产低温纤维素酶菌株的分离鉴定及产酶特征研究
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摘要
自东帕米尔高原布伦口湖群湿地10 cm草甸中分离到一株高酶活低温纤维素酶产生菌BLKC2,经16S rDNA序列鉴定为枯草芽孢杆菌(Bacillus subtilis)。BLKC2在10℃低温条件下保持繁殖能力,在20℃,pH 7.0条件下发酵72 h,酶活力达到最大值28.7 U/mL。酶学特性显示,BLKC2分泌的粗酶最适催化温度为30℃,对热敏感,最适反应pH8.0左右,在pH 7.0～9.0的偏碱性环境中酶活力较稳定,Mn~(2+)、Zn~(2+)、Mg~(2+)对酶反应有明显激活作用,而Fe~(3+)、Cu~(2+)则抑制了酶反应。
A strain BLKC2 producing cold?active cellulase was isolated from 10 cm depth under the sur?face layer at Bulunkou lakes group alpine wetland.It was identified as Bacillus subtilis by16 S rDNA sequence analysis.Strain BLKC2 could still reproduce at 10 ℃.The results of cellulose producing research showed that the optimum temperature was 20 ℃,the optimum pH value was 7.0.Under this condition,the highest cellulose activity reached 28.7 U/mL in fermentation for 72 h.Enzymatic properties showed that the optimum reaction temperature was 30 ℃,but thermal stability of the enzyme was relatively poor.The enzyme showed the highest activity at pH 8.0,and the kept its satisfactory stability at pH 7.0-9.0.Mn~(2+),Zn~(2+),Mg~(2+)were the activators and Fe~(3+),Cu~(2+)were the inhaibitiors of the enzyme.
A strain BLKC2 producing cold?active cellulase was isolated from 10 cm depth under the sur?face layer at Bulunkou lakes group alpine wetland.It was identified as Bacillus subtilis by16 S rDNA sequence analysis.Strain BLKC2 could still reproduce at 10 ℃.The results of cellulose producing research showed that the optimum temperature was 20 ℃,the optimum pH value was 7.0.Under this condition,the highest cellulose activity reached 28.7 U/mL in fermentation for 72 h.Enzymatic properties showed that the optimum reaction temperature was 30 ℃,but thermal stability of the enzyme was relatively poor.The enzyme showed the highest activity at pH 8.0,and the kept its satisfactory stability at pH 7.0-9.0.Mn~(2+),Zn~(2+),Mg~(2+)were the activators and Fe~(3+),Cu~(2+)were the inhaibitiors of the enzyme.
引文
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[20]宋文芳,胡国全,徐彦胜.一株低温厌氧纤维素降解细菌的分离、鉴定及其酶学性质的研究[J].西南农业学报,2011,24(4):1317-1322.Song W F,Hu G Q,Xu Y S.Isolation,identification and enzymic characteristics of cold-adapted,cellulytic,anaerobic bacterium[J].Southwest China Journal of Agricultural Sciences,2011,24(4):1317-1322.
[21]孔雅丽,贾辉,王生荣,等.一株东祁连山耐低温纤维素降解菌株的分离、鉴定及产酶特性[J].工业微生物,2014,44(1):60-66.,Kong Y L,Jia H,Wang S R,et al.Isolation,identification and characterization of a cold-adapted cellulose-degrading strain from eastern Qilian mountains.[J].Industrial Microbiology,2014,44(1):60-66.
[22]Akila G,Chandra T S.A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase[J].Fems Microbiology Letters,2003,219(1):63-67.
[23]尚晓瑛,程旭艳,霍培书,等.1株堆肥耐低温纤维素降解菌的筛选、鉴定及生长特性的初步研究[J].华中农业大学学报,2012,31(5):558-562.Shang X Y,Cheng X Y,Huo P S,et al.Screening,identification and growth of a cold-adapted cellulose-decomposing bacterium for composting[J].Journal of Huazhong Agricultural University,2012,31(5):558-562.
[24]谢永丽,马莉贞,徐志伟,等.冻土荒漠区分离低温适生PGPR菌的鉴定及其抗菌促生特性[J].中国生物防治学报,2014,30(1):94-100.Xie Y L,Ma L Z,Xu Z W,et al.Identification of low-temperature adapting PGPR strains isolated from frozen desert area and their antimicrobial and growth-promoting activity[J].Chinese Journal of Biological Control,2014,30(1):94-100.
[25]谢永丽,高学文.可可西里低温适生拮抗芽孢杆菌的筛选鉴定及脂肽化合物分析[J].应用生态学报,2013,24(1):149-155.Xie Y L,GAO X W.Screening and identification of low temperature-adapted antagonistic bacillus isolated from Kekexili region of west China and the analysis of the isolates lipopeptide compounds.[J].Chinese Journal of Applied Ecology,2013,24(1):149-155.
[26]孟建宇,李蘅,樊兆阳,等.低温纤维素降解菌的分离与鉴定[J].应用与环境生物学报,2014,20(1):152-156.Meng J Y,Li H,Fan Z Y,et al.Isolation and identification of cellulose-degrading bacteria under low temperature[J].Chinese Journal of Applied and Environmental Biology,2014,20(1):152-156.
[27]吕明生,吕凤霞,房耀维,等.低温纤维素酶产生菌的筛选、鉴定及酶学性质初步研究[J].食品科学,2007,28(12):235-239.,LüM S,Lv F X,Fang X W,et al.Screening and identification of a cold-adapted cellulase-producing strain and characterization of cellulase[J].Food Science,2007,28(12):235-239.
[28]王玢,汪天虹.冷活性纤维素酶性质研究[J].海洋科学,2004,28(2):40-42.Wang B,Wang T H.Characteristics of the cold-active cellulase[J].Marine Sciences,2004,28(2):40-42.
[29]黄玉兰,李征,刘晓宁,等.一株耐低温纤维素酶高产菌株的筛选、鉴定和产酶的初步试验[J].微生物学通报,2010,37(5):637-644.Huang Y L,Li Z,Liu X N,et al.Screening and identification of a cold-adapted cellulase-producing bacteria[J].Microbiology China,2010,37(5):637-644.
[30]韩龙,石家骥,石磊,等.一株耐冷玫红假裸囊菌HD1031的鉴定及其所产纤维素酶的研究[J].微生物学通报,2013,40(6):928-938.Han L,Shi J J,Shi L,et al.Identification of a psychrotroph Pseudogymnoascusroseus HD1031and the properties of its producing cellulase[J].Microbiology China,2013,40(6):928-938.
[31]邵娜娜,轩换玲,罗锋,等.一株产低温纤维素酶放线菌的分离与产酶研究[J].食品工业科技,2015,36(24):159-163.Shao N N,Xuan H L,Luo F,et al.Isolation,identification and characterization of a cold-adapted cellulase-producing psychrotrophic Streptomyces sp.[J].Science and Technology of Food Industry,2015,36(24):159-163.
[32]王继莲,李明源,宋保健,等.产低温淀粉酶菌株的筛选、鉴定及酶学性质初步研究[J].农业生物技术学报,2016,24(3):426-434.Wang J L,Li M Y,Song B J,et al.Screening,identification of a high-producing cold-active amylase strain and its amylase characterization[J].Journal of Agricultural Biotechnology,2016,24(3):426-434.
[33]Siddiqui K S,Cavicchioli R.Cold-adapted enzymes[J].Annual Review of Biochemistry,2006,75(1):403-433.
[2]于跃,张剑.纤维素酶降解纤维素机理的研究进展[J].化学通报,2016,79(2):118-122.Yu Y,Zhang J.Research progress in cellulose degradation by cellulase[J].Chemistry,2016,79(2):118-122.
[3]Wang M,Li Z,Fang X,et al.Cellulolytic enzyme production and enzymatic hydrolysis for second-generation bioethanol production[J].Advances in Biochemical Engineering/biotechn-ology,2012,128(1):1-24.
[4]Inoue H,Fujii T,Yoshimi M,et al.Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus[J].Journal of Indust rial Microbiology&Biotechnology,2013,40(8):823-830.
[5]Poszytek K.Microbial cellulose utilization[J].Postepy Mikrobiologii,2016,55(2):132-146.
[6]Shi Z,Zhang Y,Phillips G O,et al.Utilization of bacterial cellulose in food[J].Food Hydrocolloids,2014,35(1):539-545.
[7]何敏超,许敬亮,陈小燕,等.用于生物质酶解过程的纤维素酶研究进展[J].农业工程学报,2016,32(s1):290-296.He M C,Xu J L,Chen X Y,et al.Progress of cellulase that using for biomass hydrolysis process[J].Transactions of the Chinese Society of Agricultural Engineering,2016,32(s1):290-296.
[8]Fang H,Xia L.Cellulose production by recombinant Trichoderma reesei,and its application in enzymatic hydrolysis of agricultural residues[J].Fuel,2015,143:211-216.
[9]Belghith H,Ellouz-Chaabouni S,Gargouri A.Biostoning of denims by Penicillium occitanis(Pol6)cellulases[J].Journal of Biotechnology,2001,89(2/3):257-262.
[10]Pereira B M P,Alvarez T M,Delabona P D S,et al.Cellulase on-site production from sugar cane bagasse using Penicillium echinulatum[J].Bioenergy Research,2013,6(3):1052-1062.
[11]Singhania R R,Sukumaran R K,Rajasree K P,et al.Properties of a majorβ-glucosidaseBGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources[J].Process Biochemistry,2011,46(7):1521-1524.
[12]周若飞,汤斌,李松,等.匍枝根霉淀粉发酵产纤维素酶的条件研究[J].食品与发酵工业,2016,42(6):93-97.Zhou R F,Tang B,LI S,et al.Studies on the fermentation conditions of Rhizopus stolonifer to produce cellulase by starch hydrolysate[J].Food and Fermentation Industries,2016,42(6):93-97.
[13]Margesin R,Schinner F.Low-temperature bioremediation of a waste water contaminated with anionic surfactants and fuel oil[J].Applied Microbiology&Biotechnology,1998,49(4):482-486.
[14]林艳梅,生吉萍,申琳,等.适冷纤维素降解微生物研究进展[J].生物技术,2010,20(2):95-97.Lin Y M,Sheng J P,Shen L,et al.Trends in cold-adapted and cellulose-degrading microorganisms[J].Biotechnology,2010,20(2):95-96.
[15]曾磊,杨太保,田洪阵.近40年东帕米尔高原冰川变化及其对气候的响应[J].干旱区资源与环境,2013,27(5):144-150.Zeng L,Yagn T B,Tian H Z.Response of glacier variations in the eastern Pamirs plateau to climate change,during the last40 years[J].Journal of Arid Land Resources and Environment,2013,27(5):144-150.
[16]郑景雄,周尚哲,王杰,等.东帕米尔高原冰川花岗岩漂砾风化穴初步研究[J].华南师范大学学报(自然科学版),2009,1(3):109-113.Zheng J X,Zhou S Z,Wang J,et al.A preliminary study on tafoni developed on glacial granite bouldersin Eastern Pamir[J].Journal of South China Normal University(Natural Science Edition),2009,1(3):109-113.
[17]孙旭东,黄晓梅,陈秀玲,等.产纤维素酶菌株的筛选鉴定及发酵条件优化[J].基因组学与应用生物学,2014,33(6):1281-1287.Sun X D,Huang X M,Chen X L,et al.Screening and identifying the cellulase-producing bacteria and optimizing fermentation conditions[J].Genomics and Applied Biology,2014,33(6):1281-1287.
[18]沈萍,陈向东.微生物学实验[M].4版.北京:高等教育出版社,2007.Shen P,Chen X D.Experiment of microbiology[M].4th ed.Beijing:Higher Edudcation Press,2007.
[19]姚良同,丁延芹,刘尧,等.玉米秸秆低温快速腐熟菌的筛选、鉴定及效果试验[J].山东农业科学,2008,40(3):85-87.Yao L T,Ding Y Q,Liu Y,et al.Screening and identification of bacteria decomposing maize straw under low temperature and their effect test[J].Shandong Agricultural Sciences,2008,40(3):85-87.
[20]宋文芳,胡国全,徐彦胜.一株低温厌氧纤维素降解细菌的分离、鉴定及其酶学性质的研究[J].西南农业学报,2011,24(4):1317-1322.Song W F,Hu G Q,Xu Y S.Isolation,identification and enzymic characteristics of cold-adapted,cellulytic,anaerobic bacterium[J].Southwest China Journal of Agricultural Sciences,2011,24(4):1317-1322.
[21]孔雅丽,贾辉,王生荣,等.一株东祁连山耐低温纤维素降解菌株的分离、鉴定及产酶特性[J].工业微生物,2014,44(1):60-66.,Kong Y L,Jia H,Wang S R,et al.Isolation,identification and characterization of a cold-adapted cellulose-degrading strain from eastern Qilian mountains.[J].Industrial Microbiology,2014,44(1):60-66.
[22]Akila G,Chandra T S.A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase[J].Fems Microbiology Letters,2003,219(1):63-67.
[23]尚晓瑛,程旭艳,霍培书,等.1株堆肥耐低温纤维素降解菌的筛选、鉴定及生长特性的初步研究[J].华中农业大学学报,2012,31(5):558-562.Shang X Y,Cheng X Y,Huo P S,et al.Screening,identification and growth of a cold-adapted cellulose-decomposing bacterium for composting[J].Journal of Huazhong Agricultural University,2012,31(5):558-562.
[24]谢永丽,马莉贞,徐志伟,等.冻土荒漠区分离低温适生PGPR菌的鉴定及其抗菌促生特性[J].中国生物防治学报,2014,30(1):94-100.Xie Y L,Ma L Z,Xu Z W,et al.Identification of low-temperature adapting PGPR strains isolated from frozen desert area and their antimicrobial and growth-promoting activity[J].Chinese Journal of Biological Control,2014,30(1):94-100.
[25]谢永丽,高学文.可可西里低温适生拮抗芽孢杆菌的筛选鉴定及脂肽化合物分析[J].应用生态学报,2013,24(1):149-155.Xie Y L,GAO X W.Screening and identification of low temperature-adapted antagonistic bacillus isolated from Kekexili region of west China and the analysis of the isolates lipopeptide compounds.[J].Chinese Journal of Applied Ecology,2013,24(1):149-155.
[26]孟建宇,李蘅,樊兆阳,等.低温纤维素降解菌的分离与鉴定[J].应用与环境生物学报,2014,20(1):152-156.Meng J Y,Li H,Fan Z Y,et al.Isolation and identification of cellulose-degrading bacteria under low temperature[J].Chinese Journal of Applied and Environmental Biology,2014,20(1):152-156.
[27]吕明生,吕凤霞,房耀维,等.低温纤维素酶产生菌的筛选、鉴定及酶学性质初步研究[J].食品科学,2007,28(12):235-239.,LüM S,Lv F X,Fang X W,et al.Screening and identification of a cold-adapted cellulase-producing strain and characterization of cellulase[J].Food Science,2007,28(12):235-239.
[28]王玢,汪天虹.冷活性纤维素酶性质研究[J].海洋科学,2004,28(2):40-42.Wang B,Wang T H.Characteristics of the cold-active cellulase[J].Marine Sciences,2004,28(2):40-42.
[29]黄玉兰,李征,刘晓宁,等.一株耐低温纤维素酶高产菌株的筛选、鉴定和产酶的初步试验[J].微生物学通报,2010,37(5):637-644.Huang Y L,Li Z,Liu X N,et al.Screening and identification of a cold-adapted cellulase-producing bacteria[J].Microbiology China,2010,37(5):637-644.
[30]韩龙,石家骥,石磊,等.一株耐冷玫红假裸囊菌HD1031的鉴定及其所产纤维素酶的研究[J].微生物学通报,2013,40(6):928-938.Han L,Shi J J,Shi L,et al.Identification of a psychrotroph Pseudogymnoascusroseus HD1031and the properties of its producing cellulase[J].Microbiology China,2013,40(6):928-938.
[31]邵娜娜,轩换玲,罗锋,等.一株产低温纤维素酶放线菌的分离与产酶研究[J].食品工业科技,2015,36(24):159-163.Shao N N,Xuan H L,Luo F,et al.Isolation,identification and characterization of a cold-adapted cellulase-producing psychrotrophic Streptomyces sp.[J].Science and Technology of Food Industry,2015,36(24):159-163.
[32]王继莲,李明源,宋保健,等.产低温淀粉酶菌株的筛选、鉴定及酶学性质初步研究[J].农业生物技术学报,2016,24(3):426-434.Wang J L,Li M Y,Song B J,et al.Screening,identification of a high-producing cold-active amylase strain and its amylase characterization[J].Journal of Agricultural Biotechnology,2016,24(3):426-434.
[33]Siddiqui K S,Cavicchioli R.Cold-adapted enzymes[J].Annual Review of Biochemistry,2006,75(1):403-433.