吉兰泰盐湖中度嗜盐菌生物学特性的初步研究
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摘要
本论文采用传统的微生物稀释平板法从我国内蒙古自治区吉兰泰盐湖(东经105°42′,北纬39°45′)中分离筛选出23株最适生长盐浓度在12%左右的细菌菌株。对其中6株具有产酶活性的嗜盐菌株进行了表型特征(形态学观察、生理生化试验)分析。在此基础上,对其中一株JLT-15菌株进行了菌株系统鉴定及产蛋白酶特性的初步研究。结果如下:
1.分离筛选得到的23株嗜盐菌株中有6株产酶菌株,其中产淀粉酶2株,产脂肪酶3株,产蛋白酶2株,产甘露聚糖酶1株,产其它酶的菌株未筛到。其中JLT-6与JLT-15产生较低酶活性的脂肪酶和蛋白酶两种酶。
2.对其中一株JLT-15进行了菌株鉴定及产蛋白酶特性研究。该菌株呈杆状,革兰氏阴性,无芽孢,无荚膜,无鞭毛;菌落呈乳白色,圆形,表面光滑且边缘整齐。甲基红和V.P.测定阴性,柠檬酸盐试验阳性;产生吲哚,产H2S,不能利用硝酸盐及亚硝酸盐;能利用葡萄糖、蔗糖、半乳糖作为唯一碳源生长。蛋白酶、酯酶、氧化酶、过氧化氢酶、盐酸联苯胺阳性,不液化明胶,不具淀粉酶、纤维素酶、脲酶活性。通过生长限制因子实验得到该菌株最适生长盐浓度为12%左右,最适生长Mg2+浓度为2%,最适生长pH为7.5,最适培养温度为37℃。结合16S rDNA序列分析初步鉴定该菌株为海杆菌属Marinobacter的一个新菌株,被命名为Marinobacter lipolyticus JLT-15。
3.对JLT-15产生的蛋白酶的特性进行了初步的质研究。JLT-15在pH 7.5～8.5,温度32℃～37℃,接种量1%～4%,盐浓度(NaCl)8%～12%范围内,均具有较高的蛋白酶产量,最适产酶条件为pH 8.0、37℃、12% NaCl和4%接种量。JLT-15所产粗蛋白酶酶液具有以下一般性质:酶的最适pH为8.5,酶的最适作用温度为40℃,该蛋白酶在温度超过60℃时基本丧失了酶活力。
By traditional dilution plate method,the 23strains halophiles, Optimal growth occuring in the presence of 12%(w/v) total salt, are isolated from Jilantai Salt Lake (longitude 105°42 ', latitude 39°45'). we had identified 6 strains halophiles ,having enzyme activity,by phenotype (morphological, physiological and biochemical tests).On this basis, the JLT-15 strain is identified on taxology and studying protease Characteristics from it.The results were reported as follows:
1.There are 6 strains having enzyme activity in 23 strains halophiles:2 strains having amylase,3 strains having lipase,2 strains protease, 1 strains mannandse.JLT-06 and JLT-15 have lipase and protease activity.
2.Identifing JLT-15:it is rod shaped, Gram-negative.The bacterial colony is rotundity, ivory-white and glossy. Methyl red and Voges-Proskauer are negative. Simmons′citrate test is positive. Indole and H2S are positive. Benzidine hydrochloride is positive,Gelatin liquefaction is negative. Protease, esterase,oxidase, catalase, hydrochloric acid and benzidine are positive, but it had non-amylase, cellulose, urease activity. The following compounds are utilized as sole carbon and energy sources: glucose, sucrose, galactose. Optimal growth occurs in the presence of 12%(w/v) total salt, in the presence of 2%(w/v)Mg2+, at 37℃, at pH 7.5.Combining with 16s rDNA sequence analysis, JLT-15 was identified a new strain of Marinobacter and named Marinobacter lipolyticus JLT-15.
3.The results of fermentation showed that The optimal conditions for enzyme production was pH 8.0、37℃、12% NaCl and 4% inoculum rate. And the enzyme production was to follow the growth. The crude enzyme activity was optimal at pH 8.5 and 40℃,and actually, above 60℃,the enzyme lost activity.
1.分离筛选得到的23株嗜盐菌株中有6株产酶菌株,其中产淀粉酶2株,产脂肪酶3株,产蛋白酶2株,产甘露聚糖酶1株,产其它酶的菌株未筛到。其中JLT-6与JLT-15产生较低酶活性的脂肪酶和蛋白酶两种酶。
2.对其中一株JLT-15进行了菌株鉴定及产蛋白酶特性研究。该菌株呈杆状,革兰氏阴性,无芽孢,无荚膜,无鞭毛;菌落呈乳白色,圆形,表面光滑且边缘整齐。甲基红和V.P.测定阴性,柠檬酸盐试验阳性;产生吲哚,产H2S,不能利用硝酸盐及亚硝酸盐;能利用葡萄糖、蔗糖、半乳糖作为唯一碳源生长。蛋白酶、酯酶、氧化酶、过氧化氢酶、盐酸联苯胺阳性,不液化明胶,不具淀粉酶、纤维素酶、脲酶活性。通过生长限制因子实验得到该菌株最适生长盐浓度为12%左右,最适生长Mg2+浓度为2%,最适生长pH为7.5,最适培养温度为37℃。结合16S rDNA序列分析初步鉴定该菌株为海杆菌属Marinobacter的一个新菌株,被命名为Marinobacter lipolyticus JLT-15。
3.对JLT-15产生的蛋白酶的特性进行了初步的质研究。JLT-15在pH 7.5～8.5,温度32℃～37℃,接种量1%～4%,盐浓度(NaCl)8%～12%范围内,均具有较高的蛋白酶产量,最适产酶条件为pH 8.0、37℃、12% NaCl和4%接种量。JLT-15所产粗蛋白酶酶液具有以下一般性质:酶的最适pH为8.5,酶的最适作用温度为40℃,该蛋白酶在温度超过60℃时基本丧失了酶活力。
By traditional dilution plate method,the 23strains halophiles, Optimal growth occuring in the presence of 12%(w/v) total salt, are isolated from Jilantai Salt Lake (longitude 105°42 ', latitude 39°45'). we had identified 6 strains halophiles ,having enzyme activity,by phenotype (morphological, physiological and biochemical tests).On this basis, the JLT-15 strain is identified on taxology and studying protease Characteristics from it.The results were reported as follows:
1.There are 6 strains having enzyme activity in 23 strains halophiles:2 strains having amylase,3 strains having lipase,2 strains protease, 1 strains mannandse.JLT-06 and JLT-15 have lipase and protease activity.
2.Identifing JLT-15:it is rod shaped, Gram-negative.The bacterial colony is rotundity, ivory-white and glossy. Methyl red and Voges-Proskauer are negative. Simmons′citrate test is positive. Indole and H2S are positive. Benzidine hydrochloride is positive,Gelatin liquefaction is negative. Protease, esterase,oxidase, catalase, hydrochloric acid and benzidine are positive, but it had non-amylase, cellulose, urease activity. The following compounds are utilized as sole carbon and energy sources: glucose, sucrose, galactose. Optimal growth occurs in the presence of 12%(w/v) total salt, in the presence of 2%(w/v)Mg2+, at 37℃, at pH 7.5.Combining with 16s rDNA sequence analysis, JLT-15 was identified a new strain of Marinobacter and named Marinobacter lipolyticus JLT-15.
3.The results of fermentation showed that The optimal conditions for enzyme production was pH 8.0、37℃、12% NaCl and 4% inoculum rate. And the enzyme production was to follow the growth. The crude enzyme activity was optimal at pH 8.5 and 40℃,and actually, above 60℃,the enzyme lost activity.
引文
[1] Rothschild LJ,Mancinelli RL.Life in extreme environ-ments[J] .Nature,2001,409:1092~1101.
[2] Javaux EJ.Extreme life on Earth-past,present and possibly bey[J] .Nature,2006,157:37~48.
[3]曹军卫,沈萍,等.嗜极微生物[M],武汉大学出版社,2004.
[4]Michael T Madigan,Aharon Orent.Thermophilic and halophilic extremophlies[J] .Current Opinion in Microbiology,1999,2(3):265~269.
[5]张敏,东秀珠.973项目“极端微生物及其功能利用的基础研究”研究进展[J] .微生物学报,2006,46(2):336.
[6]裴凌鹏,骆海朋.极端微生物浅谈[J] .首都师范大学学报(自然科学版),2003,23(1):49~55.
[7]马延和.新的生命形式-极端微生物[J] .微生物学通报,1999,26(1):16.
[8]周德庆.微生物学教程[M],高等教育出版社,2002,251~256.
[9]任红妍.嗜热微生物[J] .生物学通报,1995,30(3):18.
[10]唐冰,唐晓峰,彭珍荣.嗜冷菌研究进展[J] .微生物学杂志,2002,22(1):51~53.
[11]张洪勋,郝春博,白志辉.嗜酸菌研究进展[J] .微生物学杂志,2006,26(2):68~72.
[12]华洋林,赵继伦,潘力.碱菌的特性及其应用前景[J] .生命的化学,2004,24(4):358~360.
[13] Kushner D J .The halobacteriaceae.In : Sokatch J R ,Ornston L N.ed.The Bacteria,a Treatise on Structure and Function,volⅧ.San Diego:Academic Press,1985,171~206.
[14]任培根,周培瑾.中度嗜盐菌的研究进展[J].微生物学报,2003,43(3):427~431.
[15] Kushner D J.Life in high salt and solute concentrations .halophilic bacteria.In:Kushner DJ.ed.Microbial Life in Extreme Environments[M] .London :Academic Press,1978.317~368.
[16] Ramos-Cormenzana A.Ecol ogy of moderately halophilic bacteria.In:Vreeland R H,Hochstein L I.ed.The Biology of Halo-philic Bacteria[M].Boca Raton:CRC Press,1993.55~86.
[17] LeFevre E.Round L A.A preliminary report upon some halophilic bacteria[J].J Bacteriol,1919,4:177~182.
[18] Baxter R M,Gibbons N E.Effects of sodium and potassium chloride on certain enzymes of Micrococcus halodenitrificans and Pseudomonas salinaria[J].Can J Microbiol,1956,2:599~606.
[19] Ventosa A,Nieto J J,Oren A.Biology of moderately halophilic aerobic bacteria[J] .Microbiol Mol Biol Rev,1998,62 (2):504~544.
[20] Bajpai P.Microbial degradation of pollutions in pulp mill effluents[J].In:Laskin A I,Bennett J W,Gadd G.ed.Advances in Applied Microbiology,vol 48.San Diego:Academic Press ,2001,79~127.
[21] Dobson S J,Franzmann P D.Unification of the genera Deleya ( Baumann,et al.1983),Halomonas (Vreeland,et al.1980),and Halovibrio(Fendrich.1988) and the secies Paracoccus halodenitrificans (Robinson,Gibbons.1952) into a single genus.Halomonas,and lacement of the genus Zymobacter in thefamily Halomonadaceae[J].Int J Syst Bacteriol,1996,46:550~558.
[22] Franzmann P D,Wehmeyer U,Stackebrandt E.Halomonadaceae fam.nov..a newfamily of the class Proteobacteria to accommodate the genera Halomonas and Deleya[J].System Appl Microbiol,1988,11:16~19.
[23] Arahal D R,Ludwig W,Schleifer K H,et al.Phylogeny of the family Halomonadaceae based on 23S and 16S rDNA sequence analysis[J].Int J Syst Evol Microbiol ,2002,52:241~249.
[24] Arahal D R,Marquez M C,Vocani B E,et al.Reclassification of Bacillus marismortui as Salibacillus marismortui comb[J].nov..Int J Syst Evol Microbiol,2000,50(4):1501~1503.
[25] Bajpai P.Microbial degradation of pollutions in pulp mill effluents[J].In:Laskin A I,Bennett J W,Gadd G.ed.Advances in Applied Microbiology,vol 48.San Diego:Academic Press ,2001,130~156.
[26] Arahal D R,Marquez M C,Vocani B E,et al.Reclassification of Bacillus marismortui as Salibacillus marismortui comb[J].nov..Int J Syst Evol Microbiol,2000,50(4):1511~1515.
[27] Arahal D R,Ludwig W,Schleifer K H,et al.Phylogeny of the family Halomonadaceae based on 23S and 16S rDNA sequence analysis[J].Int J Syst Evol Microbiol ,2002,52:257~269.
[28] Brown AD,Simpson JR.Water relations of sugar tolerant yeasts:the role of intracellular pollyols.J Gen Microbiol,1972,72:589~591.
[29] Gupta S,Kuhad R C,Bhushan B.et al.Improved xylanase production from a haloalkaphilic Staphylococcus sp.SG213 usinginexpensive agricultural residues[J].World J Microbiol Biotechnol,2001,17:5~8.
[30] Robert H,Marrec C L,Blanco C.et al. Glycine betaine,carnitine and choline enhance salinity tolerance and prevent the accunulation of sodium to a level inhibiting growth of Tetragenococcus halophila[J].Appl Environ Microbiol.2000,66 (3):509~517.
[31] Eisenberg H.et al,Structure of Halophilic System[J] .Annual Reviews of Biophysics and Biophysical Chemistry,1987,vol.16,69~92.
[32]赵百锁,杨礼富,宋蕾,王慧.中度嗜盐菌在生物技术中的应用[J].微生物学通报,2007,34(2) :359~362.
[33] Moshe M,Felix F,Lisa M.Halophilic enzymes:proteins with a grain of salt[J] .Biophysical Chemistry,2000,86:155~164.
[34]唐兵,周林峰,陈向东,等.微生物学报[J],2000,40 (2):188~192.
[35]张锐,曾润颖.极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J ].微生物学通报.2001,28 (4):5~9.
[36] De Castro RE,MaupinsFurlow JA,Giménez MI,et al.Haloarchaeal proteases and proteolytic systems[J] .FEMS Microbiol Rev,006,30:17~351.
[37]刘彦,何先祺,张义正.耐盐脱毛蛋白酶产生菌的研究[J].皮革科学与工程.1997,7(4):6~10.
[38]江津津,曾庆孝,朱志伟,晁岱秀,颜楠楠.耐盐微生物对鯷制鱼露风味形成的影响[J].食品与发酵工业.2008,34(11):25~28.
[39]张树政.酶制剂工业[M],北京,科学出版社.1984:456~484.
[40] Gibbons N E.Can J Microbiol.195,3:249~255.
[41] Good W A,Hartman P A.J Bacteriol[J].1970,140:601~603.
[42] Nachum R,Bartholomew J W.Bacteriological Proceedings[J] .1969,137~138.Ollivier B,Caumette P,Garcia J L.et al.Anaerobic bacteria from hypersaline environments[J].Microbiol Rev,1994,38 (1):27~38.
[43]董伟,郭立忠,李翠翠,王琳,卢伟东.一株高产酯酶中度嗜盐菌的分离、鉴定及酯酶部分酶学性质的研究[J].微生物学通报.2009,36(4):479~483.
[44] Maltseva O,McInerney M J,Jenneman G E,et al,Applied and Environmental Microbiology,1986,51 (6):1224~1229.Margesin R,Schinner F.Potential of halotolerant and halophilic microorganisms for biotechnology[J].Extremophiles,2001,5:73~83.
[45]东秀珠,蔡妙英,等.常见细菌系统鉴定手册[M]:第一版.北京:科学出版社,2001.
[46]沈萍,范秀容,李广武.微生物学实验[M].北京:高等教育出版社,1999.
[47]陈毓荃.生物化学实验方法和技术[M] .科学出版社,2002.
[48] Boutaiba S,Bhatnagar T,Baratti J C.et al.Preliminary characterisation of a lipolyticactivity from an extremely halophilic archaeon,Natronococcus sp. [J] .Journal of Molecular Catalysis B:Enzymatic,2006,41:21~26.
[49]石万良,钟传奇,唐兵,沈萍.极端嗜盐古生菌(Natrinema sp.)R6~5胞外嗜盐蛋白酶的纯化和性质研究[J] .微生物学报,2007,47(1):161~163.
[50]刘东波,刘建,李雁.一株生孢噬纤维细菌的16S rDNA基因序列分析,长春理工大学学报,2005,28(4):35~38.
[2] Javaux EJ.Extreme life on Earth-past,present and possibly bey[J] .Nature,2006,157:37~48.
[3]曹军卫,沈萍,等.嗜极微生物[M],武汉大学出版社,2004.
[4]Michael T Madigan,Aharon Orent.Thermophilic and halophilic extremophlies[J] .Current Opinion in Microbiology,1999,2(3):265~269.
[5]张敏,东秀珠.973项目“极端微生物及其功能利用的基础研究”研究进展[J] .微生物学报,2006,46(2):336.
[6]裴凌鹏,骆海朋.极端微生物浅谈[J] .首都师范大学学报(自然科学版),2003,23(1):49~55.
[7]马延和.新的生命形式-极端微生物[J] .微生物学通报,1999,26(1):16.
[8]周德庆.微生物学教程[M],高等教育出版社,2002,251~256.
[9]任红妍.嗜热微生物[J] .生物学通报,1995,30(3):18.
[10]唐冰,唐晓峰,彭珍荣.嗜冷菌研究进展[J] .微生物学杂志,2002,22(1):51~53.
[11]张洪勋,郝春博,白志辉.嗜酸菌研究进展[J] .微生物学杂志,2006,26(2):68~72.
[12]华洋林,赵继伦,潘力.碱菌的特性及其应用前景[J] .生命的化学,2004,24(4):358~360.
[13] Kushner D J .The halobacteriaceae.In : Sokatch J R ,Ornston L N.ed.The Bacteria,a Treatise on Structure and Function,volⅧ.San Diego:Academic Press,1985,171~206.
[14]任培根,周培瑾.中度嗜盐菌的研究进展[J].微生物学报,2003,43(3):427~431.
[15] Kushner D J.Life in high salt and solute concentrations .halophilic bacteria.In:Kushner DJ.ed.Microbial Life in Extreme Environments[M] .London :Academic Press,1978.317~368.
[16] Ramos-Cormenzana A.Ecol ogy of moderately halophilic bacteria.In:Vreeland R H,Hochstein L I.ed.The Biology of Halo-philic Bacteria[M].Boca Raton:CRC Press,1993.55~86.
[17] LeFevre E.Round L A.A preliminary report upon some halophilic bacteria[J].J Bacteriol,1919,4:177~182.
[18] Baxter R M,Gibbons N E.Effects of sodium and potassium chloride on certain enzymes of Micrococcus halodenitrificans and Pseudomonas salinaria[J].Can J Microbiol,1956,2:599~606.
[19] Ventosa A,Nieto J J,Oren A.Biology of moderately halophilic aerobic bacteria[J] .Microbiol Mol Biol Rev,1998,62 (2):504~544.
[20] Bajpai P.Microbial degradation of pollutions in pulp mill effluents[J].In:Laskin A I,Bennett J W,Gadd G.ed.Advances in Applied Microbiology,vol 48.San Diego:Academic Press ,2001,79~127.
[21] Dobson S J,Franzmann P D.Unification of the genera Deleya ( Baumann,et al.1983),Halomonas (Vreeland,et al.1980),and Halovibrio(Fendrich.1988) and the secies Paracoccus halodenitrificans (Robinson,Gibbons.1952) into a single genus.Halomonas,and lacement of the genus Zymobacter in thefamily Halomonadaceae[J].Int J Syst Bacteriol,1996,46:550~558.
[22] Franzmann P D,Wehmeyer U,Stackebrandt E.Halomonadaceae fam.nov..a newfamily of the class Proteobacteria to accommodate the genera Halomonas and Deleya[J].System Appl Microbiol,1988,11:16~19.
[23] Arahal D R,Ludwig W,Schleifer K H,et al.Phylogeny of the family Halomonadaceae based on 23S and 16S rDNA sequence analysis[J].Int J Syst Evol Microbiol ,2002,52:241~249.
[24] Arahal D R,Marquez M C,Vocani B E,et al.Reclassification of Bacillus marismortui as Salibacillus marismortui comb[J].nov..Int J Syst Evol Microbiol,2000,50(4):1501~1503.
[25] Bajpai P.Microbial degradation of pollutions in pulp mill effluents[J].In:Laskin A I,Bennett J W,Gadd G.ed.Advances in Applied Microbiology,vol 48.San Diego:Academic Press ,2001,130~156.
[26] Arahal D R,Marquez M C,Vocani B E,et al.Reclassification of Bacillus marismortui as Salibacillus marismortui comb[J].nov..Int J Syst Evol Microbiol,2000,50(4):1511~1515.
[27] Arahal D R,Ludwig W,Schleifer K H,et al.Phylogeny of the family Halomonadaceae based on 23S and 16S rDNA sequence analysis[J].Int J Syst Evol Microbiol ,2002,52:257~269.
[28] Brown AD,Simpson JR.Water relations of sugar tolerant yeasts:the role of intracellular pollyols.J Gen Microbiol,1972,72:589~591.
[29] Gupta S,Kuhad R C,Bhushan B.et al.Improved xylanase production from a haloalkaphilic Staphylococcus sp.SG213 usinginexpensive agricultural residues[J].World J Microbiol Biotechnol,2001,17:5~8.
[30] Robert H,Marrec C L,Blanco C.et al. Glycine betaine,carnitine and choline enhance salinity tolerance and prevent the accunulation of sodium to a level inhibiting growth of Tetragenococcus halophila[J].Appl Environ Microbiol.2000,66 (3):509~517.
[31] Eisenberg H.et al,Structure of Halophilic System[J] .Annual Reviews of Biophysics and Biophysical Chemistry,1987,vol.16,69~92.
[32]赵百锁,杨礼富,宋蕾,王慧.中度嗜盐菌在生物技术中的应用[J].微生物学通报,2007,34(2) :359~362.
[33] Moshe M,Felix F,Lisa M.Halophilic enzymes:proteins with a grain of salt[J] .Biophysical Chemistry,2000,86:155~164.
[34]唐兵,周林峰,陈向东,等.微生物学报[J],2000,40 (2):188~192.
[35]张锐,曾润颖.极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J ].微生物学通报.2001,28 (4):5~9.
[36] De Castro RE,MaupinsFurlow JA,Giménez MI,et al.Haloarchaeal proteases and proteolytic systems[J] .FEMS Microbiol Rev,006,30:17~351.
[37]刘彦,何先祺,张义正.耐盐脱毛蛋白酶产生菌的研究[J].皮革科学与工程.1997,7(4):6~10.
[38]江津津,曾庆孝,朱志伟,晁岱秀,颜楠楠.耐盐微生物对鯷制鱼露风味形成的影响[J].食品与发酵工业.2008,34(11):25~28.
[39]张树政.酶制剂工业[M],北京,科学出版社.1984:456~484.
[40] Gibbons N E.Can J Microbiol.195,3:249~255.
[41] Good W A,Hartman P A.J Bacteriol[J].1970,140:601~603.
[42] Nachum R,Bartholomew J W.Bacteriological Proceedings[J] .1969,137~138.Ollivier B,Caumette P,Garcia J L.et al.Anaerobic bacteria from hypersaline environments[J].Microbiol Rev,1994,38 (1):27~38.
[43]董伟,郭立忠,李翠翠,王琳,卢伟东.一株高产酯酶中度嗜盐菌的分离、鉴定及酯酶部分酶学性质的研究[J].微生物学通报.2009,36(4):479~483.
[44] Maltseva O,McInerney M J,Jenneman G E,et al,Applied and Environmental Microbiology,1986,51 (6):1224~1229.Margesin R,Schinner F.Potential of halotolerant and halophilic microorganisms for biotechnology[J].Extremophiles,2001,5:73~83.
[45]东秀珠,蔡妙英,等.常见细菌系统鉴定手册[M]:第一版.北京:科学出版社,2001.
[46]沈萍,范秀容,李广武.微生物学实验[M].北京:高等教育出版社,1999.
[47]陈毓荃.生物化学实验方法和技术[M] .科学出版社,2002.
[48] Boutaiba S,Bhatnagar T,Baratti J C.et al.Preliminary characterisation of a lipolyticactivity from an extremely halophilic archaeon,Natronococcus sp. [J] .Journal of Molecular Catalysis B:Enzymatic,2006,41:21~26.
[49]石万良,钟传奇,唐兵,沈萍.极端嗜盐古生菌(Natrinema sp.)R6~5胞外嗜盐蛋白酶的纯化和性质研究[J] .微生物学报,2007,47(1):161~163.
[50]刘东波,刘建,李雁.一株生孢噬纤维细菌的16S rDNA基因序列分析,长春理工大学学报,2005,28(4):35~38.