褐藻胶降解菌的筛选、鉴定及产酶条件优化
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摘要
【目的】筛选一株能降解褐藻胶的菌株,并优化产酶条件以提高褐藻胶裂解酶活力。【方法】从漳州海域采集到海水和海泥,以海藻酸钠为唯一碳源,通过富集培养、初筛、复筛筛选到一株能够降解褐藻胶的菌株。依据16S rRNA序列分析、生理生化特征、菌体形态及菌落特征对该菌进行鉴定。通过单因素和正交试验对该菌的产酶条件进行优化。【结果】该菌属于海科贝特氏菌,命名为Cobetiamarina HQZ08。该菌株最佳的产酶培养基组成为:海藻酸钠7.00g/L、蛋白胨3.00g/L、NaCl30.00g/L,K2HPO4·3H2O 1.25 g/L。最佳发酵条件为:接种量2%,接种龄12 h,培养基起始pH为7.0,培养温度25°C,培养时间24 h。优化后褐藻胶裂解酶活力达到68.5 U/mL,TLC法分析酶解产物为褐藻胶寡糖。【结论】HQZ08菌株可以用于降解褐藻胶,产生聚合度为2–6的褐藻胶寡糖。
[Objective] To screen an alginate-degrading strain and to improve the activity of its alginate lyase by optimizing the fermentation conditions. [Methods] A strain capable of degrading alginate was screened by the steps of enrichment culture, screening, and re-screening by using seawater and sea mud collected from the sea area of Zhangzhou(Fujian, China). Then, the strain was identified according to its 16 S rRNA sequence analysis, physiological and biochemical characteristics, mycelium morphology and colony characteristics. The conditions of enzyme production of the strain were optimized by single factor and orthogonal test. [Results] The strain, belonging to the genus Cobetia marina, was named Cobetia marina HQZ08. The optimal medium of the strain was composed of 7.00 g/L sodium alginate, 3.00 g/L peptone, 30.00 g/L NaCl, 1.25 g/L K2 HPO4·3 H2 O. The optimal fermentation conditions were as follows: the optimum inoculum was 2%, the inoculation age was 12 h, the initial pH of the culture medium was 7.0, the culture temperature was 25 °C, and the incubation time was 24 h. The enzymatic activity of alginate lyase reached 68.5 U/mL after optimization. Enzymatic hydrolysates were determined to be alginate oligosaccharides. [Conclusion] HQZ08 strain can be used to degrade alginate to produce alginate oligosaccharides with a degree of polymerization of 2–6.
[Objective] To screen an alginate-degrading strain and to improve the activity of its alginate lyase by optimizing the fermentation conditions. [Methods] A strain capable of degrading alginate was screened by the steps of enrichment culture, screening, and re-screening by using seawater and sea mud collected from the sea area of Zhangzhou(Fujian, China). Then, the strain was identified according to its 16 S rRNA sequence analysis, physiological and biochemical characteristics, mycelium morphology and colony characteristics. The conditions of enzyme production of the strain were optimized by single factor and orthogonal test. [Results] The strain, belonging to the genus Cobetia marina, was named Cobetia marina HQZ08. The optimal medium of the strain was composed of 7.00 g/L sodium alginate, 3.00 g/L peptone, 30.00 g/L NaCl, 1.25 g/L K2 HPO4·3 H2 O. The optimal fermentation conditions were as follows: the optimum inoculum was 2%, the inoculation age was 12 h, the initial pH of the culture medium was 7.0, the culture temperature was 25 °C, and the incubation time was 24 h. The enzymatic activity of alginate lyase reached 68.5 U/mL after optimization. Enzymatic hydrolysates were determined to be alginate oligosaccharides. [Conclusion] HQZ08 strain can be used to degrade alginate to produce alginate oligosaccharides with a degree of polymerization of 2–6.
引文
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[18]An QD,Zhang GL,Wu HT,Zhang ZC,Zheng GS,Luan L,Murata Y,Li X.Alginate-deriving oligosaccharide production by alginase from newly isolated Flavobacterium sp.LXA and its potential application in protection against pathogens.Journal of Applied Microbiology,2009,106(1):161-170.
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[21]Fu XY,Li JB,Han F,Lu XZ,Yu WG.Studies on Vibro sp.QY102 fermentation processes for alginate lyase production.Periodical of Ocean University of China,2007,37(3):432-436.(in Chinese)傅晓妍,李京宝,韩峰,路新枝,于文功.褐藻胶裂解酶产生菌Vibro sp.QY102的发酵条件优化.中国海洋大学学报,2007,37(3):432-436.
[22]Wei D,Dou WF,Li H,Li H,Xu ZH,Shi JS.Isolation,identification,and fermentation optimization of a high efficient novel alginate-degrading strain.Food and Fermentation Industries,2012,38(7):26-31.(in Chinese)魏丹,窦文芳,李恒,李会,许正宏,史劲松.高效降解褐藻胶新菌种的筛选、鉴定及产酶条件优化.食品与发酵工业,2012,38(7):26-31.
[23]Ando Y,Inoue K.Decomposition of alginic acid by microorganisms-Ⅳ.On the Vibro-type bacteria,newly isolated from the decaying Laminaria.Nippon Suisan Gakkaishi,1961,27(4):339-341.
[24]Wang LN.Study on oligoalginate lyases from marine bacterium Shewanella sp.Kz7.Doctor Dissertation of Ocean University of China,2014.(in Chinese)王林娜.海洋细菌Shewanella sp.Kz7的寡褐藻胶裂解酶研究.中国海洋大学博士学位论文,2014.
[25]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[26]Han JP,Huang YY,Ye J,Xiao MT.Screening and identification of a bacterium capable of converting agar to neoagaro oligosaccharides.Acta Microbiologica Sinica,2015,55(9):1126-1132.(in Chinese)韩军萍,黄雅燕,叶静,肖美添.一株转化琼胶为新琼寡糖菌株的筛选及鉴定.微生物学报,2015,55(9):1126-1132.
[27]Arahal DR,Castillo AM,Ludwig W,Schleifer KH,Ventosa A.Proposal of Cobetia marina gen.nov.,comb.nov.,within the family Halomonadaceae,to include the species Halomonas marina.Systematic and Applied Microbiology,2002,25(2):207-211.
[28]Liu XM,Li H,Jiang M,Gong JS,Xu ZH,Shi JS.Screening of alginate lyase producing strain Cobetia sp.WG-007 and optimization of fermentation.Journal of Food Science and Biotechnology,2014,33(9):903-909.(in Chinese)刘旭梅,李恒,蒋敏,龚劲松,许正宏,史劲松.产褐藻胶裂解酶菌株Cobetia sp.WG-007的筛选及发酵优化.食品与生物技术学报,2014,33(9):903-909.
[29]Wang T,Yang S,Chen YX,Hu LL,Tu Q,Zhang L,Liu XQ,Wang XG.Microbiological properties of two endophytic bacteria isolated from tea(Camellia sinensis L.).Acta Microbiologica Sinica,2014,54(4):424-432.(in Chinese)王婷,杨升,陈亚雪,胡玲玲,屠琴,张丽,刘雪芹,王行国.两株茶树内生草螺菌的微生物学特性.微生物学报,2014,54(4):424-432.
[30]Ma YX,Ji T,Li HQ,Chen W,Gao S,Liu SF.Culture optimization and characterization of an alginate-lyase from Pseudoalteromonas sp.LJ1.Acta Microbiologica Sinica,2009,49(8):1086-1094.(in Chinese)马悦欣,纪涛,李慧琼,陈炜,高嵩,刘声锋.假交替单胞菌LJ1菌株产褐藻胶裂解酶的培养条件优化及酶学性质.微生物学报,2009,49(8):1086-1094.
[31]Hou BB.Isolation of alginate-degrading bacteria,fermation and some properties of alginate lyase.Master Dissertation of Zhejiang University of Technology,2009.(in Chinese)侯宝兵.褐藻胶裂解酶产生菌的筛选、发酵优化及其酶学特性研究.浙江工业大学硕士学位论文,2009.
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[35]Wu HG,Zhang YJ,Luo FW,Yao ZA,Li Q,Gao Z.Isolation of a strain degrading sodium alginate and optimization of its fermentation conditions.Food Science and Technology,2014,39(8):31-36.(in Chinese)吴海歌,张玉娟,罗福文,姚子昂,李倩,高征.海藻酸钠降解菌的筛选及其发酵条件的优化.食品科技,2014,39(8):31-36.
[36]Yagi H,Fujise A,Itabashi N,Ohshiro T.Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp.NAP1 isolated from brown algae.Bioscience,Biotechnology,and Biochemistry,2016,80(12):2338-2346.
[2]Rahelivao MP,Andriamanantoanina H,Heyraud A,Rinaudo M.Structure and properties of three alginates from Madagascar seacoast algae.Food Hydrocolloids,2013,32(1):143-146.
[3]Rhein-Knudsen N,Ale MT,Meyer AS.Seaweed hydrocolloid production:An update on enzyme assisted extraction and modification technologies.Marine Drugs,2015,13(6):3340-3359.
[4]Zhang YJ,Luo FW,Yao ZA,Li Q,Wu HG.Research progress about biological activity of sodium alginate oligosaccharides.China Brewing,2014,33(1):5-8.(in Chinese)张玉娟,罗福文,姚子昂,李倩,吴海歌.海藻酸钠寡糖生物活性的研究进展.中国酿造,2014,33(1):5-8.
[5]Tajima S,Inoue H,Kawada A,Ishibashi A,Takahara H,Hiura N.Alginate oligosaccharides modulate cell morphology,cell proliferation and collagen expression in human skin fibroblasts in vitro.Archives of Dermatological Research,1999,291(7/8):432-436.
[6]Kawada A,Hiura N,Shiraiwa M,Tajima S,Hiruma M,Hara K,Ishibashi A,Takahara H.Stimulation of human keratinocyte growth by alginate oligosaccharides,a possible co-factor for epidermal growth factor in cell culture.FEBSLetters,1997,408(1):43-46.
[7]Xu X,Wu XT,Wang QQ,Cai N,Zhang HX,Jiang ZD,Wan M,Oda T.Immunomodulatory effects of alginate oligosaccharides on murine macrophage RAW264.7 cells and their structure-activity relationships.Journal of Agricultural and Food Chemistry,2014,62(14):3168-3176.
[8]Hu XK,Jiang XL,Hwang H,Liu SL,Guan HS.Antitumour activities of alginate-derived oligosaccharides and their sulphated substitution derivatives.European Journal of Phycology,2004,39(1):67-71.
[9]T?ndervik A,Sletta H,Klinkenberg G,Emanuel C,Powell LC,Pritchard MF,Khan S,Craine KM,Ons?yen E,Rye PD,Wright C,Thomas DW,Hill KE.Alginate oligosaccharides inhibit fungal cell growth and potentiate the activity of antifungals against Candida and Aspergillus spp.PLoS One,2014,9(11):e112518.
[10]Zhou R,Shi XY,Gao Y,Cai N,Jiang ZD,Xu X.Anti-inflammatory activity of guluronate oligosaccharides obtained by oxidative degradation from alginate in lipopolysaccharide-activated murine macrophage RAW 264.7cells.Journal of Agricultural and Food Chemistry,2015,63(1):160-168.
[11]Zhang YH,Yin H,Zhao XM,Wang WX,Du YG,He AL,Sun KG.The promoting effects of alginate oligosaccharides on root development in Oryza sativa L.mediated by auxin signaling.Carbohydrate Polymers,2014,113:446-454.
[12]Xu L,Li Z,Zhou HL,Ding YT,Liu L.Study of hygroscopic and moisturizing performance of oligosaccharides obtained from enzymolysis of algin.China Surfactant Detergent&Cosmetics,2011,41(1):42-45.(in Chinese)许雷,李湛,周火兰,丁玉庭,刘璘.酶解褐藻胶寡糖的吸湿及保湿性能研究.日用化学工业,2011,41(1):42-45.
[13]Wu HG,Wang XW,Li Q,Yao ZA.Study on the anti-oxidant activity of alginate oligosaccharides.Journal of Dalian University,2015,36(3):70-75,83.(in Chinese)吴海歌,王雪伟,李倩,姚子昂.海藻酸钠寡糖抗氧化活性的研究.大连大学学报,2015,36(3):70-75,83.
[14]Wong TY,Preston LA,Schiller NL.Alginate Lyase:review of major sources and enzyme characteristics,structure-function analysis,biological roles,and applications.Annual Review of Microbiology,2000,54(1):289-340.
[15]Cheng Z.Isolation,purifition and optimization the fermentation conditions of the polysaccharide of alginate and inulin degradation bacteria.Master Dissertation of Qingdao University of Science and Technology,2014.(in Chinese)成珍.多糖降解菌的筛选鉴定及培养条件优化-褐藻胶和菊芋降解菌的分离纯化.青岛科技大学硕士学位论文,2014.
[16]Xiao L,Han F,Yang Z,Lu XZ,Yu WG.A novel alginate lyase with high activity on acetylated alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp.QD03.World Journal of Microbiology and Biotechnology,2006,22(1):81-88.
[17]Gimmestad M,Ertesv?g H,Heggeset TMB,Aarstad O,Svanem BIG,Valla S.Characterization of three new Azotobacter vinelandii alginate lyases,one of which is involved in cyst germination.Journal of Bacteriology,2009,191(15):4845-4853.
[18]An QD,Zhang GL,Wu HT,Zhang ZC,Zheng GS,Luan L,Murata Y,Li X.Alginate-deriving oligosaccharide production by alginase from newly isolated Flavobacterium sp.LXA and its potential application in protection against pathogens.Journal of Applied Microbiology,2009,106(1):161-170.
[19]Duan GF,Han F,Yu WG.Cloning,sequence analysis,and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp.CY24.Canadian Journal of Microbiology,2009,55(9):1113-1118.
[20]Miyake O,Ochiai A,Hashimoto W,Murata K.Origin and diversity of alginate lyases of families PL-5 and-7 in Sphingomonas sp.strain A1.Journal of Bacteriology,2004,186(9):2891-2896.
[21]Fu XY,Li JB,Han F,Lu XZ,Yu WG.Studies on Vibro sp.QY102 fermentation processes for alginate lyase production.Periodical of Ocean University of China,2007,37(3):432-436.(in Chinese)傅晓妍,李京宝,韩峰,路新枝,于文功.褐藻胶裂解酶产生菌Vibro sp.QY102的发酵条件优化.中国海洋大学学报,2007,37(3):432-436.
[22]Wei D,Dou WF,Li H,Li H,Xu ZH,Shi JS.Isolation,identification,and fermentation optimization of a high efficient novel alginate-degrading strain.Food and Fermentation Industries,2012,38(7):26-31.(in Chinese)魏丹,窦文芳,李恒,李会,许正宏,史劲松.高效降解褐藻胶新菌种的筛选、鉴定及产酶条件优化.食品与发酵工业,2012,38(7):26-31.
[23]Ando Y,Inoue K.Decomposition of alginic acid by microorganisms-Ⅳ.On the Vibro-type bacteria,newly isolated from the decaying Laminaria.Nippon Suisan Gakkaishi,1961,27(4):339-341.
[24]Wang LN.Study on oligoalginate lyases from marine bacterium Shewanella sp.Kz7.Doctor Dissertation of Ocean University of China,2014.(in Chinese)王林娜.海洋细菌Shewanella sp.Kz7的寡褐藻胶裂解酶研究.中国海洋大学博士学位论文,2014.
[25]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[26]Han JP,Huang YY,Ye J,Xiao MT.Screening and identification of a bacterium capable of converting agar to neoagaro oligosaccharides.Acta Microbiologica Sinica,2015,55(9):1126-1132.(in Chinese)韩军萍,黄雅燕,叶静,肖美添.一株转化琼胶为新琼寡糖菌株的筛选及鉴定.微生物学报,2015,55(9):1126-1132.
[27]Arahal DR,Castillo AM,Ludwig W,Schleifer KH,Ventosa A.Proposal of Cobetia marina gen.nov.,comb.nov.,within the family Halomonadaceae,to include the species Halomonas marina.Systematic and Applied Microbiology,2002,25(2):207-211.
[28]Liu XM,Li H,Jiang M,Gong JS,Xu ZH,Shi JS.Screening of alginate lyase producing strain Cobetia sp.WG-007 and optimization of fermentation.Journal of Food Science and Biotechnology,2014,33(9):903-909.(in Chinese)刘旭梅,李恒,蒋敏,龚劲松,许正宏,史劲松.产褐藻胶裂解酶菌株Cobetia sp.WG-007的筛选及发酵优化.食品与生物技术学报,2014,33(9):903-909.
[29]Wang T,Yang S,Chen YX,Hu LL,Tu Q,Zhang L,Liu XQ,Wang XG.Microbiological properties of two endophytic bacteria isolated from tea(Camellia sinensis L.).Acta Microbiologica Sinica,2014,54(4):424-432.(in Chinese)王婷,杨升,陈亚雪,胡玲玲,屠琴,张丽,刘雪芹,王行国.两株茶树内生草螺菌的微生物学特性.微生物学报,2014,54(4):424-432.
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