琼胶酶生产菌的筛选、鉴定及其产酶条件的优化
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摘要
利用琼胶作唯一碳源,从厦门近海海水中及大型经济海藻江蓠表面分离琼胶降解细菌,筛选得到10余株琼胶降解菌,对菌株的生理生化性质进行了初步研究。
克隆其16S rDNA序列,进行分子鉴定并对其中一株高产琼胶酶菌株的产酶条件进行了优化。结果如下:(1)分离得到的琼胶降解菌从菌落形态上可以分为两大类:海水样品中筛选得到(QM1、QM3、QM4、QM6、QM10)的细菌在平板上生长时,分泌的胞外琼胶酶能使琼脂平板松软,在菌落周围产生凹陷或透明圈;从紫菜浸出液中筛选得到(QM2、QM5、QM7、QM8、QM9)的细菌在其菌落周围产生琼胶表面糊化现象;(2)琼胶降解菌理化性质分析结果表明:菌株QM1、QM4、QM5、QM8、QM9均能发酵葡萄糖并产酸;菌株QM5、QM8能产气;菌株QM1、QM4、QM5、QM8、QM9五个菌株均能发酵蔗糖并产酸,但是不产气,仅QM8能发酵乳糖并产酸。QM1、QM2、QM4为好氧菌;QM5和QM8厌氧菌;(3)通过16S rDNA的PCR扩增,得到长度为1.5 kb的片段,测序结果与Genbank数据库进行比对,确定其中的七株菌主要分布在Vibro、Pseudomonas及Proteobacteria等三个属中。其中,QM1与QM11分别鉴定为Vibrio sp. CF4-11和Vibrio shilonii strain SW-2,QM4为一株未培养的海洋细菌,而QM2、QM5、QM8、QM9则分别被鉴定为Pseudomonas sp. LB-2、Gamma proteobacterium B12、Pseudomonas stutzeri及Pseudomonas sp.hyss58;(4)对其中一株高产琼胶酶菌株(QM1)的最佳产酶条件(发酵时间、温度、pH、转速、琼脂浓度、NaCl浓度等)进行了优化,该菌株粗酶液的酶活为3.4 U/mL,最适发酵产酶条件为温度28°C,pH 7.5,转速150 r/m,琼脂浓度0.20%,NaCl浓度2.0%,发酵时间为36 h。
从研究结果可以看出,厦门近海域拥有丰富的琼胶降解细菌资源,为将来琼胶酶的体外表达、酶活性的分析检测及琼胶酶的开发利用奠定了基础。
A preliminary screening for agar-degrading bacteria from the coastal waters of Xiamen and the surface of laver yielded, more than ten isolated with promising agarolytic activity. To investigate the phylogenetic position of these strains, the 16S rDNA sequences were cloned, sequenced and compared with those of related strains. Physiology and biochemistry were also performed.
Clone 16S rDNA sequence for molecular identification in these strains. Then enzyme production conditions of one strain which produced more agarase enzyme were optimized. Results are as follows, (1) agar degrading bacteria isolated from the colony morphology can be divided into two categories, the bacterias isolated from the seawater samples secreted extracellular enzymes to make agar plates soft, dip in or around the colonies clear zone, the bacterias isolated from leachate of seaweed had an phenomena that the agar surface was gelatinizated around the bacterial colonies. (2) physical and chemical properties of agar degradation analysis showed that, five strains, QM1, QM4, QM5, QM8 and QM9, could ferment glucose and produce acid; QM5 and QM8 could produce gas; five strains, QM1, QM4, QM5, QM8 and QM9, could ferment sugar and produce acid, but no gas. only QM8 can ferment lactose and produce acid. Anaerobic and aerobic results showed that, QM1, QM2 and QM4 were aerobic; QM5 and QM8 anaerobic bacteria. (3) a length of 1.5 kb fragment was obtained by the PCR of 16S rDNA. And the sequencing results were compared with the Genbank database. It was confirm that the strains were classified as members of genera of Vibrio, Pseudomonas and Proteobacteria. Respectively, QM1 and QM11 were identified as Vibrio sp. CF4-11 and Vibrio shilonii strain SW-2. QM4 as an uncultured marine bacteria. While QM2, QM5, QM8 and QM9 as Pseudomonas sp. LB-2, Gamma proteobacterium B12, Pseudomonas sp. Hyss58 and Pseudomonas stutzeri. (4) on which a strain(QM1) of the best high agarase enzyme production conditions were optimized, including fermentation time, temperature, pH, speed, agar concentration, NaCl concentration on enzyme production. After optimization of fermentation, the crude enzyme activity of the 3.4 U/ml; optimal conditions for enzyme production temperature of 28°C, pH 7.5, speed 150 r/m, agar concentration of 0.20%, NaCl concentration of 2.0%, fermentation 36 h.
All the results indicated that there were rich resources of agar degrading bacterias on Xiamen coast. It is the basis for vitro agar enzyme expression, activity of the enzyme test and development and utilization of the agar.
克隆其16S rDNA序列,进行分子鉴定并对其中一株高产琼胶酶菌株的产酶条件进行了优化。结果如下:(1)分离得到的琼胶降解菌从菌落形态上可以分为两大类:海水样品中筛选得到(QM1、QM3、QM4、QM6、QM10)的细菌在平板上生长时,分泌的胞外琼胶酶能使琼脂平板松软,在菌落周围产生凹陷或透明圈;从紫菜浸出液中筛选得到(QM2、QM5、QM7、QM8、QM9)的细菌在其菌落周围产生琼胶表面糊化现象;(2)琼胶降解菌理化性质分析结果表明:菌株QM1、QM4、QM5、QM8、QM9均能发酵葡萄糖并产酸;菌株QM5、QM8能产气;菌株QM1、QM4、QM5、QM8、QM9五个菌株均能发酵蔗糖并产酸,但是不产气,仅QM8能发酵乳糖并产酸。QM1、QM2、QM4为好氧菌;QM5和QM8厌氧菌;(3)通过16S rDNA的PCR扩增,得到长度为1.5 kb的片段,测序结果与Genbank数据库进行比对,确定其中的七株菌主要分布在Vibro、Pseudomonas及Proteobacteria等三个属中。其中,QM1与QM11分别鉴定为Vibrio sp. CF4-11和Vibrio shilonii strain SW-2,QM4为一株未培养的海洋细菌,而QM2、QM5、QM8、QM9则分别被鉴定为Pseudomonas sp. LB-2、Gamma proteobacterium B12、Pseudomonas stutzeri及Pseudomonas sp.hyss58;(4)对其中一株高产琼胶酶菌株(QM1)的最佳产酶条件(发酵时间、温度、pH、转速、琼脂浓度、NaCl浓度等)进行了优化,该菌株粗酶液的酶活为3.4 U/mL,最适发酵产酶条件为温度28°C,pH 7.5,转速150 r/m,琼脂浓度0.20%,NaCl浓度2.0%,发酵时间为36 h。
从研究结果可以看出,厦门近海域拥有丰富的琼胶降解细菌资源,为将来琼胶酶的体外表达、酶活性的分析检测及琼胶酶的开发利用奠定了基础。
A preliminary screening for agar-degrading bacteria from the coastal waters of Xiamen and the surface of laver yielded, more than ten isolated with promising agarolytic activity. To investigate the phylogenetic position of these strains, the 16S rDNA sequences were cloned, sequenced and compared with those of related strains. Physiology and biochemistry were also performed.
Clone 16S rDNA sequence for molecular identification in these strains. Then enzyme production conditions of one strain which produced more agarase enzyme were optimized. Results are as follows, (1) agar degrading bacteria isolated from the colony morphology can be divided into two categories, the bacterias isolated from the seawater samples secreted extracellular enzymes to make agar plates soft, dip in or around the colonies clear zone, the bacterias isolated from leachate of seaweed had an phenomena that the agar surface was gelatinizated around the bacterial colonies. (2) physical and chemical properties of agar degradation analysis showed that, five strains, QM1, QM4, QM5, QM8 and QM9, could ferment glucose and produce acid; QM5 and QM8 could produce gas; five strains, QM1, QM4, QM5, QM8 and QM9, could ferment sugar and produce acid, but no gas. only QM8 can ferment lactose and produce acid. Anaerobic and aerobic results showed that, QM1, QM2 and QM4 were aerobic; QM5 and QM8 anaerobic bacteria. (3) a length of 1.5 kb fragment was obtained by the PCR of 16S rDNA. And the sequencing results were compared with the Genbank database. It was confirm that the strains were classified as members of genera of Vibrio, Pseudomonas and Proteobacteria. Respectively, QM1 and QM11 were identified as Vibrio sp. CF4-11 and Vibrio shilonii strain SW-2. QM4 as an uncultured marine bacteria. While QM2, QM5, QM8 and QM9 as Pseudomonas sp. LB-2, Gamma proteobacterium B12, Pseudomonas sp. Hyss58 and Pseudomonas stutzeri. (4) on which a strain(QM1) of the best high agarase enzyme production conditions were optimized, including fermentation time, temperature, pH, speed, agar concentration, NaCl concentration on enzyme production. After optimization of fermentation, the crude enzyme activity of the 3.4 U/ml; optimal conditions for enzyme production temperature of 28°C, pH 7.5, speed 150 r/m, agar concentration of 0.20%, NaCl concentration of 2.0%, fermentation 36 h.
All the results indicated that there were rich resources of agar degrading bacterias on Xiamen coast. It is the basis for vitro agar enzyme expression, activity of the enzyme test and development and utilization of the agar.
引文
[1]杜宗军,赵苑,李美菊等.青岛近海琼胶降解细菌的筛选和多样性分析[J].中国海洋大学学报, 2007, 37(2): 277-282.
[2]缪伏荣,李忠荣.琼胶的降解及其产物的开发应用[J].现代农业科技, 2007, 12(5): 156-158.
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[5] Ha J C, Kim G T, Kim S K.β-Agarase from Pseudomonas sp.W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt in its activity[J]. Biotechnol. Appl. Biochem, 1997, 26(9): 1-6.
[6] Jorge V, Raul A, Erminio M.Identification and Characterization of Its Extracellular Agarase[J]. Applied and Environmental Microbiology, 1998, 64(11): 4378-4383.
[7] Potin P, Richard C, Rochas C. Purification and characterization of theα-agarase from Alteromonas agarolyticus(Cataldi)[J]. Eur.J.Biochem, 1993, 214(13): 599-607.
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[15]刘毅,韩金祥. 16S rRNA基因在脑脊液细菌鉴定中的应用[J].临床检验杂志, 2002, 21(4): 245-246.
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[17]褚艳,于文功,韩峰.琼胶酶高产海洋假单胞菌CY24的筛选及培养条件优化[J].中国海洋药物, 2003, 5(3): 215-218.
[18]韩德权,章佳佳. DNS法在普鲁兰多糖发酵液中糖测定的研究[J].食品工业科技, 2008, 29(2):113-116.
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[21]杨贵明,蒋爱华,薛秋生.用DNS光度法测定还原糖的条件研究[J].安徽农业科学, Journal of Anhui Agri.Sci[J]. 2006, 34(14): 3258-3264.
[22]洪义国,孙谧,张云波,等. 16SrRNA在海洋微生物系统分子分类鉴定及分子检测中的应用[J].海洋水产研究, 2002, 23(1): 58-63.
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[25]陈惠源,蔡俊鹏,刘江涛.海洋细菌产琼胶酶的条件优化[J].现代食品科技, 2005, 21(3): 48-50.
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[27]韩文君,古静燕,李天东,等.一株产琼胶酶土壤细菌的筛选与分子鉴定[J].绵阳师范学院学报, 2007, 26(2): 86-89.
[28]马翠萍,石超.一种新型琼胶酶AgaB的基因克隆及生物信息学分析[J].青岛科技大学学报(自然科学版), 2007, 28(6): 512-515.
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[2]缪伏荣,李忠荣.琼胶的降解及其产物的开发应用[J].现代农业科技, 2007, 12(5): 156-158.
[3] Lora M M, Maitland W M, Frank B W.β-AgaraseⅠandⅡfrom Pesudomonas atlantica Purification and some properties[J]. Eur J Biochem, 1983, 135(1): 553-558.
[4] Sugano Y, Matsumoto T, Kodama H. Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibro sp. strain JT0107[J]. Applied and Environmental Microbiology, 1993, 59(11): 3750-3756.
[5] Ha J C, Kim G T, Kim S K.β-Agarase from Pseudomonas sp.W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt in its activity[J]. Biotechnol. Appl. Biochem, 1997, 26(9): 1-6.
[6] Jorge V, Raul A, Erminio M.Identification and Characterization of Its Extracellular Agarase[J]. Applied and Environmental Microbiology, 1998, 64(11): 4378-4383.
[7] Potin P, Richard C, Rochas C. Purification and characterization of theα-agarase from Alteromonas agarolyticus(Cataldi)[J]. Eur.J.Biochem, 1993, 214(13): 599-607.
[8] Midori K, Akira Y. Agarivorans albus gen.nov,sp.nov.aγ-proteobacterium isolated from marine animals[J]. Int J Syst Evol Microbiol, 2004, 54(6): 693-697.
[9]刘江涛,蔡俊鹏,吴冰.琼胶酶及其综合应用的研究概况[M].现代食品科技, 2005, 11(3): 15-18.
[10] Harmsen D, Karch H. 16S rDNA for diagnosing pathogensallving tree[J]. ASM News, 2004, 70(5): 19-24.
[11]苏维奇,纪迎春,姜岩. 16S rRNA基因检测在临床细菌学鉴定中的应用[J].世界感染杂志, 2005, 5(1): 118-121.
[12] Teng I J, Hsueh P R, Huang Y H. Identification of bacteroides thetaiotaom icron on the basis of an unexpected specific amplicon of universal 16Sribosomal DNA PCR[J]. J Clin Microbiol, 2004, 42(4): 1727-1730.
[13]张芳,宋力. 16S rRNA基冈检测在新生儿败血症早期诊断中的研究进展[J].医学综述, 2006, 12(7): 1035-1037.
[14]杨霞,陈陆,王川庆. 16S rRNA基因序列分析技术在细菌分类中应用的研究进展[J].西北农林科技大学(自然科学版). 2008, 36(2): 105-108.
[15]刘毅,韩金祥. 16S rRNA基因在脑脊液细菌鉴定中的应用[J].临床检验杂志, 2002, 21(4): 245-246.
[16]欧昌荣,汤海青,管斌.琼胶酶生产菌的筛选、鉴定及其酶学性质的初步研究[J].食品科学, 2005, 26(6): 25-29.
[17]褚艳,于文功,韩峰.琼胶酶高产海洋假单胞菌CY24的筛选及培养条件优化[J].中国海洋药物, 2003, 5(3): 215-218.
[18]韩德权,章佳佳. DNS法在普鲁兰多糖发酵液中糖测定的研究[J].食品工业科技, 2008, 29(2):113-116.
[19] Gussow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction[J]. Nucleic Acids Res., 1989, 17(7): 3996-4000.
[20]唐晔盛,李英.菌落PCR在大规模基因组测序中的应用[J].生物化学与生物物理进展, 2002, 29(2): 316-318.
[21]杨贵明,蒋爱华,薛秋生.用DNS光度法测定还原糖的条件研究[J].安徽农业科学, Journal of Anhui Agri.Sci[J]. 2006, 34(14): 3258-3264.
[22]洪义国,孙谧,张云波,等. 16SrRNA在海洋微生物系统分子分类鉴定及分子检测中的应用[J].海洋水产研究, 2002, 23(1): 58-63.
[23]焦振泉,刘秀梅. 16SrRNA序列同源性分析与细菌系统分类鉴定[J].国外医学:卫生学分册, 1998 (1): 12-16.
[24]付万冬,韩宝芹,王常红,等.琼胶降解菌F-6筛选、培养条件及对条斑紫菜细胞解离作用的研究[J].海洋与湖沼, 2007, 38(4): 212-215.
[25]陈惠源,蔡俊鹏,刘江涛.海洋细菌产琼胶酶的条件优化[J].现代食品科技, 2005, 21(3): 48-50.
[26]蔡俊鹏,许少丹.一株海洋细菌所产琼胶酶酶学特性的研究[J].现代食品科技, 2008, 24(12): 62-65.
[27]韩文君,古静燕,李天东,等.一株产琼胶酶土壤细菌的筛选与分子鉴定[J].绵阳师范学院学报, 2007, 26(2): 86-89.
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