一株产纤维素酶的解淀粉芽孢杆菌的分离鉴定及其酶促反应适宜条件初步研究
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摘要
为了分离、筛选产纤维素酶益生菌,确定其酶促反应适宜条件,利用羧甲基纤维素钠等作为筛选培养基,结合刚果红染色法,从玉米青贮饲料样品中筛选产纤维素酶益生菌,并通过形态学和系统进化树方法鉴定分离菌。同时对培养时间、pH和温度等酶促反应条件进行了研究。结果表明,筛选出1株可降解羧甲基纤维素,并产生清晰透明圈的菌株。经形态学观察和16SrRNA基因序列分析,鉴定该菌为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。粗酶液中滤纸酶活为1.5U/mL,外切葡聚糖酶活为1.3U/mL,内切葡聚糖酶活为6.4U/mL,β-葡萄糖苷酶活为2.3U/mL。酶促反应的适宜条件为pH 5.5,温度50℃,粗酶液为培养3d后发酵液上清。解淀粉芽孢杆菌分离株具有一定的产纤维素酶能力,在玉米秸秆生物降解与利用方面具有潜在的开发价值。
In order to isolate and screen strain which can degrade corn stalk,and study its enzyme-reaction conditions,a cellulose-producing strain was isolated from corn silage by carboxymethyl cellulose-Na(CMC-Na)medium and Congo red.Morphological and 16 SrDNA gene were analyzed to identify the taxonomic position of the strain.Meanwhile,the effects of culture time,pH and temperature on the activity of cellulase in the fermentation broth were studied.A strain which could degrade carboxymethyl cellulose-Na was obtained,with a clear hydrolysis circle around it.The morphological and Phylogenetic analysis based on 16 SrDNA sequence showed that the strain was Bacillus amyloliquefacien.In the crude enzyme liquid,the filter paper enzyme activity was 1.5U/mL,exoglucanases activity 1.3U/mL,endoglucanases activity6.4U/mL,β-glucosidases activity 2.3U/mL.The suitable conditions for enzymatic reactions were pH5.5and 50 ℃,the crude enzyme liquid is the supernatant of the fermentation liquid of 3days.The strain has cellulase-producing ability,and potential value in the biodegradation and utilization of corn stalk.
In order to isolate and screen strain which can degrade corn stalk,and study its enzyme-reaction conditions,a cellulose-producing strain was isolated from corn silage by carboxymethyl cellulose-Na(CMC-Na)medium and Congo red.Morphological and 16 SrDNA gene were analyzed to identify the taxonomic position of the strain.Meanwhile,the effects of culture time,pH and temperature on the activity of cellulase in the fermentation broth were studied.A strain which could degrade carboxymethyl cellulose-Na was obtained,with a clear hydrolysis circle around it.The morphological and Phylogenetic analysis based on 16 SrDNA sequence showed that the strain was Bacillus amyloliquefacien.In the crude enzyme liquid,the filter paper enzyme activity was 1.5U/mL,exoglucanases activity 1.3U/mL,endoglucanases activity6.4U/mL,β-glucosidases activity 2.3U/mL.The suitable conditions for enzymatic reactions were pH5.5and 50 ℃,the crude enzyme liquid is the supernatant of the fermentation liquid of 3days.The strain has cellulase-producing ability,and potential value in the biodegradation and utilization of corn stalk.
引文
[1]Mu Guang-Ying.我国主要农作物秸秆综合利用率超过8%[J].农业机械,2016(6):18.
[2]刘宇,史同瑞,朱丹丹,等.玉米秸秆利用现状及其生物发酵技术研究进展[J].中国畜牧杂志,2014,50(6):61-64.
[3]Chandra M S,Viswanath B,Reddy B R,et al.Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by Aspergillus niger[J].Indian J Microbiol,2007,47(4):323-328.
[4]解恒参,赵晓倩.农作物秸秆综合利用的研究进展综述[J].环境科学与管理,2015,40(1):86-90.
[5]王永军,郭航,田秀娥.农作物秸秆饲料化的技术路线与关键技术[J].家畜生态学报,2015,36(12):6-11.
[6]Cunha F M,EsperanaM N,Zangirolami T C,et al.Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulose[J].Bioresource Technology,2012,112(5):270-274.
[7]Gautam S P,Bundela P S,Pandey A K,et al.Optimization for the Production of Cellulase Enzyme from Municipal Solid Waste Residue by Two Novel Cellulolytic Fungi[J].Biotechnology Research International,2011.doi:10.F062//2011/810425.
[8]Sutyak K E,WirawanR E,Aroutcheva A A,et al.Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens[J].Journal of Applied Microbiology,2008,104(4):1 067-1 074.
[9]Gonzalez-ortiz G,Castillejos L,,Allo J J,et al.Effects of dietary supplementation of Bacillus amyloliquefaciens CECT 5940and Enterococcus faecium CECT 4515in adult healthy dogs[J].Archives of Animal Nutrition,2013,67(5):406-415.
[10]Mallo J J,Gracia M I,Honrubia P,et al.Use of a Bacillus amyloliquefaciens probiotic in broiler farms[C].Athens:Poultry science association annual meeting,2010:978.
[11]沈勇涛.芽孢杆菌对母猪仔猪微生态的影响及其生长条件优化[D].武汉:华中农业大学,2011:34-42.
[12]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.饲用纤维素酶活性的测定-滤纸法:GB/T23881-2009[S].北京:中国标准出版社,2009.
[13]中华人民共和国农业部.饲料添加剂纤维素酶活力的测定:NY/T 912-2004[S].北京:中国农业出版社,2005.
[14]殷中伟.秸秆纤维高效降解菌株的筛选及对秸秆降解效果初步研究[D].北京:中国农业科学院,2010.
[15]Soma M,Rangasamy M.Production of cellulose by Aspergillus niger under submerged and solid state fermentation using coir wast as a substrate[J].Brazilian Journal of Microbiology,2011,42(3):1 119-1 127.
[16]顾文杰,徐有权,徐培智.酸性土壤中高效半纤维素降解菌的筛选与鉴定[J].微生物学报,2012,52(10):1 251-1 259.
[17]史通,宋向阳,欧阳嘉,等.黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶[J].生物加工过程,2013,11(5):11-15.
[18]Souza P M,Magalhaes P O.Application of microbia-amylase in industry-A review[J].Braz J Microbiol,2010,41(41):850-861.
[19]曹涵文,吴珑韬,甘乾福,等.熊猫粪便中纤维素降解菌的筛选与鉴定[J].家畜生态学报,2015,36(6):19-25.
[20]宋贤冲,唐健,邓小军,等.产真菌纤维素酶的分离、鉴定及酶学性质分析[J].基因组学与应用生物学,2013,32(3):372-378.
[21]王芳,陈介南,张林,等.产纤维素酶里氏木霉的研究进展[J].中国酿造,2014,33(6):1-5.
[22]姜军坡,朱宝成,王世英,等.以滤纸酶活力为指标优化解淀粉芽孢杆菌Tu-115菌株产纤维素酶液体发酵条件[J].饲料工业,2014,35(20):43-47.
[23]Chandra M S,Viswanath B,Reddy B R,et al.Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by Aspergillus niger[J].Indian J.Microbiol,2007,47(4):323-328.
[2]刘宇,史同瑞,朱丹丹,等.玉米秸秆利用现状及其生物发酵技术研究进展[J].中国畜牧杂志,2014,50(6):61-64.
[3]Chandra M S,Viswanath B,Reddy B R,et al.Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by Aspergillus niger[J].Indian J Microbiol,2007,47(4):323-328.
[4]解恒参,赵晓倩.农作物秸秆综合利用的研究进展综述[J].环境科学与管理,2015,40(1):86-90.
[5]王永军,郭航,田秀娥.农作物秸秆饲料化的技术路线与关键技术[J].家畜生态学报,2015,36(12):6-11.
[6]Cunha F M,EsperanaM N,Zangirolami T C,et al.Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulose[J].Bioresource Technology,2012,112(5):270-274.
[7]Gautam S P,Bundela P S,Pandey A K,et al.Optimization for the Production of Cellulase Enzyme from Municipal Solid Waste Residue by Two Novel Cellulolytic Fungi[J].Biotechnology Research International,2011.doi:10.F062//2011/810425.
[8]Sutyak K E,WirawanR E,Aroutcheva A A,et al.Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens[J].Journal of Applied Microbiology,2008,104(4):1 067-1 074.
[9]Gonzalez-ortiz G,Castillejos L,,Allo J J,et al.Effects of dietary supplementation of Bacillus amyloliquefaciens CECT 5940and Enterococcus faecium CECT 4515in adult healthy dogs[J].Archives of Animal Nutrition,2013,67(5):406-415.
[10]Mallo J J,Gracia M I,Honrubia P,et al.Use of a Bacillus amyloliquefaciens probiotic in broiler farms[C].Athens:Poultry science association annual meeting,2010:978.
[11]沈勇涛.芽孢杆菌对母猪仔猪微生态的影响及其生长条件优化[D].武汉:华中农业大学,2011:34-42.
[12]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.饲用纤维素酶活性的测定-滤纸法:GB/T23881-2009[S].北京:中国标准出版社,2009.
[13]中华人民共和国农业部.饲料添加剂纤维素酶活力的测定:NY/T 912-2004[S].北京:中国农业出版社,2005.
[14]殷中伟.秸秆纤维高效降解菌株的筛选及对秸秆降解效果初步研究[D].北京:中国农业科学院,2010.
[15]Soma M,Rangasamy M.Production of cellulose by Aspergillus niger under submerged and solid state fermentation using coir wast as a substrate[J].Brazilian Journal of Microbiology,2011,42(3):1 119-1 127.
[16]顾文杰,徐有权,徐培智.酸性土壤中高效半纤维素降解菌的筛选与鉴定[J].微生物学报,2012,52(10):1 251-1 259.
[17]史通,宋向阳,欧阳嘉,等.黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶[J].生物加工过程,2013,11(5):11-15.
[18]Souza P M,Magalhaes P O.Application of microbia-amylase in industry-A review[J].Braz J Microbiol,2010,41(41):850-861.
[19]曹涵文,吴珑韬,甘乾福,等.熊猫粪便中纤维素降解菌的筛选与鉴定[J].家畜生态学报,2015,36(6):19-25.
[20]宋贤冲,唐健,邓小军,等.产真菌纤维素酶的分离、鉴定及酶学性质分析[J].基因组学与应用生物学,2013,32(3):372-378.
[21]王芳,陈介南,张林,等.产纤维素酶里氏木霉的研究进展[J].中国酿造,2014,33(6):1-5.
[22]姜军坡,朱宝成,王世英,等.以滤纸酶活力为指标优化解淀粉芽孢杆菌Tu-115菌株产纤维素酶液体发酵条件[J].饲料工业,2014,35(20):43-47.
[23]Chandra M S,Viswanath B,Reddy B R,et al.Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by Aspergillus niger[J].Indian J.Microbiol,2007,47(4):323-328.