高产纤维素酶真菌的筛选及鉴定
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摘要
该研究旨在从腐殖土中筛选得到高产纤维素酶的菌株。利用刚果红染色作为初筛,菌株酶活测定作为复筛得到3株高产纤维素酶的菌株。通过形态学鉴定与分子生物学鉴定,菌株B03、E15为枝孢菌属(Cladosporium sp.),D11为极细枝孢菌(Cladosporium tenuissimum)。菌株B03在以CMC为底物,28℃、180 r/min条件下培养3 d后,CMC酶活达(427. 62±2. 78) U/m L,FPA酶活为(46. 83±1. 85) U/m L,在以油菜秸秆为底物时其CMC酶活也达到(352. 50±4. 17) U/m L,培养6 d后秸秆降解率达26. 8%,具有较好的降解能力。筛选得到3株高产纤维素酶的真菌菌株,其中菌株B03的纤维素酶活较高,是一株具有研究潜力的纤维素酶菌株。
This study aimed to screen high-level cellulase-producing fungi from humus soil. By primary screening on plates with Congo red staining followed by enzyme activity assay,three isolates were obtained and identified as Cladosporium sp.( isolates B03 and E15) and Cladosporium tenuissimum( D11) based on their morphology and molecular markers. The strain B03 produced cellulase with(427. 62 ± 2. 78) U/m L against CMC and(46. 83 ± 1. 85) U/m L against FPA after being cultured for 3 d at 28℃ and at 180 r/min using CMC as the substrate. By using rape straw as the substrate,it produced(352. 50 ± 4. 17) U/m L CMC activity and degraded 26. 8% of the straw after culturing for6 d. In conclusion,strain B03 produced relatively high-level of cellulose among three selected isolates,which is worth for further study.
This study aimed to screen high-level cellulase-producing fungi from humus soil. By primary screening on plates with Congo red staining followed by enzyme activity assay,three isolates were obtained and identified as Cladosporium sp.( isolates B03 and E15) and Cladosporium tenuissimum( D11) based on their morphology and molecular markers. The strain B03 produced cellulase with(427. 62 ± 2. 78) U/m L against CMC and(46. 83 ± 1. 85) U/m L against FPA after being cultured for 3 d at 28℃ and at 180 r/min using CMC as the substrate. By using rape straw as the substrate,it produced(352. 50 ± 4. 17) U/m L CMC activity and degraded 26. 8% of the straw after culturing for6 d. In conclusion,strain B03 produced relatively high-level of cellulose among three selected isolates,which is worth for further study.
引文
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[20]王洪媛,范丙全.三株高效秸秆纤维素降解真菌的筛选及其降解效果[J].微生物学报,2010,50(7):870-875.
[21] SANDOVALDENIS M,GENE J,SUTTON D A,et al.New species of Cladosporium associated with human and animal infections[J]. Persoonia Molecular Phylogeny&Evolution of Fungi,2016,36(1):281-298.
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[23]陈丽燕,张光祥,黄春萍,等.两株高产纤维素酶细菌的筛选、鉴定及酶学特性[J].微生物学通报,2011,38(4):531-538.
[24]靳冉.枝孢菌Cladosporium sp. Bio-1的木质纤维素降解特性研究[D].长沙:湖南大学,2012.
[2] MOKATSE K P,WYK J V. Relative saccharification of various waste paper materials by cellulase from Aspergillus niger[J]. 2015,3(8):44-46.
[3] ZHENG Y,ZHANG S,MIAO S,et al. Temperature sensitivity of cellulase adsorption on lignin and its impact on enzymatic hydrolysis of lignocellulosic biomass[J]. Journal of Biotechnology,2013,166(3):135.
[4]陈英连.玉米秸秆蛋白发酵饲料中木质纤维素酶高产菌株的选育及应用[D].青岛:中国海洋大学,2014.
[5]郑梦莉,王凯军,张佩华,等.农作物秸秆饲料化技术研究进展[J].中国饲料,2017(11):5-8;14.
[6]柴秀娟.高产纤维素酶菌株的筛选、酶学性质及发酵工艺的研究[D].石河子:石河子大学,2015.
[7] CHIMTONG S,TACHAAPAIKOON C,PASON P,et al.Isolation and characterization of endocellulase-free multienzyme complex from newly isolated Thermoanaerobacterium thermosaccharolyticum strain NOI-1[J]. Journal of Microbiology&Biotechnology,2011,21(3):284.
[8] GUPTA P,SAMANT K,SAHU A. Isolation of cellulosedegrading bacteria and determination of their cellulolytic potential[J]. International Journal of Microbiology,2012,2012(6):578 925.
[9]崔登雪.产纤维素酶芽胞杆菌的筛选鉴定及产酶条件优化[D].哈尔滨:东北农业大学,2017.
[10]赵钰.产纤维素酶菌株的筛选及酶学性质研究[D].沈阳:沈阳农业大学,2017.
[11] KAUR J,CHADHA B S,SAINI H S. Regulation of cellulase production in two thermophilic fungi Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC4520[J]. Enzyme&Microbial Technology,2006,38(7):931-936.
[12] ZHANG Q,LO C M,JU L K. Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30[J]. Bioresource Technology,2007,98(4):753-60.
[13]田云,曹林友,周赓,等.一株纤维素酶高产菌的筛选、鉴定与产酶研究[J].化学与生物工程,2016,33(7):34-39.
[14]李晓秀,张盼,刘琬瑜,等.玉米秸秆降解真菌的筛选及鉴定[J].饲料工业,2017,38(3):37-42.
[15]曾思泉,凌娟,林丽云,等. 1株红树林来源枝孢属真菌的分离鉴定及纤维素酶性质分析[J].微生物学杂志,2018,38(2):37-42.
[16]韩立荣,王永宏,何军,等.枝状枝孢菌(Cladosporium cladosporioides)发酵产酶培养基的响应面法优化[J].浙江大学学报(农业与生命科学版),2010,36(1):56-61.
[17]柴秀娟,李曹龙,孔德真,等.产纤维素酶菌株的筛选、鉴定及产酶条件的优化[J].生物技术通报,2014(9):164-170.
[18]李洋.纤维素酶产生菌的筛选及产酶条件优化[D].大连:大连理工大学,2015.
[19]陆晨.高产纤维素酶菌株的筛选及其产酶条件的优化[D].长沙:中南林业科技大学,2012.
[20]王洪媛,范丙全.三株高效秸秆纤维素降解真菌的筛选及其降解效果[J].微生物学报,2010,50(7):870-875.
[21] SANDOVALDENIS M,GENE J,SUTTON D A,et al.New species of Cladosporium associated with human and animal infections[J]. Persoonia Molecular Phylogeny&Evolution of Fungi,2016,36(1):281-298.
[22] LEUNG H T C,MAAS K R,WILHELM R C,et al.Long-term effects of timber harvesting on hemicellulolytic microbial populations in coniferous forest soils[J]. The ISME Journal,2016,10(2):363-375.
[23]陈丽燕,张光祥,黄春萍,等.两株高产纤维素酶细菌的筛选、鉴定及酶学特性[J].微生物学通报,2011,38(4):531-538.
[24]靳冉.枝孢菌Cladosporium sp. Bio-1的木质纤维素降解特性研究[D].长沙:湖南大学,2012.