棘孢曲霉固态发酵α-L-鼠李糖苷酶调控机制及培养基优化
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摘要
为了提高α-L-鼠李糖苷酶的发酵产量,利用高效液相色谱法检测α-L-鼠李糖苷酶活力,考察外加碳源葡萄糖、氮源及生长因子对棘孢曲霉JMUdb058固态发酵产α-L-鼠李糖苷酶的调控机制,并以之为依据优化培养基。研究结果表明,葡萄糖和淀粉对α-L-鼠李糖苷酶的产生具有代谢调节作用;外加氮源能大幅度提高α-L-鼠李糖苷酶的活力;相比用硫酸铵为氮源,用豆饼粉为氮源时,由于其中含有淀粉而导致酶合成量降低;磷酸氢二铵比其他铵盐和硝酸盐更能促进α-L-鼠李糖苷酶的合成。添加富含氨基酸的生长因子有利于α-L-鼠李糖苷酶的合成。棘孢曲霉JMUdb058发酵产α-L-鼠李糖苷酶优化后的培养基是:柚皮5 g,磷酸氢二铵0.5 g,酵母浸膏0.075 g,水5 m L,此时α-L-鼠李糖苷酶活力达到10.60 IU/gds,比初始培养基的酶活力提高了84.67%,比其他文献报道的最高酶产量提高了1.5倍。优化后的培养基大幅度提高了棘孢曲霉固态发酵α-L-鼠李糖苷酶的活力,为该酶的发酵生产及开发利用提供了技术参考。
In this study, the activity of α-L-rhamnosidase was detected by HPLC, the regulatory effects of glucose,nitrogen source and growth factors on the induction of α-L-rhamnosidase were investigated, and the medium was improved for the production of this enzyme from Aspergillus aculeatus JMUdb058 by solid-state fermentation. The results showed that glucose had strong metabolic inhibitory effect on the induction of α-L-rhamnosidase from A. aculeatus. The addition of nitrogen source significantly enhanced the production of α-L-rhamnosidase. Furthermore, in comparison to ammonium sulfate, the addition of organic nitrogen source soybean meal showed a lower activity of α-L-rhamnosidase,which was revealed in light of it contained large amount of starch that inhibited the induction of the enzyme. Among the inorganic nitrogen sources tested, diammonium hydrogen phosphate had the most effectiveness in α-L-rhamnosidase synthesis. The production of α-L-rhamnosidase could further increase by the addition of some growth factors that enriched in amino acids. The solid-state fermentation of an improved medium that consisted of pomelo peel powder(5 g), diammonium hydrogen phosphate(0.5 g), yeast extract(0.075 g) and water(5 m L) showed an α-L-rhamnosidase activity of 10.60 IU/gds, which increased by 84.67% and 1.5 times in comparison with the initial medium and the most effective process reported by other researcher's, respectively. This paper illustrated the regulatory mechanisms of glucose, nitrogen source and growth factor on the synthesis of α-L-rhamnosidase, by which the production of α-L-rhamnosidase was, increased significantly. These results provide a practical technique for the production of α-L-rhamnosidase.
In this study, the activity of α-L-rhamnosidase was detected by HPLC, the regulatory effects of glucose,nitrogen source and growth factors on the induction of α-L-rhamnosidase were investigated, and the medium was improved for the production of this enzyme from Aspergillus aculeatus JMUdb058 by solid-state fermentation. The results showed that glucose had strong metabolic inhibitory effect on the induction of α-L-rhamnosidase from A. aculeatus. The addition of nitrogen source significantly enhanced the production of α-L-rhamnosidase. Furthermore, in comparison to ammonium sulfate, the addition of organic nitrogen source soybean meal showed a lower activity of α-L-rhamnosidase,which was revealed in light of it contained large amount of starch that inhibited the induction of the enzyme. Among the inorganic nitrogen sources tested, diammonium hydrogen phosphate had the most effectiveness in α-L-rhamnosidase synthesis. The production of α-L-rhamnosidase could further increase by the addition of some growth factors that enriched in amino acids. The solid-state fermentation of an improved medium that consisted of pomelo peel powder(5 g), diammonium hydrogen phosphate(0.5 g), yeast extract(0.075 g) and water(5 m L) showed an α-L-rhamnosidase activity of 10.60 IU/gds, which increased by 84.67% and 1.5 times in comparison with the initial medium and the most effective process reported by other researcher's, respectively. This paper illustrated the regulatory mechanisms of glucose, nitrogen source and growth factor on the synthesis of α-L-rhamnosidase, by which the production of α-L-rhamnosidase was, increased significantly. These results provide a practical technique for the production of α-L-rhamnosidase.
引文
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[14]吴升山,蔡慧农,苏文金,等.黑曲霉db056发酵α-鼠李糖苷酶和柚苷酶培养基的优化研究[J].中国食品学报,2010,10(4):193-201.
[15]Rajal VB,Cid AG,Ellenrieder G,et al.Production,partial purification and characterization ofα-L-rhamnosidase from Penicillium ulaiense[J].World journal of microbiology and biotechnology,2009,25(6):1025-1033.
[16]Puri M,Kaur A,Barrow CJ,et al.Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus mak2 in a stirred tank reactor[J].Applly Microbiol Biotechnol,2011,89(3):715-722.
[17]Elinbaum S,Ferreyra H,Ellenrieder G,et al.Production of aspergillus terreusα-L-rhamnosidase by solid state fermentation[J].Letters in Applied Microbiology,2002,34(1):67-71.
[18]王迪,倪辉,李利君,等.一株棘孢曲霉的鉴定及其柚苷酶合成规律[J].微生物学报,2013,53(7):691-701.
[19]陈华根,倪辉,李利君,等.黑曲霉α-鼠李糖苷酶高产菌株的选育[J].微生物学通报,2009,36(7):1008-1012.
[20]Ni H,Chen F,Cai H,et al.Characterization and preparation of Aspergillus niger naringinase for debittering citrus juice[J].J Food Science,2012,77(1):1-7.
[21]张龙翔,张庭芳,生物化学,等.生化实验方法和技术[M].北京:高等教育出版社,1997:1-532.
[22]魏培莲,岑沛霖,盛春琦.3种固态发酵生物量测定方法的比较[J].食品与生物技术学报,2006,25(1):60-64.
[23]Ronne H.Glucose repression in fungi[J].Trends in Genetics,1995,11(1):12-17.
[24]Bram B,Solomons G.Production of the enzyme naringinase by Aspergillus niger[J].Applied Microbiology,1965,13(6):842-845.
[25]Tamayo-Ramos JA,Flipphi M,Pardo E,et al.L-rhamnose induction of Aspergillus nidulans alpha-L-rhamnosidase genes is glucose repressed via a crea-independent mechanism acting at the level of inducer uptake[J].Microb Cell Fact,2012,11(1):26.
[26]Puri M,Banerjee A,Banerjee UC.Optimization of process parameters for the production of naringinase by Aspergillus niger mtcc 1344[J].Process biochemistry,2005,40(1):195-201.
[27]Vinoth-Kumar V,Kayambu P,Revathi Babu S.Optimization of fermentation parameters for enhanced production of naringinase by soil isolate Aspergillus niger vb07[J].Food Science and Biotechnology,2010,19(3):827-829.
[28]Koseki T,Mese Y,Nishibori N,et al.Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene[J].Applly Microbiol Biotechnol,2008,80(6):1007-1013.
[29]肖安风,倪辉,吴升山,等.黑曲霉产柚苷酶的发酵条件优化[J].中国食品学报,2011,11(6):89-97.
[30]Abbate E,Palmeri R,Todaro A,et al.Production of aα-L-rhamnosidase from aspergillus terreus using citrus solid waste as inducer for application in juice industry[J].Chemical Engineering,2012,27:253-258.
[2]Puri M,Banerjee UC.Production,purification,and characterization of the debittering enzyme naringinase[J].Biotechnology Advances,2000,18(3):207-217.
[3]Caldini C,Bonomi F,Pifferi PG,et al.Kinetic and immobilization studies on fungal glycosidases for aroma enhancement in wine[J].Enzyme and Microbial Technology,1994,16(4):286-291.
[4]Cheetham PS,Quail MA.Process for preparing l-rhamnose[M].New York:Google Patents,1991:25-120.
[5]Chang HY,Lee YB,Bae HA,et al.Purification and characterisation of Aspergillus sojae naringinase:the production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of hmg-coa reductase[J].Food Chem istry,2011,124(1):234-241.
[6]Yu H,Gong J,Zhang C,et al.Purification and characterization of ginsenoside-α-L-rhamnosidase[J].Chemical and pharmaceutical bulletin,2002,50(2):175-178.
[7]Manzanares P,Vallés S,Ramòn D,et al.Α-l-rhamnosidases:old and new insights[M].New York:Industrial Enzymes.Springer,2007:117-140.
[8]Puri M,Kaur A.Molecular identification of Staphylococcus xylosus mak2,a newα-L-rhamnosidase producer[J].World journal of microbiology and biotechnology,2010,26(6):963-968.
[9]Gallego M,Pinaga F,Ramón D,et al.Purification and characterization of anα-L-rhamnosidase from Aspergillus terreus of interest in winemaking[J].J Food Science,2001,66(2):204-209.
[10]Cui Z,Maruyama Y,Mikami B,et al.Crystal structure of glycoside hydrolase family 78α-L-rhamnosidase from Bacillus sp.Gl1[J].Journal of Molecular Biology,2007,374(2):384-398.
[11]Ni H,Xiao AF,Cai HN,et al.Purification and characterization of Aspergillus niger alpha-L-rhamnosidase for the biotransformation of naringin to prunin[J].African Journal of Microbiology Research,2012,6(24):5276-5284.
[12]Ichinose H,Fujimoto Z,Kaneko S.Characterization of anα-L-rhamnosidase from Streptomyces avermitilis[J].Bio science,Biotechnology,and Biochemistry,2013,77(1):213-216.
[13]Chen Y,Ni H,Chen F,et al.Purification and characterization of a naringinase from Aspergillus aculeatus jmudb058[J].Journal of Agricultural and Food Chemistry,2013,61(4):931-938.
[14]吴升山,蔡慧农,苏文金,等.黑曲霉db056发酵α-鼠李糖苷酶和柚苷酶培养基的优化研究[J].中国食品学报,2010,10(4):193-201.
[15]Rajal VB,Cid AG,Ellenrieder G,et al.Production,partial purification and characterization ofα-L-rhamnosidase from Penicillium ulaiense[J].World journal of microbiology and biotechnology,2009,25(6):1025-1033.
[16]Puri M,Kaur A,Barrow CJ,et al.Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus mak2 in a stirred tank reactor[J].Applly Microbiol Biotechnol,2011,89(3):715-722.
[17]Elinbaum S,Ferreyra H,Ellenrieder G,et al.Production of aspergillus terreusα-L-rhamnosidase by solid state fermentation[J].Letters in Applied Microbiology,2002,34(1):67-71.
[18]王迪,倪辉,李利君,等.一株棘孢曲霉的鉴定及其柚苷酶合成规律[J].微生物学报,2013,53(7):691-701.
[19]陈华根,倪辉,李利君,等.黑曲霉α-鼠李糖苷酶高产菌株的选育[J].微生物学通报,2009,36(7):1008-1012.
[20]Ni H,Chen F,Cai H,et al.Characterization and preparation of Aspergillus niger naringinase for debittering citrus juice[J].J Food Science,2012,77(1):1-7.
[21]张龙翔,张庭芳,生物化学,等.生化实验方法和技术[M].北京:高等教育出版社,1997:1-532.
[22]魏培莲,岑沛霖,盛春琦.3种固态发酵生物量测定方法的比较[J].食品与生物技术学报,2006,25(1):60-64.
[23]Ronne H.Glucose repression in fungi[J].Trends in Genetics,1995,11(1):12-17.
[24]Bram B,Solomons G.Production of the enzyme naringinase by Aspergillus niger[J].Applied Microbiology,1965,13(6):842-845.
[25]Tamayo-Ramos JA,Flipphi M,Pardo E,et al.L-rhamnose induction of Aspergillus nidulans alpha-L-rhamnosidase genes is glucose repressed via a crea-independent mechanism acting at the level of inducer uptake[J].Microb Cell Fact,2012,11(1):26.
[26]Puri M,Banerjee A,Banerjee UC.Optimization of process parameters for the production of naringinase by Aspergillus niger mtcc 1344[J].Process biochemistry,2005,40(1):195-201.
[27]Vinoth-Kumar V,Kayambu P,Revathi Babu S.Optimization of fermentation parameters for enhanced production of naringinase by soil isolate Aspergillus niger vb07[J].Food Science and Biotechnology,2010,19(3):827-829.
[28]Koseki T,Mese Y,Nishibori N,et al.Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene[J].Applly Microbiol Biotechnol,2008,80(6):1007-1013.
[29]肖安风,倪辉,吴升山,等.黑曲霉产柚苷酶的发酵条件优化[J].中国食品学报,2011,11(6):89-97.
[30]Abbate E,Palmeri R,Todaro A,et al.Production of aα-L-rhamnosidase from aspergillus terreus using citrus solid waste as inducer for application in juice industry[J].Chemical Engineering,2012,27:253-258.