海洋弧菌NTi产琼胶酶的发酵条件优化
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摘要
以琼胶酶活力为主要指标,采用单因素与响应面相结合的方法,对降解琼胶的海洋弧菌NTi(Vibrio natriegens NTi)发酵产琼胶酶的摇瓶发酵培养基和发酵条件进行优化。结果表明,α-乳糖对菌株产酶具有较强的抑制作用,NH+4不利于菌株生长和产酶。培养基分别以3. 3 g/L琼脂、6. 33 g/L酵母膏为唯一碳源及氮源,添加20 g/L的Na Cl,发酵海洋弧菌V. natriegens NTi产琼胶酶的效果最佳。发酵过程中的p H值、接种量、装液量、温度、发酵时间最优值分别为6. 5、2%、32 m L、27℃和24 h。优化后,该菌产琼胶酶活力达到2. 81 U/m L,比优化前增加了230%。
The single factor test and response surface methodology were used to optimize the fermentation medium and fermentation conditions of agarase produced by marine Vibrio natriegens NTi,and the agarase activity was taken as main indicator. The results showed that α-lactose had a significant inhibitory effect on the agarase production,while ammonium ion was not conducive to cell growth and enzyme production. The optimal composition of culture medium for agarase-producing was: 3. 3 g/L agar as the sole carbon source,6. 33 g/L yeast extract as the sole nitrogen source and 20 g/L NaCl. Meanwhile,the optimum conditions for agarase production were pH = 6. 5,inoculation amount 2%,liquid volume 32 m L,temperature 27 ℃ and fermentation time24 h,respectively. Under the optimum conditions,agarase activity reached 2. 81 U/m L after 24 h of fermentation and increased by 230% compared with the activity under the initial medium and conditions.
The single factor test and response surface methodology were used to optimize the fermentation medium and fermentation conditions of agarase produced by marine Vibrio natriegens NTi,and the agarase activity was taken as main indicator. The results showed that α-lactose had a significant inhibitory effect on the agarase production,while ammonium ion was not conducive to cell growth and enzyme production. The optimal composition of culture medium for agarase-producing was: 3. 3 g/L agar as the sole carbon source,6. 33 g/L yeast extract as the sole nitrogen source and 20 g/L NaCl. Meanwhile,the optimum conditions for agarase production were pH = 6. 5,inoculation amount 2%,liquid volume 32 m L,temperature 27 ℃ and fermentation time24 h,respectively. Under the optimum conditions,agarase activity reached 2. 81 U/m L after 24 h of fermentation and increased by 230% compared with the activity under the initial medium and conditions.
引文
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[14]王芳.利用茶叶梗固态发酵单宁酶及其酶制剂的制备[D].厦门:集美大学,2012.
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[19]王静雪,江晓路,牟海津,等.海洋弧菌QJH-12发酵产琼胶酶条件的优化[J].海洋科学,2007,31(7):8-14.
[20]朱启忠,朱慧文,孙艳娜,等.海洋细菌NBRC10260产琼胶酶发酵条件优化[J].中国酿造,2011,30(5):53-55.
[21]王霁宁,严孝强,杜宗军.琼胶酶高产海洋细菌QM42的发酵条件优化[J].中国酿造,2012,31(7):86-89.
[22]缪伏荣,董志岩,刘景.琼胶酶产生菌Stenotrophomonas sp. Z705的发酵条件优化[J].西北农林科技大学学报(自然科学版),2014,42(10):152-158,165.
[23] WOOD J M,BREMER E,CSONKA L N,et al. Osmosensing and osmoregulatory compatible solute accumulation by bacteria[J]. Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2001,130(3):437-460. DOI:10. 1016/S1095-6433(01)00442-1.
[24]顾芮萌,李勇昊,田朝光.粗糙脉孢菌(Neurospora crassa)纤维素酶液体发酵培养基的优化[J].中国生物工程杂志,2012,32(3):76-82.
[2] TEMUUJIN U,CHI W J,CHANG Y K,et al. Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2),an exo-and endo-typeβ-agarase-producing neoagarobiose[J]. Journal of Bacteriology,2012,194(1):142-149. DOI:10. 1128/JB. 05978-11.
[3]马芮萍,朱艳冰,倪辉,等.海洋细菌NTa发酵产琼胶酶条件的初步优化[J].集美大学学报(自然科学版),2014,19(4):259-265.
[4] ZHANG W W,SUN L. Cloning,characterization,and molecular application of a beta-agarase gene from Vibrio sp.strain V134[J]. Applied and Environmental Microbiology,2007,73(9):2825-2831.
[5] XIAO T F,SANG M K. Agarase:review of major sources,categories,purification method,enzyme characteristics and applications[J]. Marine Drugs,2010,8(1):200-218. DOI:10. 3390/md8010200.
[6] FU X T,PAN C H,LIN H,et al. Gene cloning,expression,and characterization of aβ-agarase,Aga B34,from Agarivorans albus YKW-34[J]. J Microbiol Biotechnol,2009,19(3):257-264.
[7]牟宗娟,李贵阳,茅云翔,等. 4株琼胶降解菌的分离、鉴定及产酶条件分析[J].海洋科学,2013,37(4):13-20.
[8] LIAO L,XU X W,JIANG X W,et al. Cloning,expression,and characterization of a new beta-agarase from Vibrio sp.strain CN41[J]. Applied and Environmental Microbiology,2011,77(19):7077-7079. DOI:10. 1128/AEM. 05364-11.
[9]付晓婷.海洋细菌(Agarivorans albus YKW-34)产生的褐藻胶裂解酶及琼胶酶的研究[D].青岛:中国海洋大学,2008.
[10]吴国汉.琼胶酶生产菌的筛选、鉴定及其产酶条件的优化[D].厦门:集美大学,2011.
[11] JUNG C,KWON H,PARK C,et al. Optimization of Pseudoalteromonas sp. JYBCL 1 culture conditions,medium composition and extracellularβ-agarase activity[J]. Biotechnology and Bioprocess Engineering,2012,17(5):937-945. DOI:10. 1007/s12257-012-0009-2.
[12]许彩云,姚德恒,朱艳冰,等.产琼胶酶菌株海洋弧菌NTi罐上工艺优化及中试放大[J].中国食品学报,2016,16(11):96-104.
[13] MILLER G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Biochemistry,1959,31(3):426-428.
[14]王芳.利用茶叶梗固态发酵单宁酶及其酶制剂的制备[D].厦门:集美大学,2012.
[15]赵玉萍,陈晓旺,沈鹏伟. Box-Behnken实验设计及响应面分析优化锰过氧化物酶培养基条件[J].食品工业科技,2013(4):207-211.
[16]杨哲.枯草芽孢杆菌产肌苷的发酵过程优化及其代谢机理分析[D].厦门:集美大学,2013.
[17] WANG J J,SHIH J C H. Fermentation production of keratinase from Bacillus licheniformis PWD-1 and a recombinant B. subtilis FDB-29[J]. Journal of Industrial Microbiology and Biotechnology,1999,22(6):608-616. DOI:10. 1038/sj. jim. 2900667.
[18]张炎达,王风芹,彭一丁,等. NH+4对合成气乙醇发酵过程的影响及其酶学机理[J].食品与发酵工业,2015,41(12):46-53.
[19]王静雪,江晓路,牟海津,等.海洋弧菌QJH-12发酵产琼胶酶条件的优化[J].海洋科学,2007,31(7):8-14.
[20]朱启忠,朱慧文,孙艳娜,等.海洋细菌NBRC10260产琼胶酶发酵条件优化[J].中国酿造,2011,30(5):53-55.
[21]王霁宁,严孝强,杜宗军.琼胶酶高产海洋细菌QM42的发酵条件优化[J].中国酿造,2012,31(7):86-89.
[22]缪伏荣,董志岩,刘景.琼胶酶产生菌Stenotrophomonas sp. Z705的发酵条件优化[J].西北农林科技大学学报(自然科学版),2014,42(10):152-158,165.
[23] WOOD J M,BREMER E,CSONKA L N,et al. Osmosensing and osmoregulatory compatible solute accumulation by bacteria[J]. Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2001,130(3):437-460. DOI:10. 1016/S1095-6433(01)00442-1.
[24]顾芮萌,李勇昊,田朝光.粗糙脉孢菌(Neurospora crassa)纤维素酶液体发酵培养基的优化[J].中国生物工程杂志,2012,32(3):76-82.