南极深海沉积物中产低温脂肪酶菌株的筛选与基因克隆
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摘要
在10℃恒温培养条件下,从第29次南极科学考察获得的南极深海沉积物样品中分离筛选到产脂肪酶细菌3株,产脂肪酶真菌1株,对其进行分子生物学鉴定,确定为假交替单胞菌属(Pseudoalteromonas arctica)、芽孢杆菌属(Bacillus pumilus)、Glaciecola sp.以及白冬孢酵母属(Leucosporidium sp.).通过测定各酶的最适反应温度,获得产低温脂肪酶芽孢杆菌XZ18,该菌所产脂肪酶最适反应温度为30℃,在0℃仍有部分活性.从NaCl浓度和pH值2个条件对产酶条件进行研究,最终确定了该菌最适产酶NaCl浓度5.0%、最适产酶pH值为6,最高产酶量为4.65 U/cm3.通过PCR扩增,获得了脂肪酶全长序列,对该序列进行系统发育分析,发现该脂肪酶基因处于较独立的分支.
Three lipase produced bacterial and 1 lipase produced fungi have been screened from Antarctic polar sediments,they are identified as Pseudoalteromonas arctica,Bacillus pumilus、Glaciecola sp. and Leucosporidium sp.by 16 S rDNA and ITS DNA sequencing. After detection of the enzyme activity of crude enzyme under different temperatures,we obtain a cold-active lipase producetion strain XZ18 with an optimum temperature of lipase activity at30℃ and it remains active at 0℃. By the test of NaCl concentration and pH effects on its lipase production,the optimum conditions of fermentation are found at NaCl concentration 5. 0% and pH 6 with the most productive enzyme quantity at 4. 65 U /cm3. With specific designed primers,one lipase gene in XZ18 strain was amplified and sequenced and identified as an independent branch after phylogenetic analysis.
Three lipase produced bacterial and 1 lipase produced fungi have been screened from Antarctic polar sediments,they are identified as Pseudoalteromonas arctica,Bacillus pumilus、Glaciecola sp. and Leucosporidium sp.by 16 S rDNA and ITS DNA sequencing. After detection of the enzyme activity of crude enzyme under different temperatures,we obtain a cold-active lipase producetion strain XZ18 with an optimum temperature of lipase activity at30℃ and it remains active at 0℃. By the test of NaCl concentration and pH effects on its lipase production,the optimum conditions of fermentation are found at NaCl concentration 5. 0% and pH 6 with the most productive enzyme quantity at 4. 65 U /cm3. With specific designed primers,one lipase gene in XZ18 strain was amplified and sequenced and identified as an independent branch after phylogenetic analysis.
引文
[1]陈秀兰,张玉忠,王运涛,等.深海适冷菌SM9913产生的低温蛋白酶[J].海洋科学,2001,25(1):4-8.
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[15]林容霞,马延和,谭天伟.低温脂肪酶产生菌的筛选及鉴定[J].北京化工大学学报,2006,33(1):31-35.
[16]白树猛,田黎.ITS序列分析在真菌分类鉴定和分子检测中的应用[J].畜牧与饲料科学,2009,30(1):52-53.
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[19]周丽清,李炜煊.胰蛋白酶原-2与淀粉酶、脂肪酶联合检测在急性胰腺炎诊疗中的临床研究[J].国际医药卫生导报,2004,10(8):61-62.
[20]闫云君,舒正玉,杨江科,等.细菌脂肪酶的结构和功能研究进展[J].食品与生物技术学报,2006,4(25):122-126.
[21]Cieslinski H,Kur J,Bialkowska A,et al.Cloning,expression,and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp.22b[J].Protein Expression and Purification,2005,39(1):27-34.
[22]陈贵元,季秀玲,林连兵,等.低温脂肪酶产生菌筛选与鉴定、产酶条件及酶学性质研究[J].云南大学学报:自然科学版,2010,32(1):108-113.
[23]Cieslinski H,Bialkowska A M,Dlugolecka A,et al.A cold-adapted esterase from psychrotrophic Pseudoalteromas sp.strain643A[J].Arch Microbiol,2007,188(1):27-36.
[24]de Pascale D,Angela M C,Flavia A,et al.The cold-active Lip1 lipase from the Antarctic bacterium Pseudoalteromonas haloplankis TAC125 is a member of a new bacterial lipolytic enzyme family[J].Extremophiles,2008,12(3):311-323.
[25]邵铁娟,孙谧,郑家声,等.Bohaisea-9145海洋低温碱性脂肪酶研究[J].微生物学报,2004,44(6):789-793.
[26]俞勇,李会荣,陈波,等.低温脂肪酶产生菌的筛选、鉴定及其部分酶学性质[J].高技术通讯,2003(10):89-93.
[2]曾胤新,陈波,邹扬,等.极地微生物:新天然药物的潜在来源[J].微生物学报,2008,48(5):659-700.
[3]徐国英,林学政,王能飞,等.产低温蛋白酶极地菌株的筛选及Pseudoalteromonas sp.QI21产蛋白酶粗酶性质[J].生物加工工程,2008,8(2):55-60.
[4]Deming J W.Psychrophiles and polar regions[J].Current Opinion in Microbiology,2002,5(3):301-309.
[5]Jaeger K E,Dijkstra B W,Reetz M T.Bacterial biocatalysts:molecular biology,three-dimensional structures,and biotechnological applications of lipases[J].Annual Review of Microbiology,1999,53:315-351.
[6]Hasan F,Shah A A,Hameed A.Industrial applications of microbial lipases[J].Enzyme and Microbial Technology,2006,39(2):235-251.
[7]张勇,孙新城,王开放,等.一株细菌产生的脂肪酶性质研究[J].湖北农业科学,2011(1):53-54.
[8]Zhang J W,Lin S,Zeng R Y.Cloning,expression and characterization of a cold-adapted lipase gene from an Antarctic deepsea psychrotrophic bacterium,Psychrobacter sp.7195[J].Microbiol Biotechnol,2007,17(4):604-610.
[9]Joseph B,Ramteke P W,Thomas G,et al.Standard Review Cold-active microbial Lipases:a versatile tool for industrial applications[J].Biotech Mol Biology Review Vol,2007,2(2):39-48.
[10]Lo Giudice A,Michaud L,de Pascale D,et al.Lipolytic activity of Antarctic cold-adapted marine bacteria(Terra Nova Bay,Ross Sea)[J].Appl Microbiol Biotechnol,2006,101:1 039-1 048.
[11]Rashid N,Shimada Y,et al.Low-temperature lipase from psychrotrophic Pseudomonas sp.strain KB700A[J].Applied and Environmental Microbiology,2001,67(9):4 064-4 069.
[12]Gerday C,Aittaleb M,Arpigny J L,et al.Psychrophilic enzymes:a themodynamic challenger[J].Biochim Biophys Acta,1997,1 342(2):119-131.
[13]Jean L A,Jean L,Charles G,et al.Molecular adaption to cold of Antaretic bacterial lipase[J].Journal of Molecular Catalysis B:Enzymatic,1997,3:29-35.
[14]董明奇,史岩,姜春雷,等.脂肪酶高产菌株的筛选及酶学特性研究[J].四川大学学报:自然科学版,2008,45(4):985-989.
[15]林容霞,马延和,谭天伟.低温脂肪酶产生菌的筛选及鉴定[J].北京化工大学学报,2006,33(1):31-35.
[16]白树猛,田黎.ITS序列分析在真菌分类鉴定和分子检测中的应用[J].畜牧与饲料科学,2009,30(1):52-53.
[17]Gandi N N.Application of lipase[J].Journal of the American Oil Chemists’Society,1997,74:621-634.
[18]张迎光,施晓聪,林全思,等.天然高产脂肪酶细菌的分离鉴定及脂肪酶基因的分析[J].温州医学院学报,2009,36(9):541-545.
[19]周丽清,李炜煊.胰蛋白酶原-2与淀粉酶、脂肪酶联合检测在急性胰腺炎诊疗中的临床研究[J].国际医药卫生导报,2004,10(8):61-62.
[20]闫云君,舒正玉,杨江科,等.细菌脂肪酶的结构和功能研究进展[J].食品与生物技术学报,2006,4(25):122-126.
[21]Cieslinski H,Kur J,Bialkowska A,et al.Cloning,expression,and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp.22b[J].Protein Expression and Purification,2005,39(1):27-34.
[22]陈贵元,季秀玲,林连兵,等.低温脂肪酶产生菌筛选与鉴定、产酶条件及酶学性质研究[J].云南大学学报:自然科学版,2010,32(1):108-113.
[23]Cieslinski H,Bialkowska A M,Dlugolecka A,et al.A cold-adapted esterase from psychrotrophic Pseudoalteromas sp.strain643A[J].Arch Microbiol,2007,188(1):27-36.
[24]de Pascale D,Angela M C,Flavia A,et al.The cold-active Lip1 lipase from the Antarctic bacterium Pseudoalteromonas haloplankis TAC125 is a member of a new bacterial lipolytic enzyme family[J].Extremophiles,2008,12(3):311-323.
[25]邵铁娟,孙谧,郑家声,等.Bohaisea-9145海洋低温碱性脂肪酶研究[J].微生物学报,2004,44(6):789-793.
[26]俞勇,李会荣,陈波,等.低温脂肪酶产生菌的筛选、鉴定及其部分酶学性质[J].高技术通讯,2003(10):89-93.