产褐藻胶裂解酶菌株的筛选及发酵条件优化
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摘要
为进一步探索高产褐藻胶裂解酶菌株,促进其工业化应用,以褐藻酸钠为唯一碳源筛选培养基,从鲍鱼内脏中筛选出一株高产褐藻胶裂解酶菌株B4,通过形态学观察和系统发育分析,鉴定为弧菌属细菌Vibrio sp.B4。通过单因素实验对B4菌株的发酵条件和发酵培养基进行优化,获得发酵培养基的最适碳源为褐藻酸钠10g/L,最适氮源为(NH4)2SO410g/L,最适发酵条件为温度30℃,接种量1%,培养基初始pH 6.0。在单因素实验的基础上,通过Plackett-Burman(PB)筛选出3个影响产酶活力的显著因素:(NH4)2SO4浓度、pH、接种量。通过响应面进一步分析优化,得到最佳的发酵培养基为褐藻酸钠10g/L,(NH_4)_2SO_4 10.91g/L,NaCl 10g/L,KH_2PO_4 1g/L,MgSO4·7H2O 0.5g/L,CaCl_2 0.2g/L,FeSO_4·7H_2O 0.02g/L,最佳发酵条件为温度30℃,接种量1.1%,起始pH 6.07。在最佳培养条件下,褐藻胶裂解酶活力可达19.09U/mL,比基础培养条件下提高了3.67倍。该菌经过产酶发酵条件的优化,酶活力得到较大幅度的提高,且其发酵产酶时间短,可为褐藻胶裂解酶的进一步研究应用提供参考。
In order to screen the alginate lyase-producing strain and promote its commercial application,a strain B4 producing high level alginate lyase was screened from the viscera of abalone using sodium alginate as sole carbon source on agar medium.The strain was identified as Vibrio sp.B4 according to morphological observation and phylogenetic analysis.Single factor experiments showed the optimum carbon source of its fermentation medium was 10 g/L sodium alginate and the optimum nitrogen source was 10 g/L(NH_4)_2 SO_4.The optimum fermentation conditions were temperature 30℃,initial pH 6.0,inoculation1%.On the basis of single factor experiment,3 significant factors affecting enzyme activity were screened out by Plackett-Burman(PB)design:(NH_4)_2 SO_4,pH and inoculation amount.The optimization by a response surface methodology revealed that the optimal fermentation medium of B4 was comprised of sodium alginate 10 g/L,(NH_4)_2 SO_4 10.91 g/L,NaCl 10 g/L,KH2 PO41 g/L,MgSO_4·7 H_2 O 0.5 g/L,CaCl_2 0.2 g/L,FeSO_4·7 H_2 O 0.02 g/L.The optimal fermentation conditions were temperature 30℃,inoculation amount1.1%,initial pH 6.07.The alginate lyase activity was 19.09 U/mL under the optimized condition,which was 3.67 times higher than that under basic medium and culture conditions.The enzyme activity of Vibrio sp.B4 in this work has been greatly improved by optimization,and its enzyme production time is short,which can provide reference for further research and application of alginate lyase.
In order to screen the alginate lyase-producing strain and promote its commercial application,a strain B4 producing high level alginate lyase was screened from the viscera of abalone using sodium alginate as sole carbon source on agar medium.The strain was identified as Vibrio sp.B4 according to morphological observation and phylogenetic analysis.Single factor experiments showed the optimum carbon source of its fermentation medium was 10 g/L sodium alginate and the optimum nitrogen source was 10 g/L(NH_4)_2 SO_4.The optimum fermentation conditions were temperature 30℃,initial pH 6.0,inoculation1%.On the basis of single factor experiment,3 significant factors affecting enzyme activity were screened out by Plackett-Burman(PB)design:(NH_4)_2 SO_4,pH and inoculation amount.The optimization by a response surface methodology revealed that the optimal fermentation medium of B4 was comprised of sodium alginate 10 g/L,(NH_4)_2 SO_4 10.91 g/L,NaCl 10 g/L,KH2 PO41 g/L,MgSO_4·7 H_2 O 0.5 g/L,CaCl_2 0.2 g/L,FeSO_4·7 H_2 O 0.02 g/L.The optimal fermentation conditions were temperature 30℃,inoculation amount1.1%,initial pH 6.07.The alginate lyase activity was 19.09 U/mL under the optimized condition,which was 3.67 times higher than that under basic medium and culture conditions.The enzyme activity of Vibrio sp.B4 in this work has been greatly improved by optimization,and its enzyme production time is short,which can provide reference for further research and application of alginate lyase.
引文
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[18]严芬,连燕萍,杨光,等.高产褐藻胶裂解酶菌株的筛选及发酵条件优化[J].食品工业科技,2015,36(22):287-292.
[19]Kam N,Park Y J,Lee E Y,et al.Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp.strain KS-408for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate[J].Canadian Journal of Microbiology,2011,57(12):1032-1041.
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[21]侯保兵,刘书来,张建友,等.褐藻胶裂解酶产生菌的发酵优化研究[J].水产科学,2009,28(11):667-670.
[22]Tavernier M L,Petit E,Delattre C,et al.Production of oligoglucuronans using a monolithic enzymatic microreactor[J].Carbohydrate Research,2008,343(15):2687-2691.
[2]王庭欣,王庭祥,庞佳宏.海带多糖降血糖、血脂作用的研究[J].营养学报,2007,29(1):99-100.
[3]Wang P,Jiang X,Jiang Y,et al.In vitro antioxidative activities of three marine oligosaccharides[J].Natural Product Research,2007,21(7):646-654.
[4]Meiyu G,Fuchuan L,Xianliang X,et al.The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1entry.Interaction between SPMG and HIV-1rgp120and CD4molecule[J].Antiviral Research,2003,59(2):127-135.
[5]Liu H,Geng M,Xin X,et al.Multiple and multivalent interactions of novel anti-AIDS drug candidates,sulfated polymannuronate(SPMG)-derived oligosaccharides,with rgp120and their anti-HIV activities[J].Glycobiology,2005,15(5):501-510.
[6]Iwamoto Y,Xu X U,Tamura T,et al.Enzymatically Depolymerized Alginate Oligomers That Cause Cytotoxic Cytokine Production in Human Mononuclear Cells[J].Biosci Biotechnol Biochem,2003,67(2):258-263.
[7]Fujihara M,Nagumo T.An influence of the structure of alginate on the chemotactic activity of macrophages and the antitumor activity[J].Carbohydrate Research,1993,243(1):211-216.
[8]Natsume M,Kamo Y,Hirayama M,et al.Isolation and characterization of alginate-derived oligosaccharides with root growth-promoting activities[J].Carbohydr Res,1994,258(6):187-197.
[9]Hu X K,Jiang X L,Hwang Hmliu S L,et al.Promotive effects of alginate-derived oligosaccharide on maize seed germination[J].Journal of Applied Phycology,2004,16(1):73-76.
[10]Ma L J,Li X M,Bu N,et al.An alginate-derived oligosaccharide enhanced wheat tolerance to cadmium stress[J].Plant Growth Regulation,2010,62(1):71-76.
[11]周燕霞.褐藻胶裂解酶分泌菌株的分离鉴定及Tamlana holothuriorum s12~T中褐藻胶裂解酶的研究[D].济南:山东大学,2016.
[12]罗丹丹.产褐藻胶裂解酶菌种筛选与酶学性质研究[D].大连:大连工业大学,2016.
[13]李婷婷,陈倩,石萍,等.高效褐藻胶降解菌的筛选与产酶条件优化[J].北京大学学报(自然科学版),2017,53(6):1115-1121.
[14]高王宇,黄惠琴,刘敏,等.产褐藻胶裂解酶菌株HB12274的鉴定和发酵优化[J].基因组学与应用生物学.2018,37(10):4365-4371
[15]李云涛,汪立平,张孟,等.鲍鱼来源褐藻胶裂解酶菌株的筛选及发酵条件优化[J].大连海洋大学学报,2017,32(5):574-583.
[16]李永幸,杨帆,姜泽东,等.响应面优化微泡菌ALW1产褐藻胶裂解酶的发酵条件[J].发酵科技通讯,2016,45(2):75-80.
[17]刘彩琴,王楠,陈薇青,等.一株明亮发光杆菌产褐藻胶裂解酶的培养基优化[J].食品工业科技,2017,38(8):156-160.
[18]严芬,连燕萍,杨光,等.高产褐藻胶裂解酶菌株的筛选及发酵条件优化[J].食品工业科技,2015,36(22):287-292.
[19]Kam N,Park Y J,Lee E Y,et al.Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp.strain KS-408for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate[J].Canadian Journal of Microbiology,2011,57(12):1032-1041.
[20]Fu X,Lin H,Sang M K.Optimization of culturing condition and medium composition for the production of alginate lyase by a marine Vibrio,sp.YKW-34[J].中国海洋大学学报(英文版),2008,7(1):97-102.
[21]侯保兵,刘书来,张建友,等.褐藻胶裂解酶产生菌的发酵优化研究[J].水产科学,2009,28(11):667-670.
[22]Tavernier M L,Petit E,Delattre C,et al.Production of oligoglucuronans using a monolithic enzymatic microreactor[J].Carbohydrate Research,2008,343(15):2687-2691.
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