone，soybean meal，yeast extract and corn syrup were shown to express the naringinase．The strain had an outstanding ability to yield naringinase in the solid-state fermentation，which showed an α-L-rhamnosidase activity of 5903 U/gds by HPLC，and the naringinase of 1939U/gds by HPLC and 72232 U/gds by Davis method.［Conclusion］It is the first time to report a stain of Aspergillus aculeatus can produce naringinase，carbon source containing rhamnose groups are able to induce the enzyme expression．The stain JMUdb058 is a new microorganism source for high yield of naringinase，in particularly by the solid-state fermentation.